WO2007113689A2 - Use of ww protein domain for inhibiting activation of map kinases - Google Patents

Abstract

There is provided a method of inhibiting activation of a MAP kinase comprising contacting the MAP kinase with a WW domain. There is further provided a method of inhibiting a cell having increased activation of a MAP kinase comprising administering to the cell an effective amount of a molecule that increases cellular levels of a polypeptide comprising at least a WW domain and a method of treating a disease state characterized by the presence of cells that have increased activation of a MAP kinase, in a patient in need of such treatment, comprising administering to the patient an effective amount of a polypeptide comprising at least a WW domain.

Description

USE OF A WW PROTEIN DOMAIN FOR INHIBITING ACTIVATION OF MAP KINASES

CROSS-REFERENCE TO RELATED APPLICATION

[0001 ] This application claims benefit and priority from U.S. provisional patent application No. 60/787,635, filed on March 31, 2006, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to inhibitors of MAP kinase activation, and particularly to polypeptide inhibitors of MAP kinase activation.

BACKGROUND OF THE INVENTION [0003] Rho GTPases and GAPs:

[0004] Rho GTPases constitute a distinct subfamily of the small GTPase superfamily. Principally, the Rho GTPases regulate the actin cytoskeleton, but they are also involved in the control of cell polarity, microtubule dynamics, membrane transport pathways and gene transcription (Etienne-Manneville, S. & Hall, A. Rho GTPases in cell biology. Nature 420, 629-635 (2002); Hall, A. & Nobes, CD. Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton. Philos. Trans. R. Soc. Lond B Biol Sci 355, 965-970 (2000)).

[0005] The switch like behavior of Rho GTPases is tightly regulated through binding to GTP (active 'ON' state) or GDP (inactive 'OFF' state) (Jaffe, A.B. & Hall, A. Rho GTPases in transformation and metastasis. Adv. Cancer Res 84, 57-80 (2002)). Furthermore, they possess weak intrinsic GTP hydrolysis function. In the 'ON' GTP- bound state, Rho GTPases interact with target effector proteins and generate a response until GTP hydrolysis returns the switch to the 'OFF' GDP-bound state.

[0006] The human genome contains 22 Rho GTPases, 80 Guanine nucleotide

Exchange Factors (GEFs) that serve as activators by catalyzing the GDP exchange by GTP and about 80 GTPase-Activating Proteins (GAPs) which bind to Rho proteins to increase the rate of hydrolysis of bound GTP thereby inactivating their GTPase activities (Jaffe & Hall, supra). Consequently GAPs are important negative regulators of Rho signalling (Moon, S. Y. & Zheng, Y. Rho GTPase-activating proteins in cell regulation. Trends CellBiol 13, 13-22 (2003); Bernards, A. & SettlemanJ. GAPs in growth factor signalling. Growth Factors 23, 143-149 (2005); Bernards, A. & Settleman, J. GAP control: regulating the regulators of small GTPases. Trends Cell Biol 14, 377-385 (2004); Bernards, A. GAPs galore! A survey of putative Ras superfamily GTPase activating proteins in man and Drosophila. Biochim. Biophys Acta 1603, 47-82 (2003)). In addition, four Guanine nucleotide Dissociation Inhibitors (GDIs) whose roles appear to be to block the spontaneous activation of Rho GTPase have been identified. Although the Rho switch itself is straight-forward, the regulation of its activity is finely controlled. Taking into account in the number of Rho, GEF, GAP, GDI and downstream effector proteins, as well as spatial and temporal regulation of these components.

[0007] The family of Rho GTPases are multi-functional molecular switches that have been implicated in many physiological processes. Signaling through Rho GTPases, GEFs and GAPs control a large number of biological processes including cell division, movement, gene expression and dynamic changes in shape and structure required for cell functions. The regulation of Rho GTPase activities are tightly controlled through the balance of the proportion of active GTP-bound form of the Rho GTPase to the inactive GDP-bound form. While the loading of GTP onto Rho GTPases is performed by the Rho GTPase Exchange Factors (Rho GEFs), the inactivation of the Rho GTPases is expedited by the Rho GTPase Activating Proteins (Rho GAPs). Consequently the Rho GEFs and GAPs are important signaling molecules that control Rho GTPase signaling and themselves are subjected to regulation of their activities. In addition the Rho GEFs and GAPs are usually multi- domain in structure which allows them to interact with other signaling molecules that may not directly function in Rho signaling to allow for cross-talking among different signaling pathways and formation of complex signaling networks.

[0008] Given the direct and indirect impact of the Rho GTPase signaling in normal development, it is pertinent to understand as comprehensively as possible Rho signaling mechanisms and the extensive signaling networks that are controlled by Rho GTPases and the Rho effectors and regulators (Rho GEFs, Rho GAPs and others). Importantly, several human diseases including cancer immune and neurological disorders have been found to have a basis in mis-expression or genetic mutations in either Rho GTPases or Rho effectors and regulators (Rho GEFs, Rho GAPs and others).

[0009] Although the function of the majority of Rho proteins and their regulators in various physiological processes are largely unknown, the abundance and discovery of mutations in a number of these genes in human diseases highlighted the importance of Rho GTPase signaling networks in development (Sahai, E. & Marshall, CJ. RHO-GTPases and cancer. Nat Rev Cancer 2, 133-142 (2002); Ramakers, GJ. Rho proteins and the cellular mechanisms of mental retardation. Am J Med Genet 94, 367-371 (2000)).

Table 1 - Aberrant Rho signaling in cancer

Table 2 - Aberrant Rho signaling in mental retardation

[0010] One of the established functions of Rho GTPases is the regulation of the actin cytoskeleton during neuronal migration, axon growth and guidance, and formation of synapses (Luo, L. Rho GTPases in neuronal morphogenesis. Nat Rev Neurosci. 1, 173-180 (2000)). Consistent with this function, several neuronal specific Rho GAPs have been found to play key roles in neuronal development. For example, Νadrin was shown to be involved in Ca2+-dependent exocytosis which is essential for neuronal synaptic transmission (Harada, A. et al. Νadrin, a novel neuron-specific GTPase- activating protein involved in regulated exocytosis. J Biol Chem 275, 36885-36891 (2000)). Studies in pi 90 Rho GAP knockout mice showed that the Rho GAP is required for axon outgrowth, guidance and fasciculation and neuronal morphogenesis (Tcherkezian, J., Danek, E.I., Jenna, S., Triki, I. & Lamarche-Vane, Ν. Extracellular signal regulated kinase 1 interacts with and phosphorylates CdGAP at an important regulatory site. MoI Cell Biol 25, 6314-6329 (2005); Brouns, M.R., Matheson, S.F. & Settleman, J. pi 90 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation. Nat Cell Biol 3, 361-367 (2001); Brouns, M.R. et al. The adhesion signaling molecule pl90 RhoGAP is required for morphogenetic processes in neural development. Development 127, 4891-4903 (2000)). Genetic aberrations in two neuronal Rho GAPs, namely oligophrenin and MeGAP/srGAP3, had been shown to be the underlying causes for two distinct types of human mental retardation (Billuart,P. et al. Oligophrenin 1 encodes a rho-GAP protein involved in X- linked mental retardation. Pathol. Biol (Paris) 46, 678 (1998); Endris,V. et al. The novel Rho-GTPase activating gene MEGAP/ srGAP3 has a putative role in severe mental retardation. Proc Natl Acad Sci U S A 99, 11754-11759 (2002)). Rho GTPases have also been implicated in many aspects of cell growth, differentiation, tumorigenesis and metastasis (Jaffe & Hall, supra; Sahai & Marshall, supra). The Rho GAP member DLCl (Deleted in Liver Cancer 1) had been shown to be deleted in 44% of primary hepatocellular carcinoma (HCC) and 90% of HCC cell lines (Sahai & Marshall, supra; Ng, 1.0. , Liang, Z.D., Cao, L. & Lee, T.K. DLC-I is deleted in primary hepatocellular carcinoma and exerts inhibitory effects on the proliferation of hepatoma cell lines with deleted DLC-I. Cancer Res 60, 6581-6584 (2000); Yuan, B. Z. et al. Cloning, characterization, and chromosomal localization of a gene frequently deleted in human liver cancer (DLC-I) homologous to rat RhoGAP. Cancer Res 58, 2196-2199 (1998)). In cell-based studies, DLCl overexpression significantly inhibited cell proliferation, anchorage-dependent growth, and in vivo tumourigenicity, motility and invasiveness when stably expressed in HCC cell lines (Sahai & Marshall, supra; Yuan, B. Z. et al. DLC-I gene inhibits human breast cancer cell growth and in vivo tumorigenicity. Oncogene 22, 445-450 (2003)). GRAF, the focal adhesion kinase associated Rho GAP, was identified as a fusion partner of the mixed-lineage leukemia (MLL) gene by unique chromosome translocation in juvenile myelomonocytic leukemia (Borkhardt, A. et al. The human GRAF gene is fused to MLL in a unique t(5;l l)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q. Proc Natl Acad Sci U S A 97, 9168-9173 (2000)). Deletion, point mutation and insertion of GRAF had been found in patients. The fusion of BCR with ABL oncogene is a leukemia-associated chromosomal translocation which resulted in the fusion proteins pl20 and pl90 that lacked the Rho GAP domain of BCR (Shtivelman, E., Lifshitz, B., Gale, R.P. & Canaani, E. Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature 315, 550-554 (1985)).

[0011 ] The Rho GAP family is defined by the presence of a conserved Rho

GAP domain in the primary sequences that is about 150 amino acids and shares at least 20% sequence homology amongst the family members (Moon & Zheng, supra; Shtivelman, et al., supra; Donovan, S., Shannon, K.M. & Bollag, G. GTPase activating proteins: critical regulators of intracellular signaling. Biochim. Biophys Acta 1602, 23- 45 (2002)). The binding of GTP-bound Rho proteins to the Rho GAP domain is sufficient for acceleration of Rho GTPase activity.

[0012] In addition to the Rho GAP domain, a number of Rho GAPs possess other protein or lipid interaction domains including SH2, FF, SH3, PH and WW, indicating that they are involved in molecular interactions and may control signal transduction in ways other than just activating the Rho GTPases through the Rho GAP domain. These interactions could also provide additional mechanisms by which the Rho GAP activity is being regulated through interactions with other molecules.

[0013] One subfamily of recently discovered Rho GAP proteins, which includes

ArhGAP9 and ArhGAP12, has been found to contain an interesting combination of functional protein domains, including Rho GAP, SH3, WW and PH domains (Zhang, Z. et al. Cloning and characterization of ARHGAP 12, a novel human rhoGAP gene. Int. J Biochem Cell Biol 34, 325-331 (2002); Furukawa,Y. et al. Isolation of a novel human gene, ARHGAP9, encoding a rho- GTPase activating protein. Biochem Biophys Res Commun 284, 643-649 (2001)).

[0014] In mammals, ArhGAP12 is highly expressed in brain tissue and neuronal stem cells, while ArhGAP9 is predominantly expressed in the haematopoietic compartments, including thymus, bone marrow and spleen. Reports also have shown that ArhGAP9 is expressed predominantly in T-cell leukemia and myeloid or Hodgkin Lymphoma cell lines. ArhGAP9 was reported to be active towards cdc42 and Racl but not RhoA and to repress the adhesion of a human leukemia cell line KG-I to fibronectin and collagen (Furukawa, Y. et al, supra).

[0015] ArhGAP15 is possibly a distantly related isoform of ArhGAP9 and

ArhGAP12 that lacks the SH3 and WW domains, while ArhGAP27 possibly represents another distant isoform lacking the SH3 domain (Seoh, M. L., Ng, C. H., Yong, J., Lim, L. & Leung, T. ArhGAP15, a novel human RacGAP protein with GTPase binding property. FEBS Lett 539, 131-137 (2003); Katoh, Y. & Katoh, M. Identification and characterization of ARHGAP27 gene in silico. Int. JMoI Med 14, 943-947 (2004)).

[0016] It was recently reported that CAMGAPl, which also contains the Rho

GAP, SH3, WW and PH domains and closely related to ArhGAP15 and ArhGAP27, may be involved in clathrin-mediated endocytosis of transferring and membrane receptors in a signal-dependent manner through association with the adaptor protein CIN85 (Sakakibara,T., Nemoto,Y., Nukiwa,T. & Takeshima,H. Identification and characterization of a novel Rho GTPase activating protein implicated in receptor- mediated endocytosis. FEBS Lett 566, 294-300 (2004)).

[0017] The Rho GAP proteins have been known to be key negative regulators of Rho GTPase activities. Given the importance and functional diversity of Rho GTPase signaling networks in cell and developmental biology, it is not surprising that the Rho GAPs are subjected to multiple mechanisms of control of their GAP activities, as well as cross-talking with other signaling pathways to allow a fine spatial and temporal coordination of signal outcomes. One way that Rho GAPs are able to achieve these fine tuning mechanisms of control and cross-talk appears to be acquiring multiple protein interaction domains that associate with other signaling molecules. Through these interactions, different proteins could be organized into the same complex to allow for allosteric control or covalent modification to alter enzyme activities, as well as spatial and temporal coordination of different signals towards a final cellular outcome.

[0018] MAP Kinases

[0019] Mitogen-activated protein kinases (MAPKs) are important mediators of signal transduction and play a key role in the regulation of many cellular processes, such as cell growth and proliferation, differentiation, and apoptosis. In mammalian cells, three major MAPK signaling cascades have been identified, namely extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (Jnk) and p38 MAPK. It is well documented that Erk is typically stimulated by growth-related signals, whereas the Jnk and p38 MAPK cascades are activated by various stress stimuli.

[0020] The MAP (Mitogen Activated Protein) kinase pathways, which include the Erk, p38 and Jnk/Sapk pathways have a major role in cellular growth, differentiation, movement and gene transcription. The various MAP kinases (MAPKs) are involved in transducing signals from the cell surface to the nucleus, resulting in regulation of transcription of particular genes in response to an external signal.

[0021] The Erk family of MAPKs has been implicated to play a role in initiating cellular changes in response to a wide variety of extracellular signals and stimuli and has been linked to mitogen independence for oncogenes including Ras, Raf, Jun and Myc. The p38 family of MAPKs is involved in regulating cellular response to stress stimuli, and regulates genes involved in the production and regulation of various pro-inflammatory cytokines, including TNF-α, IL-I, IL-6 and IL-8. p38 MAPKs may also play a role in promoting apoptosis, in negative regulation of the cell cycle and in mediating inflammatory responses. The role of Jnk MAPKs is less well understood, but Jnk MAPKs are known to play a role in cellular growth, differentiation, apoptosis and transformation.

[0022] Not surprisingly, constitutive activation of MAPKs has been reported in various diseases, including in various cancers and in rheumatoid arthritis. Thus, MAPKs are frequently the target for inhibition in treatment in a variety of different diseases and disorders.

[0023] The 3 main classes of MAPKs:

[0024] MAPKs signal to affect gene expression as well as non-transcriptional processes such as cell structural changes and are implicated in a very large number of physiological processes. Defective MAPK signaling cascades have been implicated in various diseases such as cancer, inflammatory and nervous system disorders. (Lluis et al. 2005 Trends in cell Biology 16 : 1: 36; Engel et al 2005 Genes and Development

19(10): 1175-87; Sharma-Walia et al 2005 Jounral of virology 79:16: 10308). [0025] MAPK in hematologic malignancies and inflammation: Numerous studies have shown that the MAPK pathway is essential for the hematopoietic growth factors to regulate normal hematopoiesis. The most well-established is Erythropoietin (Epo) which activates Erk, p38 and Jnk. Together with the stem cell factor (SCF), both factors synergistically activate Erkl and 2 to induce cell growth as well as the production of many hematopoietic growth factors including Epo itself. Disruption in p38 alpha gene in mice will result in diminished erythropoietin gene expression. Hence, p38 appears to be important in the development of erythropoiesis through regulation of erythropoietin expression. On the other hand, p38 is also involved in erythropoietin- dependent differentiation of erythroid cells. Apart from the activation by hematopoietic growth factors, the MAPK are also activated by cytokines that negatively regulate normal hematopoiesis such as type I interferons which act as strong inhibitors of the growth of hematopoietic progenitors. p38 plays an important role in type I interferon signaling. Inhibition with p38 pharmacologic inhibitors can reverse the suppressive effect of type I interferon, indicating the importance of p38 as a signaling mediator for growth inhibitory signals generated by different myelosuppressive cytokines.

[0026] Studies have also shown the importance of the MAPK pathways in the pathogenesis and development of leukemia. Leukemia can be broadly categorized into 4 different types, namely acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemis (CML) and chronic lymphocytic leukemia (CLL). The Raf/Mek/Erk signaling cascade has been known to be implicated in acute and chronic human leukemias. It is clearly established that in majority of primary acute leukemia cases (AML and ALL), the Erk pathway is constitutively activated and mediates mitogenic signals. Inhibition of this pathway may spell a possible effective therapy for the acute leukemia. As for chronic myelogenous leukemis (CML), Erk signaling cascade is also involved. Inhibition of the signaling cascade of Erk appears to be effective in inducing apoptosis of the cancer cells. In chronic lymphocytic leukemia (CLL), however, there is no constitutive activation of Erkl or 2 and the kinases do not appear to play a role in maintaining cell survival.

[0027] Tumor necrosis factor alpha (TNF alpha) has been shown to induce autocrine regulation of the growth of several lymphoma or leukemia cell lines, alone or in combination with other growth factors. Numerous studies have shown that p38 can both regulate TNF alpha production and also, upon its activation by the latter, mediate signals and regulate growth of lymphoma/leukemia cells. In Burkitt lymphoma cell lines, p38 pathway has been implicated in the regulation of interleukin-10 (IL-IO). This cytokines normally regulates growth and differentiation of B cells. The Epstein-Barr virus latent membrane proteinl (LMPl) induces expression of IL-IO in Burkitt lymphoma cells lines in a p38 dependent manner. Together with other evidence, MAPK pathways are suggested to be involved in the pathogenesis of Epstein Barr virus-related lymphomas. Hence, this raises the possibility that selective pharmacologic inhibitors of MAPK may find clinical application in the treatment of these lymphomas types in the future. Beside p38, the Erk pathway (Raf/Mek/Erk) in cooperation with PI-3' kinase pathway has also been found to be implicated in the suppression of Fas-induced apoptosis in lymphoma cells. In addition to the above, various studies have shown that the MAPK pathways also play roles in growth factor loops that promote cell proliferation of the malignant cells in Hodgkin disease. Aberrant expression of c-Jun and JunB, which are the downstream effectors of the MAPK, are seen in the proliferation of malignant Hodgkin lymphomas cells indicating the importance of the kinases in the development of the disease. Furthermore, many investigations in the Hodgkin diseases cell lines have shown that Erk, p38 and Jnk are activated in response to the receptor activator NF-κB ligand (RANKL).

[0028] MAPK is also known to be implicated in multiple myeloma. The most important myeloma growth factor is interleukin-6 (IL-6), a cytokine that is produced by myeloma cells in an autocrine or paracrine manner and promotes their survival in vitro and in vivo. IL-6 activates multiple signaling cascades which includes the Erk pathway. The Erk pathway functions to mediate signals that promote malignant myeloma cell proliferation. Other than interleukin-6, insulin-like growth factor 1 (IGF-I) also play roles in the growth of malignant myeloma. Like IL-6, IGF-I also activates the Erk pathway in multiple myeloma cell lines which further strengthening the importance of MAPK pathway in the development of myeloma.

[0029] The p38 MAPK is strongly implicated in cytokine-related inflammatory response. One of its roles is to regulate cytokine expression and signaling. Thus efforts have been taken to design inhibitors for this MAPK so as to provide therapeutics for inflammatory diseases. Currently, a number of p38 pharmacologic inhibitors have been discovered and many of these are still undergoing clinical trials.

[0030] MAPK signaling inhibition in oncology: In tumour cells, constitutive

MAPK activation confers increased proliferation and resistance to apoptotic stimuli, including classical cytotoxic drugs. In most instances, however, MAPK inhibition has cytostatic rather than cytotoxic effects, which may explain the lack of objective responses observed in early clinical trials of MEK inhibitors. Nevertheless, amenability of the MAPK pathway to pharmacodynamic evaluation and negligible clinical toxicity make MEK inhibitors an ideal platform to build pharmacological combinations with synergistic anti-tumour activity. In the case of AML, the MEK/MAPK pathway is constitutively activated in the majority of cases (75%), conferring a uniformly poor prognosis; in preclinical models of AML, MEK blockade profoundly inhibits cell growth and proliferation and down-regulates the expression of several anti-apoptotic players, thereby lowering the apoptotic threshold. Apoptosis induction, however, requires concentrations of MEK inhibitors much higher than those required to inhibit proliferation. Nevertheless, MEK blockade efficiently and selectively sensitizes leukemic cells to sub-optimal doses of other apoptotic stimuli, including classical cytotoxics (nucleoside analogs, microtubule-targeted drugs, γ-irradiation), biologicals (retinoids, interferons, arsenic trioxide), and, most interestingly, other signal transduction/apoptosis modulators (UCN-01, STI571, Bcl-2 antagonists). In most instances, these MEK inhibition-based combinations result in a significant pro- apoptotic synergism in preclinical models. Therefore MAPK pathway inhibition could play a prominent role in the development of integrated therapeutic strategies aimed at synergistic anti-leukemic effects. Since the MAPK pathway is hyperactivated in a large number of cancers, it is anticipated that the results in AML can be extrapolated to other cancers. Indeed, there are numerous studies and clinical trials in various cancers that have shown promising results using MAPK inhibition as a mode of anti-cancer therapy. (Platanias L 2003 Blood 101:12:4667; Kaminska B 2005 Biochinica et Biophysica 1754:253; Salvador et al 2005 Nature immunology; Singh and Zhang 2004 Journal of immunology 173:7299; Kumar et al 2003 Nature Reviews 2 : 717). [0031] MAPK in brain diseases: The MAPKs are expressed abundantly in the central nervous system (CNS) and Erk is involved in long-lasting neuronal plasticity, including long-term potentiation and memory consolidation. The role of Erk in neuronal development, plasticity and behavioral adaptation is beginning to emerge, as well as the role of MAPK signal transduction cascades in brain disorders, including schizophrenia. Evidence from human post-mortem studies, as well as from the phencyclidine model of schizophrenia, that different MAPK cascades may be involved in the pathogenesis of schizophrenia, and potentially in other psychiatric disorders. Therefore, while Erk signaling plays a beneficial, neuroprotective role in many systems, there is growing evidence implicating these kinases in the promotion of cell death in both neurons and other cell types in the disease states.

[0032] A growing number of recent studies in models of cerebral ischemia, brain trauma and neurodegenerative diseases implicate a detrimental role for Erk signaling during oxidative neuronal injury. Neurons undergoing oxidative stress-related injuries typically display a biphasic or sustained pattern of Erk activation. A variety of potential targets of reactive oxygen species (ROS) and reactive nitrogen species (RNS) could contribute to Erk activation. These include cell surface receptors, G proteins, upstream kinases, protein phosphatases and proteasome components, each of which could be direct or indirect targets of ROS or RNS, thereby modulating the duration and magnitude of Erk activation. Neuronal oxidative stress also appears to influence the subcellular trafficking and/or localization of activated Erk. Differences in compartmentalization of phosphorylated Erk have been observed in diseased or injured human neurons and in their respective animal and cell culture model systems. We propose that differential accessibility of Erk to downstream targets, which is dictated by the persistent activation of Erk within distinct subcellular compartments, underlies the neurotoxic responses that are driven by this kinase.

[0033] Initial indications that Erk activation may contribute to central nervous system (CNS) disease pathogenesis have been observed from diseased human brain tissues using antibodies that recognize the active, phosphorylated form of both Erk 1 and 2. Aberrant neuronal expression of phosphorylated Erk and other MAPKs in Alzheimer's disease patients' brains in association with markers of oxidative stress and as well as aberrant MAPK phosphorylation was also noted in a variety of sporadic and familial neurodegenerative diseases characterized by Tau protein deposits. Phospho-Erk 1 and 2 are increased in substantia nigra neurons of patients with Parkinson's disease and other Lewy body diseases, and the midbrains of these patients show elevated Erk activity. In addition to chronic neurodegenerative diseases, increased Erk phosphorylation has been noted in the vulnerable penumbra following acute ischemic stroke in humans. (Colucci-D'Amato et al 2003 BioEssays 25: 1085; Chu et al Eur J Biochem 2004 FEBS 271 : 2060-2066; Alessandrini et al 1999: 96:2: 12866; Thomas and Huganir 2004 Nature Reviews : Neuroscience 5 : 173; Adams and Sweatt Annu Rev. Pjarmacol Toxicol 2002 42: 135; Pasterkamp et al 2003 Nature 424:398.)

SUMMARY OF INVENTION

[0034] In one aspect, there is provided a method of inhibiting activation of a

MAP kinase comprising contacting the MAP kinase with a WW domain.

[0035] In further aspect, there is provided a method of inhibiting a cell having increased activation of a MAP kinase comprising administering to the cell an effective amount of a molecule that increases cellular levels of a polypeptide comprising at least a WW domain.

[0036] In a further aspect, there is provided a method of treating a disease state characterized by the presence of cells that have increased activation of a MAP kinase, in a patient in need of such treatment, comprising administering to the patient an effective amount of a polypeptide comprising at least a WW domain.

[0037] In a further aspect, there is provided a composition comprising a polypeptide comprising at least a WW domain or comprising a nucleic acid encoding the polypeptide.

[0038] In a further aspect, there is provided a kit comprising a polypeptide comprising at least a WW domain, the WW domain having two basic amino acids at the C-terminus, or a nucleic acid molecule encoding the polypeptide, and instructions for inhibiting a cell having increased activation of a MAP kinase or for treating a disease state characterized by the presence of cells that have increased activation of a MAP kinase.

[0039] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In the figures, which illustrate, by way of example only, embodiments of the present invention,

[0041 ] FIGURE IA is a schematic representation of ArhGAP9 and ArhGAP12 domain structures from different species a s predicted by SMART domain prediction tool;

[0042] FIGURE IB is a sequence alignment of human and mouse ArhGAP9 proteins;

[0043] FIGURE 1C is a schematic depicting the phylogenetic relationship of

ArhGAP9 and ArhGAP12 protein sequences;

[0044] FIGURE 2A is an SDS-PAGE gel demonstrating interaction between the WW domain of ArhGAP9 and Erk2;

[0045] FIGURE 2B is an immunoblot of the results of an in vitro binding assay between the WW domain of ArhGAP9 and Erk2, p38α and Jnkl ;

[0046] FIGURE 2C is an immunoblot of the results of an in vitro binding assay between the WW domain of ArhGAP9, ArhGAP12 or Nedd4 and Erk2, p38α and Jnkl;

[0047] FIGURE 2D is an immunoblot of in vivo expression of ArhGAP9 and

Erk2, p38α and Jnkl in 293T cells followed by immunoprecipation;

[0048] FIGURE 2E is an immunoblot demonstrating a reduction in binding of

ArhGAP9 to the MAP kinases upon EGFR activation; [0049] FIGURE 2F is an immunoblot demonstrating that the activation loop of the MAP kinase was not involved in the binding interaction with ArhGAP9;

[0050] FIGURE 2G is a sequence alignment of the protein sequence of the

WW domains of ArhGAP9 and ArhGAP12;

[0051 ] FIGURE 3A is an immunoblot demonstrating that the common docking

(CD) domain of MAP kinase is involved in the interaction with ArhGAP9;

[0052] FIGURE 3B is an immunoblot demonstrating that the acidic residues in the CD domain of MAP kinases are involved in the interaction with ArhGAP9;

[0053] FIGURE 3C is an immunoblot demonstrating that the di-arginine motif in the WW domain of ArhGAP9 are involved in the interaction with the CD domain of the MAP kinases;

[0054] FIGURE 3D is an immunoblot demonstrating that the binding of Erk2 and p38α to the WW domain of ArhGAP9 was disrupted by R246A and R247A mutations;

[0055] FIGURE 3E is an immunoblot demonstrating that in vivo binding of

ArhGAP9 to Erk2 and p38α is reduced with R246A and R247A mutations;

[0056] FIGURE 3F is an immunoblot demonstrating that co-expression of

MEK2 reduced the binding of Erk2 to the WW domain of ArhGAP9 in vitro;

[0057] FIGURE 3G is an immunoblot demonstrating reduction in the binding of ArhGAP9 to Erk2 upon co-expression of MEK in vivo;

[0058] FIGURE 4A is an immunoblot demonstrating that MAP kinase binding

ArhGAP9 had no significant effect o on RhoGAP activity;

[0059] FIGURE 4B is an immunoblot demonstrating that ArhGAP9 binding to p38α suppressed activation of p38α by the EGF receptor;

[0060] FIGURE 5 is immunofluorescence photographs demonstrating that expression of ArhGAP9 R246,247A mutant disrupted stress fibres in Swiss 3T3 cells; [0061 ] FIGURE 6A Full length Erk2 and p38α interact with ArhGAP9 in vitro.

(i), (ii) Flag-tagged ArhGAP9 was transiently expressed in 293T cells. The lysates were subjected to in vitro binding assay with immobilized (i) GST-Erk2, (ii) GST-p38α or (iii) GST control. Bound proteins were resolved by SDS-PAGE followed by western blotting with α-Flag. (iv) SDSPAGE and western blotting of whole cell lysate with α- Flag;

[0062] FIGURE 6B Interaction of ArhGAP9 and the MAP kinases in vivo.

Cotransfection of ArhGAP9 and the Flag-tagged MAP kinases Erk2 or p38α was carried out in 293T cells. The lysates were subjected to immunoprecipitation with α- Flag, followed by Western blotting with (i) α-ArhGAP9 and (ii) α-Flag. (iii, iv) SDS- PAGE and western blotting of whole cell lysates with α-ArhGAP9 or α-Flag;

[0063] FIGURE 6C Erk2 and p38α interact specifically with the WW domain of ArhGAP9 but not ArhGAP12 or Nedd4. Flag-tagged MAP kinases (i) Erk2, (ii) p38α or (iii) Jnkl were transiently expressed in 293T cells and the lysates were incubated with GST fusion proteins of the WW domains of ArhGAP9, ArhGAP12 or Nedd4, or GST alone immobilized on glutathione Sepharose beads, (iv) No DNA was transfected for the control lysate. Specifically bound proteins were resolved by SDS- PAGE, followed by western blotting with α-Flag. (v) Whole cell lysates were resolved by SDS-PAGE followed by western blotting with α-Flag;

[0064] FIGURE 6D Erk2 and p38α do not interact with mouse ArhGAP9 in vitro. Flag-tagged Erk2, p38α or Jnkl plasmid was transfected into 293T cells. No DNA was transfected for control. Immobilized GST proteins for (i) WW domain of human ArhGAP9, (ii) N- terminal fragment of mouse ArhGAP9 (residues 1-XXX) or (iii) GST alone were incubated with the lysates. Bound proteins were resolved by SDS- PAGE, followed by western blotting with α-Flag. (iv) Total cell lysates were resolved by SDS-PAGE followed by western blotting with α-Flag;

[0065] FIGURE 6E Erk2 interacted with human ArhGAP9 (hArhGAP9) but not mouse ArhGAP9 (mArhGAP9). (i) Flag-tagged human and mouse ArhGAP9 plasmids were transiently transfected into 293T cells, individually or together with HA- tagged Erk2. The lysates were subjected to immunoprecipitation using α-Flag. Bound proteins were resolved by SDSPAGE, followed by western blotting with (i) α-Flag or (ii) α-HA.. Total cell lysates were resolved by SDS-PAGE and immunoblotted with (iii) α-Flag or (iv) α-HA;

[0066] FIGURE 7A The activation loop region of MAP kinases was not involved in binding with ArhGAP9. Flag-tagged Erk2 (wild type or T 183, Y 185 A mutant) and p38α (wild type or T 180, Y 182 A mutant) plasmids were transfected in 293T cells. The lysates were subjected to in vitro binding assays with the (i) GST fusion of the WW domain of ArhGAP9 or (ii) GST alone immobilized on glutathione Sepharose beads. Bound proteins were resolved by SDS-PAGE followed by western blotting with α-Flag. (iii) Total cell lysates were separated by SDSPAGE and immunoblotted with α-Flag;

[0067] FIGURE 7B R246 and R247 in the WW domain of ArhGAP9 are required for binding to MAP kinase. Flag-tagged Erk2, p38α or Jnkl plasmids was transfected into 293T cells and the lysates were subjected to in vitro binding assay with the GST-WW-ArhGAP9, GST-WWArhGAP9(RR) mutant where the R246A and R247A mutations had been introduced, or GST alone as a control. Specifically bound proteins were eluted and resolved by SDS-PAGE, followed by western blotting with α- Flag. (ii) The expression levels of Erk2, p38α and Jnkl were shown to be equivalent by SDS-PAGE and western blotting of the total cell lysates with α-Flag;

[0068] FIGURE 7C In vivo binding of ArhGAP9 to Erk2 and p38α was abrogated by R246A and R247A mutations. Flag-tagged Erk2 or p38α were transiently expressed either individually or together with full-length ArhGAP9 [wildtype or the R246,247A mutant (RR)] in 293T cells, as indicated in the figure. Immunoprecipitation was conducted on the lysates with α-Flag or α-ArhGAP9. The immunocomplexes were resolved by SDS-PAGE followed by western blotting with (i, iv) α-ArhGAP9 or (ii, iii) α-Flag. Total cell lysates were separated by SDSPAGE and immunoblotted with (v) α-ArhGAP9 or (vi) α-Flag;

[0069] FIGURE 7D The Common Docking (CD) domain of MAP kinase mediated the binding to the WW domain of ArhGAP9. Flag-tagged full-length ArhGAP9 (wild type or the R246,247A mutant, RR) was transiently expressed 293T cells and lysates were subjected to in vitro binding assay with GST fusion of the (i) CD domain of Erk2 (residues XX-YY), a fragment of Erk2 deleted of the CD domain, (ii) Erk2-ΔCD (XX-YY) or (iii) GST alone as a control. The protein complexes were resolved by SDS-PAGE followed by western blotting with α-Flag. (iv) Total cell lysates were resolved by SDS-PAGE and immunoblotted with α-Flag;

[0070] FIGURE 7E Acidic residues in the CD domain of MAP kinases important for interaction with ArhGAP9. Alignment of the Common Docking (CD) domains of Erk2, p38α and Jnkl, the acidic residues that were mutated to alanine are indicated in superscript;

[0071 ] FIGURE 7F Far-UV CD spectra of WW of ArhGAP9 in complex with

MAP kinase CD domain peptides, (i) The CD spectra of the WW domain of ArhGAP9 indicating that the protein folded properly and has mostly β-sheets and random coils, (ii), (iii) and (iv) CD spectra of ArhGAP9 WW domain in complex with Jnkl, p38α and Erk2 peptides, respectively. Conformational changes were observed for the case of ArhGAP9 WW domain in complex with Erk2 and p38α peptides. As for Jnkl, little effect in the spectra profile was observed when compared with WW domain alone, indicating that no significant binding of the Jnkl peptide had occurred;

[0072] FIGURE 8A and 8B Coexpression of MKK6 abrogated the binding of p38α to the WW domain of ArhGAP9 in vitro, a. Flag-tagged p38α was transfected alone or together with HA-tagged activated mutant of MKK6 in 293T cells and the lysates were subjected to in vitro binding assay with (i) GST- WW- ArhGAP9 or (ii) GST alone as a control. Bound p38α was detected by SDS-PAGE followed by western blotting with α-Flag. Total cell lysates were separated by SDS-PAGE and immunoblotted with (iii) α-phospho-p38, (iv) α-Flag or (v) α-HA. b. p38α was transfected alone or with HA-tagged MKK6 wildtype. This time round we included the mutant MKK6 as a control. This mutant will abolish the binding between the two kinases, MKK6 and p38α. The lysates were subjected to in vitro assay with WW GST of ArhGAP9 and with GST as a control to detect non-specific interaction. The binding results were resolved with western blotting using α-Flag. With the mutant MKK6 not able to bind to p38α, we observed that the magnitude of the interaction of ArhGAP 9 WW GST and p38α is greatly enhanced when compared to the control (p38α with wildtype). This confirmed the competitive interaction of these 3 proteins, (iii) and (iv) show the expression level of p38α and MKK6 using α-Flag and α-HA respectively;

[0073] FIGURE 8C Abrogation of ArhGAP9 binding to p38α by MKK6 in vivo Flag-tagged p38α, HA-tagged MKK6 and non-tagged ArhGAP9 were transiently transfected in 293T cells as indicated in the figure. Immunoprecipitation was carried out with α-Flag, followed by western blotting with (i) α-ArhGAP9 and (ii) α-Flag. Western blotting of the total lysates with (iii) α-phospho-p38 showed that p38α was activated when coexpressed with MKK6. Western blotting of the total lysates with (iv) α-ArhAGP9 and (v) α-Flag showed the equal expression of ArhGAP9 and p38α in the total cell lysates;

[0074] FIGURE 8D MAP kinase binding to ArhGAP9 had no significant effect on RhoGAP activity ArhGAP9 (wild type or the GAP-inactive mutant, R578K) was transfected with myc-tagged cdc42 with or without Flag-p38α in 293T cells as indicated in the figure, (i) The lysates were incubated with immobilized PBD-GST to assess the relative amount of active cdc42, reflected by the amount of myc-cdc42 associated with PBD-GST by western blotting with α-myc. Total cell lysates were separated by SDS-PAGE and immunoblotted with (ii) α-ArhGAP, (iii) α-Flag or (iv) α- myc;

[0075] FIGURE 9 Expression of ArhGAP9 R246,247A mutant disrupted stress fibres in Swiss 3T3 fibroblasts, (i) Swiss 3T3 cells were microinjected with full-length ArhGAP9 (wild type or the or R246,247A (RR) mutant) together with GFP-actin. The cells were imaged for GFP fluorescence, (ii) Quantification of the number of stress fibers; and

[0076] FIGURE 10 Proposed mechanism of negative regulation of MAP kinase by ArhGAP9. a. ArhGAP9 contains a WW domain which possesses a basic di- Arginine motif while MAP kinase (MAPK) contains a Common Docking (CD) domain that contains conserved acidic residues, b. In quiescent state, ArhGAP9 interacts with MAPK through electrostatic interaction between the complementary basic and acidic residues in the WW and CD domains, blocking the access of MAPK by other docking proteins therefore negatively regulating MAPK activation, c, d. In the induced state, the presumable increase in local concentration of active upstream MAPK kinase (MAPKK) displaces ArhGAP9 by docking onto CD domain of MAPK, causing the diminished binding of ArhGAP9 to MAPK. The interaction between MAPK and MAPKK results in the phosphorylation of MAPK in the kinase activation loop to activate the latter.

DETAILED DESCRIPTION

[0077] ArhGAP9 is a recently identified evolutionary conserved RHO GTPase activating protein (GAP) with a combination of SH3, WW, PH and PH GAP signalling domains. The biochemical function, regulation and role in development of ArhGAP9 and signalling mechanisms of ArhGAP9 are generally unknown. This protein is highly expressed in haematopoietic compartments including spleen, thymus and bone marrow. ArhGAP9 has been shown to be highly expressed in T-cell and B-cell leukemias and myeloid or Hodgkin Lymphoma cell lines.

[0078] The present invention relates to the surprising discovery that the WW domain of ArhGAP9 binds to the common docking (CD) domain of MAPKs, including Erk2 and p38α, and inhibits activation of the MAPKs by upstream kinases. This interaction occurs via interaction of two basic residues at the C-terminal end of the WW domain, and conserved acidic residues located in the CD domain. The binding of the WW domain of ArhGAP9 with MAP kinases, including Erk2 and p38α, was mediated through complementarity charged residues in the C-terminal end of the WW domain of ArhGAP9 and the conserved Common Docking (CD) domain in the MAP kinase.

[0079] Without being limited to any particular theory, it is possible that the electrostatic interaction of the basic residues of the WW domain and the acidic residues of the MAPK CD domain provide an electrostatic interaction that can be adjusted depending on the particular residues involved in the interaction, resulting in control of the rate of association and/or that contribute to the specificity of the binding interaction. As well, it is possible that the interaction has a biological function, sequestering the MAP kinases in their inactive state in quiescent cells by docking of a protein having a relevant WW domain on the CD domain of the MAPKs. [0080] MAPKs are implicated in the regulation of a diverse number of processes in the normal cells such as cell survival, proliferation and differentiation. In diseases such as cancer, MAP kinase signaling pathways are constitutively active. Thus, the present invention takes advantage of the above-discovery to provide a method of inhibiting the activation of MAPKs using a polypeptide comprising a WW domain that contains two basic amino acids at the C-terminus of the domain.

[0081] The present invention provides a method of inhibiting activation of a

MAP kinase comprising contacting the MAP kinase with a WW domain having two basic amino acids at the C-terminus of the domain.

[0082] The MAP kinase is any mitogen activated protein kinase. A MAP kinase is a serine/threonine protein kinase, meaning that when activated the MAP kinase phosphorylates serine and/or threonine residues in its target substrate. MAP kinases are activated in response to extracellular stimuli and mediate signal transduction from the cell surface to the nucleus of a cell in which the MAP kinase is activated, as is understood in the art. The term MAP kinase as used herein includes members of the Erk family of MAPKs, members of the p38 family of MAPKs and members of the Jnk family of MAPKs. In particular embodiments, the MAP kinase is Erkl, Erk2, Erk5, p38α, p38β, p38γ, Jnkl, Jnk2 or Jnk3. In one particular embodiment, the MAP kinase is Erk2. In another particular embodiment, the MAP kinase is p38α. In yet another particular embodiment, the MAP kinase is Jnkl.

[0083] MAP kinase activation refers to phosphorylation of a MAP kinase by an upstream MAP kinase kinase, which phosphorylation results in the ability or in the increased ability of the MAP kinase to phosphorylate its target substrate or substrates.

[0084] The WW domain is any WW domain, as will be understood in the art, which possesses two basic residues at the C-terminus of the WW domain. The WW domain possessing the two C-terminal amino acid residues has the ability to bind to a MAP kinase, for example through an interaction with the common docking (CD) domain of the MAP kinase, and inhibit, reduce, block partially or completely, or prevent activation of the MAP kinase by an upstream kinase. [0085] A WW domain is a 30-40 amino acid protein interaction domain with two signature tryptophan residues spaced by 20-22 amino acids. The three-dimensional structure of WW domains shows that they generally fold into a three-stranded, antiparallel β sheet with two ligand-binding grooves.

[0086] WW domains bind a variety of distinct peptide ligands including motifs with core proline-rich sequences, such as PPDY [SEQ ID NO: 1], PPLP [SEQ ID NO: 2], PPPPP [SEQ ID NO: 3], PPXPPXR [SEQ ID NO: 4], PPRXXP [SEQ ID NO: 5] (X: any amino acid), PR motifs or phosphorylated threonine or serine-proline (pTVpS)-P sites. WW domains have been classified into four groups on the basis of their binding to peptide ligands. Group I WW domains have been shown to recognize PY motifs, Group II WW domains recognize PPLP motifs, Group III WW domains recognize PR motifs and Group IV WW domains recognize (pS/pT)-P motifs. Group II and III WW domains can be rather versatile in their binding properties, since they not only recognize both PPLP and PR containing peptides (with varied affinities) but also polyproline stretches often containing glycine, methionine, or arginine.

[0087] WW domains are found in many eukaryotes and are present in approximately 50 human proteins (Bork, P. & Sudol, M. The WW domain: a signalling site in dystrophin? Trends Biochem Sci 19, 531-533 (1994)). WW domains may be present together with several other interaction domains, including phosphotyrosine- binding (PTB) domain in FE65 protein, FF domains in CA150 and FBPIl, as well as membrane targeting domains, such as C2 in the NEDD4 family proteins and pleckstrin homology (PH) domains in PLEKHA5. WW domains are also linked to a variety of catalytic domains, including HECT E3 protein-ubiquitin ligase domains in NEDD4 family proteins, rotomerase or peptidyl prolyisomerase domains in Pinl, and Rho GAP domains in ArhGAP9 and ArhGAP12.

[0088] In the present method, the WW domain may be a WW domain that naturally possesses two basic amino acids at the C-terminus, for example the WW domain from human ArhGAP9. In one embodiment, the WW domain comprises the sequence QRLD AWEQHLDPNSGRCFYINSLTGCKS WKPPRR [SEQ ID NO: 6]; QRLDAWEQYLDPNSGRCFYINSLTGCKSWKPPRR [SEQ ID NO: 7]; QRPDAWEQHLDPNSGRCFYINSLTGCKSWKPPRR [SEQ ID NO: 8]; QRLDSWEQHLDLNSGRCFYIHSLTGCKSWKPPRR [SEQ ID NO: 9]; or QVLELWEQYLDPATGRSFYVNTITKEKSWKPPRR [SEQ ID NO: 10]. In another embodiment, the WW domain consists of the sequence of SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10. In another embodiment, the WW domain consists essentially of the sequence SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10. "Consists essentially of means that a domain, peptide or polypeptide consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 10 or so additional residues, typically from 1 to about 5 additional residues in the domain, peptide or polypeptide.

[0089] Alternatively, the WW domain may be a WW domain that has been engineered or modified to include two basic amino acids at the C-terminus of the domain. Molecular biology, cloning and recombinant protein techniques are known in the art and are described in the art, for example in Sambrook et al. ((2001) Molecular Cloning: a Laboratory Manual, 3^rd ed., Cold Spring Harbour Laboratory Press). Thus, a skilled person could readily alter, engineer or modify an existing WW domain that does not normally have two C-terminal basic residues so as to include two basic residues at the C-terminus.

[0090] Basic amino acids are amino acids that possess a side-chain functional group that has a pKa of greater than 7 and include lysine, arginine and hisiidine, as well as basic amino acids that are not included in the twenty α-amino acids commonly included in proteins. The two basic amino acids at the C-terminus of the WW domain may be the same basic amino acid or may be different basic amino acids. In one particular embodiment, the two basic amino acids are both arginine.

[0091] The term WW domain includes homologs, fragments, derivatives or variants of a WW domain provided that any such homolog, fragment derivative or variant possesses two basic amino acids at its C-terminus and maintains the ability of the WW domain to bind to the MAP kinase and to inhibit activation of the MAP kinase by an upstream kinase. [0092] A polypeptide sequence is a "homolog" of, or is "homologous" to another sequence if the two sequences have substantial identity over a specified region and the functional activity of the sequences is conserved (as used herein, the term "homologous" does not imply evolutionary relatedness). Two polynucleotide sequences or polypeptide sequences are considered to have substantial identity if, when optimally aligned (with gaps permitted), they share at least approximately 50% sequence identity, or if the sequences share defined functional motifs. In alternative embodiments, optimally aligned sequences may be considered to be substantially identical (i.e. to have substantial identity) if they share at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity over a specified region. An "unrelated" or "non-homologous" sequence shares less than 40% identity, and possibly less than approximately 25% identity, with a polypeptide of the invention over a specified region of homology. The terms "identity" and "identical" refer to sequence similarity between two peptides or two polynucleotide molecules. Identity can be determined by comparing each position in the aligned sequences. A degree of identity between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences, i.e. over a specified region. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, as are known in the art, including the ClustalW program, available at http://clustalw.genome.ad.jp, the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. MoI. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. ScL USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. MoI. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis are available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/). As used herein, "homologous amino acid sequence" includes any polypeptide which is encoded, in whole or in part, by a nucleic acid sequence which hybridizes at 25-35°C below critical melting temperature (Tm), to any portion of a nucleic acid sequence encoding a WW domain having two basic amino acids at the C-terminus of the domain.

[0093] A variant or derivative of such a WW domain refers to a WW domain which retains the inhibiting of MAP kinase activation activity and that has been mutated at one or more amino acids, including point, insertion or deletion mutations, but still retains the inhibiting of MAP kinase activation activity. A variant or derivative therefore includes deletions, including truncations and fragments; insertions and additions, for example conservative substitutions, site-directed mutants and allelic variants; and modifications, including peptoids having one or more non-amino acyl groups (q.v., sugar, lipid, etc.) covalently linked to the peptide and post-translational modifications. As used herein, the term "conserved amino acid substitutions" or "conservative substitutions" refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without substantial loss of the relevant function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.

[0094] The WW domain may form part of a longer polypeptide. Thus, the polypeptide, in various different embodiments, comprises the WW domain, consists of the WW domain or consists essentially of the WW domain, as defined herein. The polypeptide may be a protein that includes a WW domain as a functional domain within the protein sequence. In a particular embodiment, the polypeptide is the human ArhGAP9 protein. In other particular embodiments, the polypeptide comprises the sequence set forth in SEQ ID NO: 11 (below), consists of SEQ ID NO: 12 or consists essentially of SEQ ID NO: 11.

[0095] MLSSRWWPSSWGILGLGPRSPPRGSQLCALYAFTYTGADGQQV

SLAEGDRFLLLRKTNSDWWLARRLEAPSTSRPIFVPAAYMIEESIPSQSPTTVIPG QLLWTPGPKLFHGSLEELSQALPSRAQASSEQPPPLPRKMCRSVSTDNLSPSFLK PFQEGPSGRSLSQEDLPSEASASTAGPQPLMSEPPVYCNLVDLRRCPRSPPPGPA CPLLQRLDAWEQHLDPNSGRCFYINSLTGCKSWKPPRRSRSETNPGSMEGTQT

LKRNNDVLQPQAKGFRSDTGTPEPLDPQGSLSLSQRTSQLDPPALQAPRPLPQL

LDDPHEVEKSGLLNMTKIAQGGRKLRKNWGPSWVVLTGNSLVFYREPPPTAPS

SGWGPAGSRPESSVDLRGAALAHGRHLSSRRNVLHIRTIPGHEFLLQSDHETEL

RAWHRALRTVIERLVRWVEARREAPTGRDQGSGDRENPLELRLSGSGPAELSA

GEDEEEESELVSKPLLRLSSRRSSIRGPEGTEQNRVRNKLKRLIAKRPPLQSLQE

RGLLRDQVFGCQLESLCQREGDTVPSFLRLCIAA VDKRGLD VDGIYRVSGNLA

VVQKLRFLVDRERAVTSDGRYVFPEQPGQEGRLDLDSTEWDDIHVVTGALKL

FLRELPQPLVPPLLLPHFRAALALSESEQCLSQIQELIGSMPKPNHDTLRYLLEH

LCRVIAHSDKNRMTPHNLGIVFGPTLFRPEQETSDPAAHALYPGQLVQLMLTN

FTSLFP [SEQ ID NO: 11]

[0096] The MAP kinase is contacted with the WW domain under conditions that are suitable for protein-protein binding interactions. That is, a skilled person will appreciate that the contacting should occur under conditions that do not disrupt protein- protein interactions, and that do not generally denature proteins. For example, the WW domain may be added to a physiological salt buffer to which the MAP kinase is also added. Alternatively, the contacting may also take place within a cell, or in a cell lysate.

[0097] Inhibiting activation of a MAP kinase can be used to inhibit pathways in which the MAP kinase transduces a cellular signal. Such inhibition can inhibit cell growth or differentiation, or other cellular functions. Thus, the present invention also provides a method of inhibiting a cell comprising administering to the cell an effective amount of molecule that increases cellular levels of a polypeptide comprising at least a WW domain, the WW domain having two basic amino acids at the C-terminus. In some embodiments, the cell is a cell having increased activation of a MAP kinase.

[0098] Thus, the cell may be a cell having increased activation of a MAP kinase, which may be any cell in which a MAP kinase is expressed at greater levels, phosphorylated by an upstream kinase at greater levels, has an increased ability to phosphorylate target substrate including at normal expression and/or phosphorylation levels, is expressed and/or phosphorylated at points in the cell cycle at which the MAP kinase is not normally expressed and/or phosphorylated, or is constitutively expressed, all when compared to a normal, healthy cell, including for example, a non-transformed cell or a non-cancerous cell. The increased activation may be the result of a mutation, including a deletion, insertion or substitution in the MAP kinase gene, including the open reading frame encoding the MAP kinase or in the regulatory region controlling expression of the MAP kinase. Alternatively, the increased activation may be the result of de-regulation, up-regulation, increased expression or a mutation, including a deletion, insertion or substitution, upstream in the pathway that regulates the expression and/or phosphorylation of the MAP kinase, including with respect to an upstream kinase that phosphorylates the MAP kinase or with respect to a transcription factor that regulates expression of the MAP kinase.

[0099] Inhibiting the cell refers to preventing, inhibiting, slowing or reducing cell growth, differentiation, release of cell signalling molecules such as cytokines, or other cellular functions that are regulated, effected or controlled by pathways that involve MAP kinase activity. Inhibiting includes rendering the cell incapable of growing or dividing or reducing or retarding cell growth or division, in addition to inducing cell death by lysis or apoptosis or other mechanisms of cell death. Inhibiting also includes inhibiting, reducing, blocking partially or completely, or preventing MAP kinase activation in the cell.

[00100] The cell may be an in vitro cell or it may be an in vivo cell, meaning that the cell may be a cell in culture, or it may be a cell in an animal. The cell may be explanted for administration of the polypeptide and then may be replaced in an animal following administration of the polypeptide, using standard methods known to a skilled person. The term "cell" includes a single cell as well as a plurality or population of cells.

[00101 ] A molecule that increases cellular levels of a polypeptide comprising at least a WW domain, the WW domain having two basic amino acids at the C-terminus is any molecule that raises cellular levels of the polypeptide. Increasing the cellular levels of the polypeptide refers to increasing the levels within a cell of the polypeptide to any extent, including introducing the polypeptide where none existed in the cell previous to the increasing. Thus, the cellular levels of the polypeptide comprising a WW domain having two basic amino acids at the C-terminus may be zero or negligible prior to the increasing, or the cellular levels of the polypeptide comprising a WW domain having two basic amino acids at the C-terminus may be measurable or significant, and may in some embodiments be those of a normal healthy cell.

[00102] The term "effective amount" as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired result. In the present method, the effective amount of the molecule that increases cellular levels of the polypeptide comprising the WW domain having two basic amino acids at the C- terminus of the domain is the amount required of same to inhibit the cell having increased MAP kinase activity.

[00103] Thus, the effective amount of the molecule that increases cellular levels of the polypeptide comprising the WW domain having two basic amino acids at the C- terminus of the domain is administered to the cell that is to be inhibited.

[00104] hi one embodiment, the molecule that increases cellular levels of the polypeptide comprising a WW domain having two basic amino acids at the C-terminus is the polypeptide itself. The polypeptide may be administered as a polypeptide by exposing the cell to the polypeptide, for example, by addition of the polypeptide to the growth medium or the extracellular environment. Inclusion of a sequence such as a membrane-translocating sequence that allows the polypeptide in which it is included to be transported into a cell, for example the penetratin sequence derived from the Drosophila melanogaster antennapedia homeodomain protein, facilitates the uptake of the polypeptide by the cell. Known methods of delivering protein to a cell may also be used, for example using microinjection or using a delivery vehicle such as liposomes. Liposome delivery of peptides and proteins to cells is known, and is described for example in US 6,372,720 and US 20030108597, which are incorporated herein by reference.

[00105] In an alternative embodiment, the molecule that increases cellular levels of the polypeptide comprising a WW domain having two basic amino acids at the C- terminus is a nucleic acid encoding the polypeptide. The nucleic acid molecule will include any necessary regulatory elements required for expression of the polypeptide within the cell to which the nucleic acid is administered, including any necessary promoter region and enhancer elements, as will be understood in the art. The promoter may be chosen to direct constitutive or inducible expression of the polypeptide, at low or high levels in the cell. The nucleic acid molecule is administered using known techniques, including transfection techniques such as calcium phosphate or liposome transfection, viral delivery methods, gene gun or microinjection techniques.

[00106] As stated above, molecular cloning and recombinant protein techniques are known, and a skilled person can readily prepare a suitable nucleic acid molecule that is designed to express the polypeptide comprising the WW domain in the type of cell in which the activation of a MAP kinase is to be inhibited.

[00107] In another embodiment, the polypeptide may be an endogenous polypeptide comprising a WW domain having two basic amino acids at the C-terminus and thus the cell may already express some level of the polypeptide. In this case, the molecule that increases cellular levels of the polypeptide may be a molecule that up- regulates the expression of the endogenous polypeptide comprising a WW domain having two basic amino acids at the C-terminus, including a transcription factor or activator of the gene encoding the endogenous polypeptide, or an inhibitor of a negative regulator or repressor of the gene encoding the endogenous polypeptide. The molecule may also be a nucleic acid that increases expression of the gene encoding the endogenous polypeptide, for example a nucleic acid encoding a transcription factor that increases expression from the gene encoding the endogenous polypeptide, or a nucleic acid designed to integrate into the regulatory region of the gene encoding the endogenous polypeptide so as to increase the expression levels of the gene, for example an enhancer element.

[00108] Inhibiting a cell that has an increased activation of a MAP kinase by the above method may result in the blocking or reducing of the signal transduced by the activated MAP kinase, and even in slowing or stopping of cell growth or differentiation, or even in cell death, since MAP kinases are involved in the growth signalling cascades of the cell. Thus, the above method can be used to treat a disorder that involves or is characterized by the presence of cells having an increased activation of a MAP kinase.

[00109] The present invention also provides a method of inhibiting activation of a MAP kinase comprising contacting the MAP kinase with an inhibitor that binds electrostatically to the CD domain of MAPK.

[00110] The inhibitor may be a WW domain, a peptide that mimics or competitively binds to the CD domain of MAPK, or a small molecule that binds to the CD domain of MAPK to block or prevent activation of the MAPK.

[00111] There is also presently provided a method for treating a disease state characterized by the presence of cells that have increased activation of a MAP kinase in a patient in need of such treatment comprising administering to the patient an effective amount of a polypeptide comprising a WW domain that has two basic amino acids at the C-terminus of the domain. The patient may be any animal, including a mammal, including a human.

[00112] "A disease state characterized by the presence of cells that have increased activation of a MAP kinase" as used herein refers to any disease, disorder or condition which is associated with, related to, or a characteristic of which is, the presence of cells that have increased activation of a MAP kinase and which disease, disorder, condition or symptoms thereof may be treated by killing or inhibiting the growth of these cells. For example, the disease state may be cancer or rheumatoid arthritis.

[00113] "Treating" a disease state refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilization of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression, delay or slowing of disease onset, amelioration or palliation of the disease state, and remission (whether partial or total). "Treating" can also mean prolonging survival of a patient beyond that expected in the absence of treatment. "Treating" can also mean inhibiting the progression of disease, slowing the progression of disease temporarily, although more preferably, it involves halting the progression of the disease permanently.

[00114] In one embodiment, the disease state is cancer. The cancer may be any type of cancer wherein at least some of the cells, although not necessarily all of the cells have increased activation of a MAP kinase. As used herein, the terms "tumour", "tumour cells", "cancer" and "cancer cells", (used interchangeably) refer to cells that exhibit abnormal growth, characterized by a significant loss of control of cell proliferation or cells that have been immortalized. The term "cancer" or "tumour" includes metastatic as well as non-metastatic cancer or tumours. As used herein, "neoplastic" or "neoplasm" broadly refers to a cell or cells that proliferate without normal growth inhibition mechanisms, and therefore includes benign tumours, in addition to cancer as well as dysplastic or hyperplastic cells.

[00115] A cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor.

[00116] Types of cancer that may be treated according to the present invention include, but are not limited to, hematopoietic cell cancers including leukemias and lymphomas, colon cancer, lung cancer, kidney cancer, pancreas cancer, endometrial cancer, thyroid cancer, oral cancer, ovarian cancer, laryngeal cancer, hepatocellular cancer, bile duct cancer, squamous cell carcinoma, prostate cancer, breast cancer, cervical cancer, colorectal cancer, melanomas and any other tumours. Solid tumours such as sarcomas and carcinomas include but are not limited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).

[00117] When administered to a patient, an effective amount of polypeptide comprising a WW domain having two basic amino acids at the C-terminus of the domain is the amount required, at the dosages and for sufficient time period, for the polypeptide to alleviate, improve, mitigate, ameliorate, stabilize, prevent the spread of, slow or delay the progression of or cure the disease. For example, it may be an amount sufficient to achieve the effect of reducing the number of or destroying cancerous cells or neoplastic cells, or reducing the number of or destroying cells chronically infected with a virus, or inhibiting the growth and/or proliferation of such cells. As stated above, administration of the polypeptide includes administration of a nucleic acid molecule encoding the polypeptide and which is capable of expressing the polypeptide in a cell that has increased activation of a MAP kinase.

[00118] The effective amount to be administered to a patient can vary depending on many factors such as the pharmacodynamic properties of the polypeptide or the nucleic acid encoding the polypeptide, the modes of administration, the age, health and weight of the patient, the nature and extent of the disease state, the frequency of the treatment and the type of concurrent treatment, if any.

[00119] One of skill in the art can determine the appropriate amount of polypeptide or nucleic acid encoding the polypeptide for administration based on the above factors. The polypeptide or nucleic acid encoding the polypeptide may be administered initially in a suitable amount that may be adjusted as required, depending on the clinical response of the patient. The effective amount of polypeptide or nucleic acid encoding the polypeptide can be determined empirically and depends on the maximal amount of the polypeptide or nucleic acid encoding the polypeptide that can be administered safely, and the minimal amount of the polypeptide or nucleic acid encoding the polypeptide that produces the desired result.

[00120] The polypeptide comprising a WW domain having two basic amino acids at the C-terminus, or the nucleic acid encoding same, may be administered to the patient using standard methods of administration. In one embodiment, the polypeptide or nucleic acid encoding the polypeptide is administered systemically. In another embodiment, the polypeptide or nucleic acid encoding the polypeptide is administered by injection at the disease site. In a particular embodiment, the disease state is a solid tumour and the polypeptide or nucleic acid encoding the polypeptide is administered by injection at the tumour site, hi various embodiments, the polypeptide or nucleic acid encoding the polypeptide may be administered orally or parenterally, or by any standard method known in the art. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

[00121] To produce the same clinical effect when administering the polypeptide or nucleic acid encoding the polypeptide systemically as that achieved through injection of the polypeptide or nucleic acid encoding the polypeptide at the disease site, administration of significantly higher amounts of polypeptide or nucleic acid encoding the polypeptide may be required. However, the appropriate dose level should be the minimum amount that would achieve the desired result.

[00122] Effective amounts of the polypeptide or nucleic acid encoding the polypeptide can be given repeatedly, depending upon the effect of the initial treatment regimen. Administrations are typically given periodically, while monitoring any response.

[00123] The polypeptide or nucleic acid encoding the polypeptide may be administered as a sole therapy or may be administered in combination with other therapies, including chemotherapy, radiation therapy or other anti-viral therapies. For example, the polypeptide or nucleic acid encoding the polypeptide may be administered either prior to or following surgical removal of a primary tumour or prior to, concurrently with or following treatment such as administration of radiotherapy or conventional chemotherapeutic drugs.

[00124] To aid in administration, the polypeptide or nucleic acid encoding the polypeptide may be formulated as an ingredient in a pharmaceutical composition. Therefore, in a further embodiment, there is provided a pharmaceutical composition comprising a polypeptide comprising a WW domain having two basic amino acids at the C-terminus of the domain or a nucleic acid encoding the polypeptide, and optionally a pharmaceutically acceptable diluent. The invention in one aspect therefore also includes such pharmaceutical compositions for use in inhibiting a cell that has increased activation of a MAP kinase or treating a disease state characterized by the presence of cells that have increased activation of a MAP kinase.

[00125] The compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives and various compatible carriers. For all forms of delivery, the polypeptide or nucleic acid encoding the polypeptide may be formulated in a physiological salt solution.

[00126] Solutions of the polypeptide or nucleic acid encoding the polypeptide may be prepared in a physiologically suitable buffer. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms, but that will not denature the polypeptide. A person skilled in the art would know how to prepare suitable formulations. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences and in The United States Pharmacopeia: The National Formulary (USP 24 NF 19) published in 1999.

[00127] The pharmaceutical composition may additionally contain additional therapeutic agents, such as additional anti-cancer agents. In one embodiment, the composition includes a chemotherapeutic agent. The chemotherapeutic agent, for example, may be substantially any agent which exhibits an oncolytic effect against cancer cells or neoplastic cells of the patient and that does not inhibit or diminish the effect of the polypeptide comprising a WW domain having two basic amino acids at the C-terminus of the domain or a nucleic acid encoding the polypeptide. For example, the chemotherapeutic agent may be, without limitation, an anthracycline, an alkylating agent, an alkyl sulfonate, an aziridine, an ethylenimine, a methylmelamine, a nitrogen mustard, a nitrosourea, an antibiotic, an antimetabolite, a folic acid analogue, a purine analogue, a pyrimidine analogue, an enzyme, a podophyllotoxin, a platinum-containing agent or a cytokine. Preferably, the chemotherapeutic agent is one that is known to be effective against the particular cell type that is cancerous or neoplastic.

[00128] The proportion and identity of the pharmaceutically acceptable diluent is determined by chosen route of administration, compatibility the chemical stability of the polypeptide or nucleic acid encoding the polypeptide, and standard pharmaceutical practice. Generally, the pharmaceutical composition will be formulated with components that will not significantly impair the biological properties of, or cause degradation of or reduce the stability or efficacy of the polypeptide or nucleic acid encoding the polypeptide.

[00129] The pharmaceutical composition can be prepared by known methods for the preparation of pharmaceutically acceptable compositions suitable for administration to patients, such that an effective quantity of the active substance or substances is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).

[00130] On this basis, the compositions include, albeit not exclusively, solutions of the polypeptide or nucleic acid encoding the polypeptide, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffer solutions with a suitable pH and iso-osmotic with physiological fluids.

[00131 ] The pharmaceutical composition may be may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets. For oral therapeutic administration, the polypeptide or nucleic acid encoding the polypeptide may be incorporated with an excipient and be used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like.

[00132] In different embodiments, the composition is administered by injection

(subcuteanously, intravenously, intramuscularly, etc.) directly at the disease site, such as a tumour site, or by oral administration, alternatively by transdermal administration. The forms of the pharmaceutical composition suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.

[00133] The dose of the pharmaceutical composition that is to be used depends on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and other similar factors that are within the knowledge and expertise of the health practioner. These factors are known to those of skill in the art and can be addressed with minimal routine experimentation.

[00134] The polypeptide comprising a WW domain having two basic amino acids at the C-terminus of the domain or a nucleic acid encoding the polypeptide, or pharmaceutical compositions comprising the polypeptide or nucleic acid encoding the polypeptide, may also be packaged as a kit, containing instructions for use of the polypeptide or nucleic acid encoding the polypeptide to inhibit a cell that has increased activation of a MAP kinase, or use of the polypeptide or nucleic acid encoding the polypeptide to treat a disease state characterized by the presence of cells that have increased activation of a MAP kinase, in a patient in need thereof. The disease state may be cancer.

[00135] The present invention also contemplates the use of a polypeptide comprising a WW domain having two basic amino acids at the C-terminus of the domain or a nucleic acid encoding the polypeptide, for inhibiting a cell that has increased activation of a MAP kinase. There is further provided use a polypeptide comprising a WW domain having two basic amino acids at the C-terminus of the domain or a nucleic acid encoding the polypeptide for treating a disease state characterized by the presence of cells that have increased activation of a MAP kinase, in a patient in need thereof. In one embodiment the disease state is cancer. There is also provided use of a polypeptide comprising a WW domain having two basic amino acids at the C-terminus of the domain or a nucleic acid encoding the polypeptide, in the manufacture of a medicament, for inhibiting a cell that has increased activation of a MAP kinase, or for treating a disease state characterized by the presence of cells that have increased activation of a MAP kinase, in a patient in need thereof.

[00136] The above methods may be practised in combination with other known inhibitors of MAPK, for example inhibitors that bind or target the kinase domain of MAPK, for example, ATP analogue inhibitors.

[00137] The invention is further illustrated by the following non-limiting examples.

EXAMPLES

[00138] EXAMPLE l

[00139] Materials and methods

[00140] Antibodies: Mouse monoclonal anti-Flag (M2) and anti-phospho- specific p38 were obtained from Sigma, mouse phospho-Erk was from Cell Signaling Technology, mouse monoclonal anti-HA (12CA5) was from Boerhinger, rabbit polyclonal anti-ArhGAP9 was generated by standard immunization procedure with full-length human ArhGAP9-GST as the immunogen. Mouse monoclonal anti- phosphotyrosine conjugated to horse radish peroxidase (PY20-HRP) was from Transduction Laboratories, rabbit polyclonal EGFR was from Santa Cruz Biotechnology.

[00141] Cell lines: Human 293T cells were cultured in Dulbecco's minimal essential medium (DMEM) containing 4.5g/l D-glucose and supplemented with 10% fetal bovine serum (HyClone), 10 mM L-glutamine, and 100 μg each of penicillin and streptomycin/ml from Sigma. Swiss 3T3 were cultured in DMEM containing 4.5g/l D- glucose and supplemented with 10% COSMIC™ calf serum (HyClone), 10 mM L- glutamine, and 100 μg each of penicillin and streptomycin per ml.

[00142] Expression constructs: The human ArhGAP9 (wild-type and mutants), and EGFR coding sequences were cloned into the mammalian expression vector pRK5. The Erk2, p38, Jnkl were cloned in pxJ40-Flag vector which allowed N-terminal Flag epitope tagging. HA-tagged activated MEK2(S222D, S226D) was cloned in pUSEamp vector. Activated MKK6(S207E, T211E) was cloned in pxJ40-HA vector. Plasmid encoding Pak-binding domain (PBD)-GST was obtained from Dr. E Manser. Cdc42- myc was cloned in pcDNA3. For transient expression in mammalian cells, Lipofectamine (Gibco BRL) was used for transfection, following the protocol recommended by the manufacturer.

[00143] For expression of glutathione S-transferase (GST) fusion proteins, PCR- amplified sequences corresponding to the regions of interest of the proteins were cloned into the pGEX4Tl vector (Pharmacia). The GST fusion proteins were expressed in E. coli BL21 (DE3) and purified with glutathione-conjugated agarose beads (Pharmacia).

[00144] Proteomics and mass spectrometry analysis: Protein complexes of the

WW domain of ArhGAP9 were eluted and resolved by 10% SDS-PAGE, followed by detection of binding proteins by staining with Colloidal Coomassie Blue (Pierce). Specific bands were excised and subjected to in-gel reduction, S-alkylation and trypsin hydrolysis. Liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis of the peptides was performed on a Finnigan LCQ DECA™ ion trap mass spectrometer (Thermo Finnigan) fitted with a NANOSPRAY™ source (MDS Proteomics). Chromatographic separation was conducted using a FAMOS™ autosampler and an ULTIMATE™ gradient system (LC Packings) over ZORB AX™ SB-C 18 reverse phase resin (Agilent) packed into 75μm PICOFRIT™ columns (New Objective). Protein identifications were made using the search engines MASCOT™ (Matrix Sciences) and SONAR™ (ProteoMetrics).

[00145] Site-directed mutagenesis: Point mutations were introduced with the

Quickchange sitedirected mutagenesis kit (Stratagene). The mutations were confirmed by DNA sequencing.

[00146] Binding assays: Lysates for binding assays and immunoprecipitation were prepared by lysis of cells in the cell lysis buffer (20 mM HEPES [pH 7.5], 137 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgC12, 1 mM EGTA, Complete Protease inhibitors (Boehringer) and 0.1 mM Na3VO4), followed by centrifugation 13000g for 15 min at 4°C and collecting the supernatant. For in vitro pulldown assays, GST fusion proteins immobilized on glutathione SEPHAROSE™ beads were incubated with the lysates for 1 hour at 4°C with rotation, followed by washing of the beads with specifically bound proteins with cell lysis buffer for 5 times, each time 5 min with rotation at 4⁰C. The protein complexes were then eluted with Laemmli Buffer and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), followed by detection by blotting onto PVDF membranes and detection with specific antibodies. For immunoprecipitation, antibodies were added to the lysates for 1 hour at 4°C with rotation, followed by addition of Protein A PLUS G™ beads (Calbiochem) to capture the immunocomplexes. The immuno-complexes were washed with cell lysis buffer for 5 times, each time 5 min with rotation at 4°C. The immunocomplexes were then eluted with Laemmli Buffer and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by blotting onto PVDF membranes detection by with specific antibodies.

[00147] Microinjection and live cell imaging: Swiss 3T3 cells were plated at

1x105 per glass bottom dish and grown overnight at 37°C in DMEM with 10% Cosmic calf serum. DNA was injected at 0.5 μg/ml into the nucleus using a custom setup microinjector and OLYMPUS™ microscope (Olympus Number) and the cells returned to incubation at 37°C to allow for protein expression for 4 hours. For DIC and fluorescence time-lapse analysis, cells were incubated on a heated stage at 37°C and imaged with a monochromator on a Zeiss AXIOVERT™ 200 microscope enclosed in an incubator with COOLSNAP™ CCD camera.

[00148] Figure Legends

[00149] Figure IA: Evolutionary conservation of ArhGAP9 and ArhGAP12 in different organisms. Schematic representation of ArhGAP9 and ArhGAP12 domain structures of different species as predicted by SMART domain prediction tool reveals conserved domains amongst orthologs. Conservation of the RhoGAP and PH domains provided the distinctive features for this sub-family of RhoGAPs. In contrast with its human orthologs, mouse ArhGAP9 does not contain a WW domain.

[00150] Figure IB: Sequence alignment of human and mouse ArhGAP9. The sequence alignment of human and mouse ArhGAP9 indicates the absence of the WW domain in mouse. The human ArhGAP9 WW domain as predicted by SMART prediction tool is underlined. [00151] Figure 1C: Phylogenetic relationship of protein sequences of ArhGAP9 and ArhGAP12. Phylogenetic analysis of protein sequences of ArhGAP9 and ArhGAP12 of different species generated from TreeView after ClustalW analysis. The analysis shows the evoluntionary relationship of this sub-family of RhoGAPs. The accession number of each gene is noted below of the gene name.

[00152] Figure 2A: Identification of Erk2 as an interacting protein of the WW domain of ArhGAP9. GST-WW-ArhGAP9 was immobilized on glutathione Sepharose beads and incubated with rat brain lysate. Specifically bound proteins were eluted, resolved by SDSPAGE and detected by Colloidal Coomassie Blue. The bands were excised for in-gel reduction, S-alkylation and trypsin hydrolysis. The peptides were identified by mass spectrometry. Bands f and g generated 15 unique peptides covering approximately 40% of the MAP kinase, Erk2 sequence.

[00153] Figure 2B: Erk2 and p38α interacted with ArhGAP9 WW domain.

(i) Flagtagged MAP kinases (Erk2, p38α and Jnkl) were transiently transfected into 293T cells and lysates were subjected to in vitro binding assay with GST-WW- ArhGAP9, followed by western blotting with α-Flag. (ii) No binding was observed with GST control, (iii) Western blotting with a-Flag showed that the expression levels of Erk2, p38α and Jnkl in the total cell lysate of the transfected cells were equal.

[00154] Figure 2C: Erk2 and p38a interacted specifically with the WW domain of ArhGAP9 but not ArhGAP12 or Nedd4. (i) Flag-tagged MAP kinases Erk2, p38α or Jnkl were transiently transfected into 293T cells and the lysates were incubated with GST-WW domains of ArhGAP9, ArhGAP12, Nedd4 or GST alone (control) immobilized on glutathione Sepharose beads. Specifically bound proteins were resolved by SDS-PAGE, followed by western blotting with α-Flag. (ii) The expression levels of Erk2, p38α and Jnkl were shown to be equal in the total cell lysates of the transfected cells by western blotting with α-Flag.

[00155] Figure 2D: Interaction of ArhGAP9 and the MAP kinases in vivo.

Cotransfection of ArhGAP9 and the MAPKs (Erk2, p38α and Jnkl) was carried out in 293T cells. The lysates were subjected to immunoprecipitation with α-Flag, followed by Western blotting with (i) α- Flag and (ii) α-ArhGAP9. Western blotting with α- ArhGAP9 or α-Flag of the total cell lysates from the transfected cells showed equal expression levels of (Hi) ArhGAP9 and (iv) Erk2, p38α and Jnkl.

[00156] Figure 2E: Reduction of binding of ArhGAP9 to the MAP kinases upon EGFR activation. Flag-tagged Erk2, p38α or Jnkl were transfected individually or together with wildtype EGFR in 293T cells. The lysates were subjected to in vitro binding assay with the WW domain of ArhGAP9 immobilised on glutathione Sepharose beads and Western blot was performed with (i) α-Flag. (ii)There was no binding with GST alone (control). Total cell lysate from the transfected cells showed (iii) the auto- activation status of EGFR and (iv) equal expression of Erk2, p38α and Jnkl.

[00157] Figure 2F: The activation loop region of MAP kinase was not involved in binding with ArhGAP9. Flag-tagged p38α, wildtype or mutants at TXY motif in the activation loop were cotransfected with full-length ArhGAP9 in 293T cells. Immunoprecipitation was conducted with α-Flag and followed by western blotting with (i) α-ArhGAP9 and (ii) α-Flag. Total cell lysates showed equal expression of (iii) ArhGAP9 and (iv) p38.

[00158] Figure 2G: Alignment of the WW domain of ArhGAP9 and ArhGAP12. WW domains of ArhGAP9 and ArhGAP12 were aligned using ClustalW. The di-Arginine motif (shown in bold and indicated with solid squares) at the extreme C terminal were shown to be important for binding of ArhGAP9 with the MAP kinases.

[00159] Figure 3A: Mapping binding sites of the interaction between MAPKs Erk2 and p38α and ArhGAP9. GST fusion proteins of the C-terminal regions of Erk2 and p38α containing the common docking (CD) domain GST were expressed and purified. Flagtagged ArhGAP9 was transiently transfected in 293T cells and the lysates obtained were subjected to in vitro binding assay with CD-GST of the MAP kinases. Specifically bound proteins were eluted and resolved by SDS-PAGE, followed by western blotting with α-Flag.

[00160] Figure 3B: Acidic residues in the CD domain of MAP kinases important for interaction with ArhGAP9. Flag-tagged wildtype or CD domains mutants of Erk2 (D316A, D319A or E320A) were transfected into 293T cells. In vitro binding assay was carried out on the lysates with (i) GST-WW-ArhGAP9 or (ii) the GST control. Western blotting with α-Flag was performed and the results showed significant reduction in the binding of the Erk2 D316A mutant. Similarly, for p38α, the D312A, D315A and E316A mutants showed reduction in binding to ArhGAP9 WW domain, (ii) There was no binding with GST alone, (iii) Western blotting of the total cell lysates with α-Flag showed equal expression of the wildtype and mutant MAP kinases.

[00161] Figure 3C: The di-Arginine motif (R246 and R247) was important for ArhGAP9 WW domain binding to CD domain of the MAP kinases, (i) Flag- tagged wildtype ArhGAP9 or mutants at R246A, R247A or R246,247A were expressed in 293T cells and the lysates are subjected to in vitro binding assay with (i) GSTCD of Erk2 or the (ii) control of GST alone. Western blotting was carried out with α-Flag. Specific interaction occurred between GST-CD-Erk2 and wildtype ArhGAP9 but not the R246A, R247A or R246,247A mutants. The GST control showed no binding, (iii) Western blotting of total cell lysates with α-Flag showed equal expression of wildtype ArhGAP9 and R246A, R247A and R246,247A mutants.

[00162] Figure 3D: Binding of Erk2 and p38α to the WW domain of

ArhGAP9 was disrupted by R246A and R247A mutations Flag-tagged Erk2, p38α or Jnkl was transfected in 293T cells and the lysates were subjected to in vitro binding assay with the (i) GST- WW- ArhGAP9 and (ii) GST-WWArhGAP9( RR) mutant where the R246A and R247A mutations had been introduced, (iii) GST alone was used as a control. Specifically bound proteins were eluted and resolved by SDS-PAGE, followed by western blotting with α-Flag. Significant reduction of binding was detected for Erk2 and p38α. (iii) No binding with the GST control was detected, (iv) The expression levels of Erk2, p38α and Jnkl were shown to be equivalent by western blotting of the total cell lysates with α-Flag.

[00163] Figure 3E: In vivo binding of ArhGAP9 to Erk2 and p38α reduced with R246A and R247A mutations. Flag-tagged Erk2 or 38α were transiently individually or together with wildtype ArhGAP9 or the R246,247A mutant (RR) in 293T cells, as indicated in the figure. Immunoprecipitation was conducted on the lysates with α-Flag and followed by western blotting with (i) α-Flag or (ii) α-ArhGAP9. Reduction in binding of Erk2 and p38α with ArhGAP9 R246,247A mutant was observed. Western blotting of the total cell lysates with (iii) α-ArhGAP9 or (iv) α-Flag showed equal expression levels of ArhGAP9 and the MAP kinases.

[00164] Figure 3F: Coexpression of MEK2 reduced the binding of Erk2 to the WW domain of ArhGAP9 in vitro. Flag-tagged Erk2 was transfected alone or together with HA-tagged MEK2 in 293T cells and the lysates were subjected to in vitro binding assay with (i) GST-WW-ArhGAP9 or (ii) the control of GST alone. Bound Erk2 was detected by western blotting with α-Flag. Reduction of binding between the WW domain of ArhGAP9 and Erk2 in the presence of MEK2 was detected. Western blotting of the total lysates with (iii) α-phospho-Erk showed that Erk2 was activated when coexpressed with MEK2. Western blotting of the total lysates with (iv) α-Flag and (v) α-HA showed the expression of Erk2 and MEK2 in the total cell lysates.

[00165] Figure 3G: Reduction of ArhGAP9 binding to Erk2 by MEK in vivo. Flag-tagged Erk2, HA-tagged MEK2 and ArhGAP9 were transiently transfected in 293T cells as indicated in the figure. Immunoprecipitation was carried out with α- ArhGAP9, followed by by western blotting with (i) α-ArhGAP9α-Flag and (ii) α-HA. Western blotting of the total lysates with (iii) α-phospho-Erk showed that Erk2 was activated when coexpressed with MEK2. Western blotting of the total lysates with (iv) α-ArhAGP9, (v) α-Flag and (vi) α-HA showed the expression of ArhGAP9, Erk2 and MEK2 in the total cell lysates.

[00166] Figure 4A: MAP kinase binding to ArhGAP9 had no significant effect on RhoGAP activity. ArhGAP9 was transfected with myc-tagged cdc42 with or without Flag-p38α in 293T cells as indicated in the figure. The R578K GAP-inactive mutant coexpressed with myc-cdc42 as a control. The lysates were incubated with purified PBD-GST to assess the amount of active cdc42, reflected by the amount of myc-cdc42 associated with PBD-GST by western blotting with α-myc (i). The presence of ArhGAP9 but not the R578K mutant showed significant reduction in the amount of active cdc42. In the presence of p38α, there was no significant reduction of cdc42 activity. Western blotting of total cell lysates with (ii) α-ArhGAP, (iii) α- Flag and (iv) α-myc showed the expression levels of ArhGAP9, p38α and cdc42, respectively.

[00167] Figure 4B: ArhGAP9 suppressed MAP kinase activation by EGF receptor. Wildtype ArhGAP9 or the mutants WW-I (W219K), WW-2 (W242K), PH4 (R339,349A), PH9 (R342,K343A), SH3* (W181,182K) were cotransfected with EGFR and p38α in 293T cells, (i) The activity of EGFR was indicated by western blotting of the total cell lysates with α- phosphotyrosine (pTyr). The expression of ArhGAP9 and p38α in the lysates was shown to be equal by western blotting with (ii) α-ArhGAP9 and (iii) α-Flag. The activity of p38α was detected by western blotting α-phospho- specific p38 (iv). Coexpression of EGFR with p38α induced the activity of the MAP kinase (iv, lane 3) and the presence of wildtype ArhGAP9 caused a significant suppression of this activation (iv, lane 4). The coexpression of the WW domain mutants of ArhGAP9 (WW-I and WW-2) caused a partial restoration of the ability of ArhGAP9 to suppress EGFR- induced p38α activity.

[00168] Figure 5: Expression of ArhGAP9 R246.247A mutant disrupted stress fibres in Swiss 3T3 cells. Swiss 3T3 cells were microinjected with wildtype ArhGAP9 or the ArhGAP9 R246,257A (RR) mutant together with GFP-actin. The cells were imaged and the GFP fluorescence showed that while wildtype ArhGAP9 (left, top and bottom panels) maintained the actin stress fibres in the fibroblasts, the expression of the ArhGAP9 R246,257A (RR) mutant (right, top and bottom panels) defective in MAP kinase-binding resulting in the loss of stress fibers.

[00169] Results

[00170] Domain structure and phylogenetic analysis of ArhGAP9 and

ArhGAP12: ArhGAP9 and ArhGAP12 are multi-domain polypeptides consisting of an interesting combination of protein interaction domains, including the Src Homology 3 (SH3) and WW, a phospholipid binding Pleckstrin Homology (PH) domain and the catalytic Rho GTPase Activating Protein (Rho GAP) domain. The ArhGAP9 and ArhGAP12 genes found in different species of organisms with the domain structures predicted by the SMART domain prediction program are shown in Figure IA. We have cloned the full-length mouse ArhGAP9 cDNA and observed that interestingly, although it shared 64% sequence identity with human ArhGAP9, it lacked the WW domain (GeneBank accession no. XXX, availability pending submission of sequence to NCBI). This is shown in the amino acids alignment in Figure IB. However, we could not rule out the possibility that SMART did not identify a WW domain in mouse ArhGAP9 due to low sequence homology to other WW domains. The ArhGAP9 ortholog in the C. elegans and C. Briggsae lacked an SH3 domain, which could be due to the SH3 domain being a result of evolutionary accretion of the SH3 domain. The radial tree depicted highlights the evolutionary relationship of ArhGAP9 and ArhGAP12 for different species. The phylogenetic analysis was carried out using ClustalW and visualized with Tree View. The mammalian ArhGAP9 and ArhGAP12 genes showed high evolutionary homology separately. There is strong probability that the worms ArhGAP12 gene may be the evolutionary ancestor of the mammalian ArhGAP9 and ArhGAP12.

[00171] Identification of the Erk and p38 MAP kinases as novel binding proteins to the WW domain of ArhGAP9: To identify interacting protein partners for the WW domain of ArhGAP9, the GST fusion protein of the WW domain of human ArhGAP9 was purified and immobilized on glutathione SEPHAROSE™ beads and used as a bait to isolate interacting proteins from rat brain lysate. The protein complex was resolved by 10% SDS-PAGE, followed by detection with Colloidal Coomassie Blue (Figure 2A). Several specific binding proteins were identified, including the MAP kinase Erk2 from bands f and g that generated 15 unique peptides covering 40% of the protein sequence of Mitogen-activated protein kinase 1, also known as extracellular signal-regulated kinase 2, Erk2.

[00172] To confirm that Erk2 bound to the WW domain of ArhGAP9, in vitro pulldown and co-immunoprecipitation assays were performed and the results are shown in Figure 2B. Flag-tagged Erk2 was expressed by transfection of the cDNA into 293T cells and the lysates were incubated with GST fusion protein of the WW domain of ArhGAP9 immobilized on glutathione SEPHAROSE™ beads. The protein complex was washed and eluted, followed by SDS-PAGE and western blotting with anti-Flag. The binding of two closely related MAP kinases, p38α-Flag and Jnkl-Flag, were also tested. Consistent with the proteomics data, Erk2 bound to the WW domain of ArhGAP9 with high affinity (Figure 2B(i)). While p38α also showed binding albeit to a lesser extent than Erk2, the binding of Jnkl was not detectable in the pulldown assay (Figure 2B(i)). The control of GST alone immobilized on glutathione SEPHAROSE™ beads did not bind the MAP kinases, indicating that the binding the binding of Erk2 and p38α to ArhGAP9 WW domain was specific (Figure 2B(H)). The expression levels of Erk2, p38α and Jnkl were comparable as shown by western blotting with anti-Flag of the whole cell lysates (Figure 2B(Hi)).

[00173] To further determine the specificity of Erk2 and p38α binding to the WW domain of ArhGAP9, we tested the binding of Erk2, p38α and Jnkl to the two WW domains of ArhGAP12 (ArhGAP12-WWl and ArhGAP12-WW2) individually and the first WW domain of Nedd4 (Nedd4-WWl). The lysates of 293T cells transfected with Flag-tagged Erk2, p38α and Jnkl were incubated with GST fusion proteins of ArhGAP12-WWl, ArhGAP12-WW2 and Nedd4-WWl immobilized on glutathione SEPHAROSE™ beads. The protein complexes were washed and eluted, followed by SDS-PAGE and western blotting with anti-Flag. Erk2, p38α and Jnkl showed no detectable binding to ArhGAP12-WWl, ArhGAP12-WW2 and Nedd4- WWl (Figure 2C(i)). The expression levels of Erk2, p38α and Jnkl were comparable as shown by western blotting with anti-Flag of the whole cell lysates (Figure 2C(U)).

[00174] To demonstrate the binding of Erk2 to ArhGAP9 in vivo, 293T cells were transfected with ArhGAP9, Erk2-Flag, p38α-Flag or Jnkl-Flag alone, or ArhGAP9 with Erk2-Flag, p38α-Flag or Jnkl-Flag. The lysates were subjected to immunoprecipitation with anti-Flag and the protein complexes washed and eluted for SDS-PAGE. Western blotting with anti-ArhGAP9 showed that ArhGAP9 associated with both Erk2 and p38α equally, and to Jnkl to a relatively lesser extent as shown in Figure 2D(i). Western blotting of the immunoprecipitates with anti-Flag showed that the relative amounts of the Erk2, p38α and Jnkl were equivalent (Figure 2D(ii)).

[00175] The expression of Erk2, p38α and Jnkl were shown to be comparable by western blotting of the whole cell lysates with anti-Flag (Figure 2D(Hi)), and similarly the expression of ArhGAP9 was shown to be comparable with anti-ArhGAP9 western blotting (Figure 2D(iv)). [00176] From the pulldown and coimmunoprecipitation results, we concluded that the three MAP kinases Erk2, p38α and Jnkl were able to bind to the WW domain of ArhGAP9 albeit with varying affinities. Our results show that the affinity is highest with Erk2, followed by p38α and lowest with Jnkl. The binding of p38α and Jnkl to full-length ArhGAP9 may be more readily detectable than binding to the WW domain alone, explaining why Jnkl could be seen as a coimmunoprecipitating protein with full- length ArhGAP9 but not detectable in the pulldown assay with ArhGAP9 WW-GST.

[00177] Binding of Erk and p38α to ArhGAP9 diminished with activation of the MAP kinases: The MAP kinases have been known to be activated downstream of growth factor receptor activation. To assess the importance of upstream activating signals to the binding of the MAP kinases to ArhGAP9, the binding of the inactive and EGFR-activated MAP kinases to ArhGAP9 WW-GST was compared. 293T cells were transfected with Erk2-Flag, p38α-Flag or Jnkl-Flag alone or in combination with EGFR. When over-expressed by transient transfection in 293T cells, EGFR was active due to auto-phosphorylation on tyrosine residues required for kinase activation. The activation of the EGFR was confirmed by western blotting of total cell lysates with anti-phosphotyrosine (Figure 2E(Ui)). The activation of the Erk2, p38α and Jnkl by co- expression with EGFR was confirmed by western blotting with phosphospecific antibodies (data not shown). The lysates prepared were incubated with ArhGAP9- WW-GST immobilized on glutathione SEPHAROSE™ beads. The protein complexes were washed and eluted, followed by SDS-PAGE and western blotting with anti-Flag. Interestingly, it was observed that EGFR activation caused the binding of Erk2 and p38α to ArhGAP9-WW to be significantly reduced (Figure 2E(i)). GST alone did not show detectable binding, confirming the specificity of the interaction of Erk2 and p38α to ArhGAP9 WW domain (Figure 2E(U)). The expression levels of Erk2, p38α and Jnkl were confirmed to be comparable by western blotting of whole cell lysates with anti-Flag (Fig 2E(iv)).

[00178] One possibility that EGFR activation might result in the reduction of

Erk2 and p38α binding to the WW domain of ArhGAP9 could be that the binding motif for ArhGAP9-WW domain resided around the activation loop of the inactive MAP kinases, such that when these sites became phosphorylated upon activation as a consequence of EGFR activation, the structural changes in the activation loop became unfavourable for the binding of ArhGAP9-WW domain. To test this hypothesis, in the context of p38α, the two residues whose phosphorylation resulted in structural changes required for phosphoryl transfer onto substrate proteins, namely ThrlδO and Tyrl82 were mutated to Ala or Phe. The binding of wild type p38α and mutants were compared. Flag-tagged p38α (wildtype, Thrl80Ala, Thrl80Phe, Tyrl82Ala, Tyrl82Phe or the double mutants Thrl80Ala,Tyrl82Ala or Thrl80Phe,Tyrl82Phe) were coexpressed with ArhGAP9 in the presence or absence of EGFR in 293T cells by transient transfection. The lysates were subjected to immunoprecipitation with anti- Flag. The immunoprecipitates were washed and resolved by SDS-PAGE, followed by western blotting with anti-ArhGAP9 or anti-Flag. As shown in Figure 2F(i), the binding of ArhGAP9 to the different p38α mutants were not significantly reduced compared to the wildtype protein, indicating that the binding motif resides outside the activation loop. The amounts of immunoprecipitated wildtype p38α and the different mutants were equivalent, providing a valid comparison of ArhGAP9 binding (Figure 2F(U)). The expression levels of ArhGAP9 and p38α (wildtype and the different mutants) were shown to be comparable by western blotting of whole cell lysates with anti-ArhGAP9 and p38α, respectively (Figure 2F(iii, iv)).

[00179] These results indicated that the binding of the MAP kinases Erk2 and p38α to the WW domain of ArhGAP9 was negatively regulated by upstream activating signals of these MAP kinases suggesting that the binding sites most likely resided outside the activation loops of the latter. The structural changes that resulted upon activation of the MAP kinases by phosphorylation at the activation loop most likely also would not alter the binding of Erk2 and p38α to the WW domain of ArhGAP9.

[00180] ArhGAP9 and MAP kinase binding was mediated by complementarity charged residues in WW and CD domains, respectively: From the sequence alignment of ArhGAP9-WW domain with the WW domains of all the other proteins that contain the domain, it was observed that the C-terminal end of ArhGAP9- WW domain contained a unique basic di-Arginine motif (R246 and R247) that were not present in all other WW domains compared (data not shown). Figure 2G showed the sequence alignment of the WW domains of ArhGAP12 and ArhGAP9. Notably in the first and second WW domains of ArhGAP12 which did not bind Erk2 and p38α, the residues in alignment with R247 are W and Y, respectively. Such short basic motifs had been shown to be present in a large number of MAP kinase docking proteins that bind to the common docking (CD) domains of MAP kinases. The CD domains of Erk2, p38 and Jnk consisted of acidic residues which form electrostatic interactions with the basic residues of the target docking proteins (Tanoue, T., Adachi, M., Moriguchi, T. & Nishida, E. A common docking motif in MAP kinases common to substrates, activators and regulators. Nat Cell Biol 2, 110-116(2000)). We postulated that complementarity charged residues on the CD domains of Erk2 and p38α and WW domain of ArhGAP9 could be the mechanism which enhanced or mediated the binding of these MAP kinases and ArhGAP9. Consequently, we tested whether the WW domain of ArhGAP9 alone could be binding to a C-terminal fragment of Erk2 and p38α containing the CD domain.

[00181 ] A C-terminal fragment of Erk2 and p38α containing their respective CD domains, termed Erk2-CD-GST, p38α-CD-GST were expressed as GST fusion proteins, purified and immobilized on glutathione SEPHAROSE™ beads. Lysates from 293T cells transfected with Flag-tagged ArhGAP9 were incubated with immobilized Erk2-CD-GST, p38α-CD-GST or GST alone. The protein complexes were washed and eluted, followed by SDS-PAGE and western blotting with anti-Flag. As shown in Figure 3A, ArhGAP9 specifically precipitated with Erk2-CDGST or p38α-CD-GST but not the GST alone control, indicating that the CD domain of Erk2 and p38α were most likely sufficient to mediate their binding with ArhGAP9. To further confirm that the CD domains of Erk2 and p38α were involved in binding to the WW domain of ArhGAP9, the acidic residues (Asp or GIu) in the CD domain of Erk2 and p38α known to be involved in binding to the basic residues of other docking proteins were mutated to Ala and expressed in 293T cells then tested for binding to the WW domain of ArhGAP9. As shown in Figure 3B(i), Erk2-Asp316Ala interacted with ArhGAP9-WW domain poorly, while the mutations Asp312Ala, Asp315Ala, and Glu316Ala of p38α all caused a significant reduction in interaction with ArhGAP9-WW domain. These results indicated that the acidic residues in the CD domains of Erk2 and p38α were indeed important in their interactions with ArhGAP9. The control of GST alone did not bind to wildtype or mutant Erk2 and p38α, indicating that there was no non-specific interactions (Figure 3B(ii)). The expression of wildtype or mutant Erk2 and p38α were equivalent and allowed for valid comparison of their binding to ArhGAP9 WW domain (Figure 3B(Ui)). We then proceeded to determine if the di-basic motif in the C-terminal end of the WW domain of ArhGAP9 was important in mediating the binding with the CD domains of Erk2 and ρ38α. R246A (Rl), R247A (R2) and R246,247A (RR) mutants of ArhGAP9 were generated and expressed in 293T cells and used for testing of interaction with the CD domain of Erk2. Lysates containing wildtype ArhGAP9 or the Rl, R2 and RR mutants were incubated with immobilized Erk2-CD-GST. The protein complexes were washed and eluted for SDS-PAGE, followed by western blotting with anti-ArhGAP9. As shown in Figure 3C(i), while wildtype ArhGAP9 formed a complex with Erk2-CD-GST, the binding of the Rl, R2 and RR mutants were not detectable. GST control alone showed no binding (Figure 3C(U)) and the expression levels of wildtype ArhGAP9 or the Rl, R2 and RR mutants in whole cell lysates were confirmed to be equal (Figure 3C(Ui)).

[00182] To verify that R246 and R247 in the WW domain of ArhGAP9 were required for interaction with the CD domain of Erk2 and p38α, the GST fusion proteins of the Rl, R2 and RR mutants of the WW domain of ArhGAP9 were expressed, purified and immobilized on glutathione SEPHAROSE™ beads and used for binding assays. Lysates from 293T cells transfected with Flag-tagged Erk2, p38α or Jnkl were incubated with immobilized wildtype, Rl, R2 or RR mutant WW domain of ArhGAP9. The protein complexes were washed and eluted, followed by SDS-PAGE and western blotting with anti-Flag. As shown in Figure 3D(ii), the binding of Erk2 and p38α to Rl, R2 and RR mutants of the WW domain of ArhGAP9 was significantly reduced compared to the wildtype WW domain (Figure 3D(i)). GST control alone showed no binding (Figure 3D(Ui)) and the expression of Erk2, p38α and Jnkl in whole cell lysates were confirmed to be equal (Figure 3D(iv)).

[00183] To compare the binding of full-length wildtype and RR mutant of

ArtiGAP9 in vivo, ArhGAP9 or RR mutant was co-transfected with Erk2-Flag or p38α- Flag in 293T cells. Lysates were prepared and subjected to immunoprecipitation with anti-Flag. The protein complexes were washed and eluted for SDS-PAGE, followed by western blotting with anti-ArhGAP9, Flag or phospho-p38. As shown in Figure 3E(i), the amount of RR mutant that coimmunoprecipitated with wildtype ArhGAP9 was almost non-detectable. The amounts of Erk2 or p38α that were immunoprecipitated were shown to be equivalent to allow valid comparison of ArhGAP9 binding (Figure 3E(ii)). The expression of Erk2, p38α, wildtype and RR mutant of ArhGAP9 was shown to be equivalent in whole cell lysates by anti-Flag and anti-ArhGAP9 western blotting (Figure 3E(Ui) and (iv)).

[00184] These results strongly supported the hypothesis that the binding between the WW domain of ArhGAP9 and Erk2 or p38α was mediated by complementarily charged residues in WW and CD domains, respectively.

[00185] Co-expression of ME K 2 and MKK6 reduced binding of Erk2 and p38α to WW domain of ArhGAP9: Since the CD domain of MAP kinases had been established as the binding sites for their upstream activating kinases, namely MEKl and 2 for Erk and MKK3, 4 and 6 for p38α29, and our data showed that upon EGFR stimulation the binding of ArhGAP9 to Erk2 and p38α was reduced, we postulated that the binding of MEK to Erk and MKK to p38α may compete with the binding of ArhGAP9 to these MAP kinases. To investigate if this is indeed the case, the binding of Erk2 or p38α to ArhGAP9 in the absence or presence of overexpressed MEK2 or MKK, respectively was compared. Lysates from 293T cells were transfected with Erk2-Flag alone or Erk2-Flag with MEK2-HA were incubated with immobilized WW- ArhGAP9-GST. The protein complexes washed and eluted for SDS-PAGE, followed by western blotting with anti- Flag. As shown in Figure 3F(i), the amount of Erk2 that precipitated with the WW domain ArhGAP9 was significantly reduced in the presence of MEK2. It was also shown that the coexpression of MEK2 strongly induced phosphorylation and activation of Erk2, as indicated by western blotting with phospho- specific Erk antibodies (Figure 3F(U)). The control of GST alone showed no binding (Figure 3F(Ui)) and the expression levels of Erk2 and MEK2 in whole cell lysates were shown by western blotting with anti-Flag and anti-HA, respectively (Figure 3F(iii, iv)).

[00186] Consistent with the above in vitro data, we observed that the binding of full-length ArhGAP9 to Erk2 in vivo was reduced in the presence of MEK2. 293T cells were transfected with Erk2- Flag alone, Erk2-Flag with ArhGAP9, Erk2-Flag with MEK2-HA or all the three plasmids together. Erk2-Flag was immunoprecipitated from the lysates with anti-Flag and the binding of ArhGAP9 was detected by western blotting with anti-ArhGAP9. It was observed that the amount of ArhGAP9 that co- immunoprecipitated with Erk2 from lysates of transfected 293T cells was reduced when MEK2 was cotransfected to cause the phosphorylation and activation of Erk2 (Figure 3G(i)). The amount of Erk2 that were immunoprecipitated was shown to be equivalent by western blotting with anti-Flag (Figure 3G(H)). The activation of Erk2 by coexpression of MEK2 was confirmed by western blotting with phospho-specific Erk2 antibodies (Figure 3G(Ui)). The expression levels of ArhGAP9, Erk2 and MEK in whole cell lysates were shown by western blotting with anti-ArhGAP9, Flag and HA, respectively (Figure 3G(iv, v and vi)). Similarly, with the cotransfection of MKK6, the amount of p38α that precipitated with the ArhGAP9 both in vitro and in vivo was significantly reduced (data not shown).

[00187] Binding of ArhGAP9 inhibited Erk2 and p38α activation by upstream signals: Since both ArhGAP9 and the MAP kinases have catalytic activity, we proceeded to investigate whether their interaction could mutually influence their activities. Furukawa et al. (Isolation of a novel human gene, ARHGAP9, encoding a rho- GTPase activating protein. Biochem Biophys Res Commun 284, 643-649 (2001)) had shown that ArhGAP9 functioned as an active GTPase activating protein towards cdc42 and Racl but not RhoA and we had confirmed the data (data not shown). To determine whether MAP kinase binding had any effect on the Rho GAP activity of ArhGAP9, the activation status of cdc42 in the lysates of cells transfected with ArhGAP9 in the presence or absence of Erk2 was compared. Recombinant Pak-binding domain (PBD) GST fusion protein which would bind preferentially to active cdc42 over the inactive form immobilized on glutathione SEPHAROSE™ beads was used as a probe. Lysates from 293T cells transfected with myc-tagged cdc42, with and without ArhGAP9 and Flag-tagged p38α in different combinations shown in Figure 4 A were prepared and incubated with the immobilized PBD-GST. The GAP-inactive mutant of ArhGAP9 (R578K) was used as a negative control for the assay. The protein complexes were washed and eluted for SDS-PAGE, followed by western blotting with anti-myc to ascertain the relative amount of active cdc42 in the lysate. As shown in Figure 4A(i), there was no significant effect in the activity with or without the presence of p38α. The expression levels of cdc42, ArhGAP9 or p38α were shown by western blotting with anti-myc, ArhGAP9 or Flag, respectively (Figure 4A(ii,iii and iv)). Similar results were obtained which indicated that Erk2 binding to ArhGAP9 did not influence the Rho GAP activity of the latter (data not shown).

[00188] We next investigated whether the binding of ArhGAP9 to the Erk2 and p38α had an effect on the MAP kinase activities. 293T cells were transfected with p38α alone, or p38α with EGFR to induce the activation of p38α, or p38α with EGFR as well as ArhGAP9 to anlayse whether the activation of p38α was affected by the presence of ArhGAP9 or various ArhGAP9 mutants. The ArhGAP9 mutants tested included the R578K GAP-inactive mutant, the W219K and W242K mutants which caused the structure of the WW domain to be disrupted and unable to bind to its targets, the R339A,K340A and R342A,K343A mutants which disrupted the ability of the PH domain to bind to its phospholipid targets and the W181,182K mutant which would render the SH3 domain defective in targeting its proline-containing targets. The mutants transfected in each sample were as indicated in the figure legend of Figure 4B. Equal amounts of the whole cell lysates prepared from these transfected cells were resolved by SDS-PAGE followed by western blotting with anti-phosphotyrosine which predominantly detected the activated EGFR that was the major tyrosine phosphorylated protein in these lysates (Figure 4B(i)). It could be concluded that the expression and activation of EGFR in each sample were equivalent. The expression levels of wildtype and the different ArhGAP mutants were shown to be comparable by anti-ArhGAP9 western blotting (Figure 4B(U)). The expression Flag-tagged p38α was confirmed to be comparable by anti-Flag western blotting (Figure 4B(Ui)). The activation status of p38α in each of the samples was analysed by anti-phospho-p38 western blotting (Figure 4B(iv)). From Figure 4B(iv), it could be seen that p38α that was transfected alone showed a slight detectable level of activation (lane 2) which increased with the coexpression of the EGFR (lane 3). With the expression of wildtype ArhGAP9 (lane 4), we observed a reduction of p38α activation by EGFR, indicating that ArhGAP9 could suppress the activation of p38α downstream of EGFR. With the coexpression of all the ArhGAP9 mutants (lanes 5- 10), there was also reduction of p38α activation by EGFR albeit to varying extents. Most interestingly, we observed that the W219K and W242K mutants (lanes 6 and 7) which would cause the structure of the WW domain to be disrupted and therefore bound MAP kinases poorly, were able to allow for activation of p38α to a level higher than wildtype ArhGAP9 (lane 3). The ability of the WW domain mutants to allow partial restoration of p38α activation by EGFR compared to wildtype ArhGAP9 strongly indicated that the binding of ArhGAP9 to MAP kinases would prevent the activation of the latter by their upstream kinases.

[00189] Taken together, the above data suggests that the binding of ArhGAP9 through its WW domain to the MAP kinase p38α, did not have a significant effect on the Rho GAP activity of ArhGAP9, but caused the inhibition of p38α activation by upstream signals.

[00190] ArhGAP9 binding prevented MAP kinase-induced loss of actin stress fibres: Rho GTPase and MAP kinase signaling have both been implicated in the control of the actin cytoskeleton. Specifically it was reported that activation of the MAP kinases Erkl, 2 and 5 pathways caused disruption of the actin cytoskeleton28. To investigate if the interaction of ArhGAP9 and MAP kinases had an effect on the actin cytoskeleton, wildtype or the RR mutant of ArhGAP9 which was defective in binding to Erk2 and p38α were expressed in Swiss 3T3 cells by microinjection of the respective cDNAs together with GFP-actin, followed by imaging of the actin structure. As shown in Figure 5(i, ii) in the presence of wildtype ArhGAP9, the cells showed clear bundles of stress fibres whereas in the presence of the RR mutant (Figure 5(iii, iv)), the stress fibres were dissolved and showed a diffuse pattern of actin. This supports the hypothesis that binding of wildtype ArhGAP9 to MAP kinases sequestered them in the inactive forms allowing the formation and maintenance of actin stress fibres. Conversely, the RR mutant being unable to bind the MAP kinases, should potentially allow activation of the latter to cause the dissolution of the actin stress fibres. This result was consistent with the notion that the binding of ArhGAP9 to the MAP kinases prevented the activation of the latter.

[00191] EXAMPLE 2

[00192] Materials and methods

[00193] Antibodies. Mouse monoclonal anti-Flag (M2) and anti-phospho- specific p38 were obtained from Sigma, mouse phospho-Erk was from Cell Signaling Technology, mouse monoclonal anti-HA (12CA5) was from Boerhinger, rabbit polyclonal anti-ArhGAP9 was generated by standard immunization procedure with full-length human ArhGAP9-GST as the immunogen. Mouse monoclonal anti- phosphotyrosine conjugated to horse radish peroxidase (PY20-HRP) was from Transduction Laboratories, rabbit polyclonal EGFR was from Santa Cruz Biotechnology.

[00194] Peptides and Far UV Circular Dichroism (CD) spectroscopy.

Peptides for Erk2: LEQYYDPSDEPIAE, p38α: FAQYHDPDEPVAD and Jnkl: INVWYDPSEAEAPP were custom synthesized by NeoSystems (Strasbourg). CD experiments were performed with a Jasco J-715 spectropolarimeter equipped with a Peltier cell holder and a PTC-348WI temperature controller. Far-UV CD spectra were measured in a 1-mm rectangular quartz cell. The buffer used was 20 mM Tris-HCl (pH 7.5), 500 mM NaCl and 1 mM DTT. Protein concentration of approximately 0.5 mg/ml (determined by the Bradford assay method) was used for wavelength scans. Wavelength scans were made at a scan rate of 10 nm/min and the average value of 3 scans of the same solution were obtained. Data were collected at 25°C over a wavelength range of 190-260 nm with a bandwidth of 1 nm. To determine the conformational changes in WW domain upon peptide binding, the ellipticity of the corresponding peptides at equimolar concentration with the solvent were subtracted from the ellipticity of the WW domain-peptide complex and similarly the solvent spectrum were subtracted from the spectrum of WW domain before analysis. The far UV CD spectra were analyzed by using the secondary structure analysis program CDNN, version 2.1 (Ref).

[00195] Cell lines. Human 293T cells were cultured in Dulbecco's minimal essential medium (DMEM) containing 4.5g/l D-glucose and supplemented with 10% fetal bovine serum (HyClone), 10 mM L-glutamine, and 100 μg each of penicillin and streptomycin/ml from Sigma. Swiss 3T3 were cultured in DMEM containing 4.5g/l D- glucose and supplemented with 10% Cosmic calf serum (HyClone), 10 mM L- glutamine, and 100 μg each of penicillin and streptomycin/ml.

[00196] Protein expression and purification. The human ArhGAP9 (wild type and mutants), and EGFR coding sequences were cloned into the mammalian expression vector pRK5. The Erk2, p38, Jnkl were cloned in pxJ40-Flag vector which allowed N- terminal Flag epitope tagging. HA-tagged activated MEK2(S222D, S226D) was cloned in pUSEamp vector. Activated MKK6(S207E, T211E) was cloned in pxJ40-HA vector. Plasmid encoding Pakbinding domain (PBD)-GST was obtained from Dr. E Manser. Cdc42-myc was cloned in pcDNA3. For transient expression in mammalian cells, Lipofectamine (Gibco BRL) was used for transfection, following the protocol recommended by the manufacturer.

[00197] For expression of glutathione S-transferase (GST) fusion proteins, PCR- amplified sequences corresponding to the regions of interest of the proteins were cloned into the pGEX4Tl vector (Pharmacia). The GST fusion proteins were expressed in E. coli BL21 (DE3) cells and purified using glutathione sepharose 4B column chromatography (Amersham). For Far-UV and ITC measurements, the GST tag was cleaved with the thrombin protease by overnight on-column digestion at 4oC. The resulting protein preparation was then further purified by using ion-exchange Mono Q Sepharose column (Amersham) which had been pre-equilibrated with buffer A (20 mM Tris-HCl pH 7.5). The protein was then eluted from the column with a linear gradient to buffer B (20 mM Tris-HCl pH 7.5, 1 M NaCl). The protein was further purified using Superdex-75 gel filtration column chromatography in Biologic Duoflow FPLC system.

[00198] Site-directed mutagenesis. Point mutations were introduced with the Quikchange sitedirected mutagenesis kit (Stratagene). The mutations were confirmed by DNA sequencing.

[00199] Proteomics and mass spectrometry analysis. Protein complexes of the

WW domain of ArhGAP9 isolated from rat brain lysate were eluted and resolved by 10% SDS-PAGE, followed by detection of bound proteins by staining with Colloidal Coomassie Blue (Pierce). Specific bands were excised and subjected to in-gel reduction, S-alkylation and trypsin hydrolysis. Liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis of the peptides was performed on a Finnigan LCQ Deca ion trap mass spectrometer (Thermo Finnigan) fitted with a Nanospray source (MDS Proteomics). Chromatographic separation was conducted using a Famos autosampler and an Ultimate gradient system (LC Packings) over Zorbax SB-C 18 reverse phase resin (Agilent) packed into 75μm PicoFrit columns (New Objective). Protein identifications were made using the search engines Mascot (Matrix Sciences) and Sonar (ProteoMetrics).

[00200] Binding assays. Lysates for binding assays and immunoprecipitation were prepared by lysis of cells in the cell lysis buffer [20 mM HEPES (pH 7.5), 137 niM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgC12, 1 mM EGTA, 0.1 mM Na3VO4 and Complete Protease inhibitors (Boehringer)], followed by centrifugation 13,000g for 15 min at 4oC and collecting the supernatant. For in vitro pulldown assays, GST fusion proteins immobilized on glutathione beads were incubated with the lysates for 1 hour at 4oC with rotation, followed by washing of the beads with specifically bound proteins with cell lysis buffer for 5 times, each time for 5 min with rotation at 4oC. The protein complexes were then eluted with Laemmli Buffer and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by detection by blotting onto PVDF membranes and detection with specific antibodies. For immunoprecipitation, antibodies were added to the lysates for 1 hour at 4oC with rotation, followed by addition of Protein A PLUS G beads (Calbiochem) to capture the immunocomplexes. The immuno-complexes were washed with cell lysis buffer for 5 times, each time for 5 min with rotation at 4oC. The immuno-complexes were then eluted with Laemmli Buffer and resolved by SDS-PAGE, followed by western blotting with specific antibodies.

[00201] Microinjection and live cell imaging. Swiss 3T3 fibroblasts were plated at 5x104 per glass bottom dish and grown overnight at 37oC in DMEM with 10% Cosmic calf serum. DNA was injected at 0.5 μg/ml into the nuclei using a custom setup microinjector and Olympus microscope (Olympus IMT-2) and the cells were returned to incubation at 37oC overnight to allow for protein expression. For DIC and fluorescence time-lapse analysis, cells were incubated on a heated stage at 37oC and imaged with a monochromator on a Zeiss Axiovert 200M microscope enclosed in an incubator with CoolSNAP CCD camera. For statistical analysis of actin stress fibres, the values were given as a mean +/- S. D. Experiments were repeated 3 times, with n = 7 to 10.

[00202] Results

[00203] Domain structure and phylogenetic analysis of ArhGAP9 and

ArhGAP12. ArhGAP9 and ArhGAP12 are multi-domain polypeptides consisting of an interesting combination of protein interaction domains, including the Src Homology 3 (SH3) and WW, a phospholipid binding Pleckstrin Homology (PH) domain and the catalytic Rho GTPase Activating Protein (RhoGAP) domain. The ArhGAP9 and ArhGAP12 genes found in different species of organisms with the domain structures predicted by the SMART domain prediction program [Ref] are shown in Fig. Ia.

[00204] We have cloned the full-length mouse ArhGAP9 cDNA and observed that interestingly, although it shared 64% sequence identity with human ArhGAP9, it lacked the WW domain (GeneBank accession no. XXX, availability pending submission of sequence to NCBI). This is shown in the amino acid alignment in Fig. Ib. However, we could not rule out the possibility that SMART did not identify a WW domain in mouse ArhGAP9 due to low sequence homology to other WW domains. The ArhGAP9 ortholog in the C. elegans and C. Briggsae lacked an SH3 domain, which could be due to the SH3 domain being a result of recent evolutionary accretion.

[00205] The radial tree depicted in Fig. Ic showed the evolutionary relationship of ArhGAP9 and ArhGAP12 for different species. The phylogenetic analysis was carried out using ClustalW and visualized with Tree View. The mammalian ArhGAP9 and ArhGAP12 are distinct genes but showed high structural and sequence homology. There is a strong probability that the worm ArhGAP12 gene may be the evolutionary ancestor of the mammalian ArhGAP9 and ArhGAP12.

[00206] Identification of the Erk and p38 MAP kinases as novel binding proteins to the WW domain of human ArhGAP9. To identify interacting proteins for the WW domain of human ArhGAP9, the GST fusion protein of the WW domain of human ArhGAP9 was purified and immobilized on glutathione Sepharose beads and used as a bait to isolate interacting proteins from rat brain lysate. The protein complex isolated was resolved by 10% SDS-PAGE, followed by detection with Colloidal Coomassie Blue (Fig. 2a). Several specific binding proteins were identified, including the MAP kinase Erk2 from bands f and g that generated 15 unique peptides covering 40% of the protein sequence of Mitogen-activated protein kinase 1 (MAPKl), also known as extracellular signal-regulated kinase 2, Erk2.

[00207] (i) Binding of Erk2 and p38α to human ArhGAP9 in vitro and in vivo To confirm that Erk2 bound to the WW domain of human ArhGAP9, in vitro pulldown assays were performed. Flag-tagged Erk2 was expressed by transfection of the cDNA into 293T cells and the lysates were incubated with GST fusion protein of the WW domain of ArhGAP9 immobilized on glutathione Sepharose beads. The protein complex was washed and eluted, followed by SDS-PAGE and western blotting with anti-Flag (Fig. 2b). The binding of two closely related MAP kinases, p38α and Jnkl, were also tested. Consistent with the proteomics data, Erk2 bound to the WW domain of ArhGAP9 with high affinity [Fig. 2b(i)].

[00208] While p38α also showed binding albeit to a lesser extent than Erk2, the binding of Jnkl was not detectable in the pulldown assay. The control of GST alone immobilized on glutathione Sepharose beads did not show any binding to the MAP kinases, indicating that the binding of Erk2 and p38α to ArhGAP9 WW domain was specific [Fig. 2b(ii)]. The expression levels of Erk2, p38α and Jnkl were comparable as shown by western blotting with anti-Flag of the whole cell lysates [Fig. 2b(iii)]. We also demonstrated that full-length ArhGAP9 from lysates of 293T cells transfected with ArhGAP9 cDNA could be precipitated by full-length Erk2 or p38α expressed as a GST fusion protein immobilized on glutathione Sepharose beads (Fig. 6A).

[00209] To demonstrate the binding of Erk2 to ArhGAP9 in vivo, 293T cells were transfected with ArhGAP9, Erk2-Flag or p38α-Flag alone, ArhGAP9 with Erk2- Flag or ArhGAP9 with p38α- Flag. The lysates were subjected to immunoprecipitation with anti-Flag and the protein complexes washed and eluted for SDS-PAGE. Western blotting with anti-ArhGAP9 showed that ArhGAP9 associated with both Erk2 and p38α, as shown in Fig. 6B(i). Western blotting of the immunoprecipitates with anti- Flag showed that equivalent amounts of Erk2 and p38α were immunoprecipitated [Fig. 6B(H)]. The expression of ArhGAP9 was shown to be comparable by western blotting of the whole cell lysates with anti-ArhGAP9 [Fig. 6B(Ui)], and similarly the expression levels of Erk2 and p38α were shown to be comparable by western blotting with anti- Flag [Fig. 6B(iv)].

[00210] From the pulldown and coimmunoprecipitation results, we concluded that the MAP kinases Erk2 and p38α were able to bind to the WW domain of ArhGAP9. Our results show that the binding affinity in vitro is higher for Erk2 than p38α and there was no detectable binding for Jnkl. The binding of p38α to full-length ArhGAP9 in vivo may be more readily detectable than its binding to the WW domain alone in the in vitro assay, providing a possible explanation that the amount of ArhGAP9 that coimmunoprecipitated with both Erk2 and p38α appeared equal.

[00211] To further determine the specificity of Erk2 and p38α binding to the

WW domain of ArhGAP9, we tested the binding of Erk2, p38α and Jnkl to the two WW domains of ArhGAP12 (ArhGAP12-WWl and ArhGAP12-WW2) individually and the first WW domain of Nedd4 (Nedd4-WWl). The lysates of 293T cells transfected with Flag-tagged Erk2, p38α and Jnkl were incubated with GST fusion proteins of ArhGAP12-WWl, ArhGAP12-WW2 and Nedd4- WWl immobilized on glutathione Sepharose beads. The protein complexes were washed and eluted, followed by SDS-PAGE and western blotting with anti-Flag. Erk2, p38α and Jnkl showed no detectable binding to ArhGAP12-WWl, ArhGAP12-WW2 and Nedd4-WWl [Fig. 6C(i)]. The expression levels of Erk2, p38α and Jnkl were comparable as shown by western blotting with anti-Flag of the whole cell lysates [Fig. 6C(U)] .

[00212] (ii) Mouse ArhGAP9 lacks the WW domain and is unable to bind to

MAP kinases We had cloned the mouse homolog of ArhGAP9 from the cDNA libraries prepared from mouse thymus, bone marrow and spleen, we noted that the open reading frame did not contain a WW domain according to the SMART protein domain prediction tool. We therefore tested whether the mouse protein would be able to interact with the MAP kinases Erk2 and p38α like the human polypeptide. For this purpose, we constructed a GST fusion of an Nterminal fragment of the mouse ArhGAP9 protein comprising amino acids (mArhGAP9-N, 1- XXX) which included the SH3 and PH domain and the intervening sequence between these two domains that may have low homology or any functional conservation to the human WW domain not determined by SMART. Flag-tagged Erk2, Jnkl or p38α was expressed by transfection of the cDNA into 293T cells and the lysates were incubated with GST fusion protein of the WW domain of ArhGAP9 or mArhGAP9-N immobilized on glutathione Sepharose beads. The protein complexes were washed and eluted, followed by SDS-PAGE and western blotting with anti-Flag (Fig. 6D). It could be seen that whereas the binding of Erk2 and p38α to human ArhGAP9 WW domain was significant [Fig. 6D(i)], the binding of all the MAP kinase proteins Erk2, Jnkl and p38α to mArhGAP9-N was not detectable [Fig. 6D(ii)], as in the GST control [Fig. 6D(Ui)]. We also confirmed that full-length mouse ArhGAP9 could not interact with Erk2 in vivo. 293T cells were transfected with Flag-tagged full-length human or mouse ArhGAP9, individually or together with HA- tagged Erk2. The lysates were subjected to immunoprecipitation with anti-Flag and the protein complexes washed and eluted for SDSPAGE.

[00213] Western blotting with anti-HA showed that while human ArhGAP9 associated with Erk2, mouse ArhGAP9 did not [Fig. 6E(i)]. Western blotting of the immunoprecipitates with anti-Flag showed that equivalent amounts of both human and mouse ArhGAP9 were immunoprecipitated [Fig. 6E(U)] . The expression of human and mouse ArhGAP9 was shown to be comparable by western blotting of the whole cell lysates with anti-Flag [Fig. 6E(Ui)], and similarly the expression levels of Erk2 in each transfection was shown to be comparable by western blotting with anti-HA [Fig. 6E(iv)]. Taken together, we concluded that human ArhGAP9 interacted with the MAP kinases Erk2 and p38α specifically with it WW domain. The mouse homologue which lacked the WW domain was unable to bind to MAP kinases.

[00214] Binding of Erk and p38α to ArhGAP9 diminished with activation of the MAP kinases The MAP kinases have been known to be activated downstream of growth factor receptor activation, amongst many other stimuli. To assess the importance of upstream activating signals to the binding of the MAP kinases to ArhGAP9, the binding of the inactive and EGFRactivated MAP kinases to ArhGAP9 WW-GST was compared. 293T cells were transfected with Flag-tagged Erk2, p38α or Jnkl alone or in combination with EGFR. When overexpressed by transient transfection in 293T cells, EGFR was active due to autophosphorylation on tyrosine residues required for kinase activation. The activation of the EGFR was confirmed by western blotting of total cell lysates with anti-phosphotyrosine [Fig. 2E(Ui)] . The auto- activation of the Erk2, p38α and Jnkl by co-expression with EGFR was confirmed by western blotting with phospho-specific antibodies (data not shown). The lysates prepared were incubated with ArhGAP9-WW-GST immobilized on glutathione Sepharose beads. The protein complexes were washed and eluted, followed by SDS- PAGE and western blotting with anti-Flag. Interestingly, we observed that EGFR activation caused the binding of Erk2 and p38α to ArhGAP9-WW to be significantly reduced [Fig. 2E(i)]. GST alone did not show detectable binding [Fig. 2E(U)] . The expression levels of Erk2, p38α and Jnkl were confirmed to be comparable by western blotting of whole cell lysates with anti-Flag [Fig 2E(iv)]. One possibility that EGFR activation might result in the reduction of Erk2 and p38α binding to the WW domain of ArhGAP9 could be that the binding motif for ArhGAP9-WW domain resided around the activation loop of the inactive MAP kinases, such that when these sites became phosphorylated upon activation as a consequence of EGFR activation, the structural changes in the activation loop became unfavourable for the binding of ArhGAP9-WW domain.

[00215] To test this hypothesis, the two residues in Erk2 and p38α whose phosphorylation resulted in structural changes required for phosphoryl transfer onto substrate proteins, namely Thrl83 and Tyrl85 in Erk2 and Thrl80 and Tyrl82 in p38α were mutated to Ala. The binding of wild type Erk2 and Erk2(T183A,Y185A), as well as wild type p38α and p38α(T180A,Y182A) binding to ArhGAP9-WW domain was compared. Flag-tagged Erk2 or p38α (wild type and the TY mutants) were expressed in 293T cells by transient transfection. The lysates were incubated with ArhGAP9-WW- GST immobilized on glutathione Sepharose beads. The protein complexes were washed and eluted, followed by SDS-PAGE and western blotting with anti- Flag to detect the bound MAP kinases. As shown in Fig. 7 A, for both Erk2 and p38α, the binding of the wild type and mutant proteins to ArhGAP9 WW domain was comparable, indicating that the binding motif resides outside the activation loop. The expression levels of the Erk2 and p38α (wildtype and mutants) were shown to be comparable by western blotting of whole cell lysates with anti-Flag, providing a valid comparison of

ArhGAP9-WW domain binding [Fig. 7A(Ui)] . [00216] The above results indicated that the binding of the MAP kinases Erk2 and p38α to the WW domain of ArhGAP9 was negatively regulated by upstream activating signals of these MAP kinases and that the binding sites most likely resided outside the activation loops of the latter. The structural changes that resulted upon activation of the MAP kinases by phosphorylation at the activation loop most likely also would not alter the binding of Erk2 and p38α to the WW domain of ArhGAP9.

[00217] ArhGAP9 and MAP kinase binding was mediated by complementarity charged residues in WW and CD domains, respectively. From the sequence alignment of human ArhGAP9-WW domain with all the known WW domains, we observed that the C-terminal end of human ArhGAP9-WW domain contained a unique basic di-Arginine motif (R246 and R247) that were not present in all other WW domains compared (data not shown). Fig. 2G showed the sequence alignment of the WW domains of human ArhGAP12 and ArhGAP9. Notably in the first and second WW domains of ArhGAP12 which did not bind Erk2 and p38α, the residues in alignment with R247 are W and Y, respectively. Such short basic motifs had been shown to be present in a large number of MAP kinase docking proteins that bind to the conserved docking (CD) domains of MAP kinases. The CD domains of Erk2, p38 and Jnk consisted of conserved acidic residues which form electrostatic interactions with the basic residues of the target docking proteins29. We postulated that complementarily charged residues on the CD domains of Erk2 and p38α and WW domain of ArhGAP9 could be the mechanism which enhanced or mediated the binding of these MAP kinases and ArhGAP9.

[00218] We tested the hypothesis that the basic residues in the WW domain of human ArhGAP9 mediated the binding to Erk2 and p38α through interaction with the acidic residues in the CD domain, as in the examples of other MAP kinase docking proteins (Ref). We substituted R246 and R247 in the WW domain of ArhGAP9 with alanine (WW-ArhGAP9-RR) and generated the GST fusion protein of the WW domain bearing these mutations and tested its binding to the MAP kinases. Lysates from 293T cells expressing Flag-tagged Erk2, p38α or Jnkl were incubated with GST fusion protein of wild type ArhGAP9 WW domain or the R246,247A (RR) mutant. Bound MAP kinase was detected by SDS-PAGE and western blotting with anti-Flag. As shown in Fig. 7B(i), for both Erk2 and p38α, the binding to the WW domain with R246,247A mutation was significantly reduced compared to binding to wild type ArhGAP9 WW domain, confirming that these basic residues played a role in MAP kinase interaction. Single mutationof R246A orR247A were also defective in binding to Erk2 (data not shown). To compare the binding of full-length wild type and R246,247A mutant of ArhGAP9 in vivo, wild type ArhGAP9 or the R246,247A mutant was cotransfected with Erk2- Flag or p38α-Flag in 293T cells. Lysates were prepared and subjected to immunoprecipitation with anti-Flag. The protein complexes were washed and eluted for SDS-PAGE, followed by western blotting with anti-ArhGAP9 or anti- Flag. As shown in Fig. 7C(i), the amount of ArhGAP9 R246,247A mutant protein that coimmunoprecipitated with Erk2 or p38α was almost non-detectable. In the immunoprecipitation with anti-ArhGAP9 followed by detection of Erk2 or p38α by western blotting with anti-Flag [Fig. 7C(Ui)], it was observed that the amounts of Erk2 or p38α that associated with the R246,247A mutant of ArhGAP9 was barely detectable compared to wild type. The amounts of Erk2 or p38α or ArhGAP9 that were immunoprecipitated were shown to be equivalent to allow for valid comparison of the binding [Fig. 7C(ii, iv)]. From these data, we concluded that R246 and R247 in the WW domain of ArhGAP9 were important in mediating the binding to Erk and p38 MAP kinases.

[00219] We proceeded to test whether the CD domain in the MAP kinases Erk2 and p38α were the regions that the WW domain of ArhGAP9 interacted with. For this purpose, a GST fusion of a C-terminal fragment of Erk2 containing the CD domain (residues XX-XX, Erk2-CD-GST) and a deleted version of Erk2 lacking the CD domain (residues XX-XX, Erk2-ΔCD-GST) were constructed. Lysates from 293T cells transfected with Flag-tagged ArhGAP9 (wild type or R246,247A mutant) were incubated with immobilized Erk2-CD-GST, Erk2-ΔCD-GST or GST alone. The protein complexes were washed and eluted, followed by SDS-PAGE and western blotting with anti-Flag. As shown in Fig. 7D(i), wild type ArhGAP9 but not the R246,247A mutant, specifically precipitated with Erk2-CD-GST, indicating that the CD domain of Erk2 was most likely sufficient to mediate the binding to ArhGAP9. Consistent with this notion, we observed that Erk2-ΔCD-GST did not bind wild type ArhGAP9 [Fig.

7D(U)] . To determine whether the conserved acidic residues [Fig. 7E(i)] in the CD domains of the MAP kinases were involved in binding to the WW domain of ArhGAP9, point mutants of Erk2 and p38α were generated and tested for their binding to ArhGAP9. As shown in Fig. 3B(ii), Erk2-Asp316Ala interacted with ArhGAP9-WW domain with reduced affinity compared to wild type Erk2. The mutations Asp312Ala, Asp315Ala, and Glu316Ala of p38α all caused a significant reduction in interaction with ArhGAP9-WW domain. The expression of wild type or mutant Erk2 and p38α were equivalent and allowed for valid comparison of their binding to ArhGAP9 WW domain [Fig. 3B(Ui)]. These results clearly indicated that the acidic residues in the CD domains of Erk2 and p38α were important for their interactions with ArhGAP9. To provide biophysical evidence that the basic residues in the WW domain of human ArhGAP9 interacted with the acidic residues in the CD domain of MAP kinases, we conducted Far-UV CD spectroscopy. The WW domain is a small three-stranded β-sheet stabilized by the stacking of several conserved aromatic and proline residues (Ref).

[00220] Crystal and solution structures of WW domains had shown that WW domain undergoes conformational changes upon binding to its corresponding peptides of its binding partners (Ref). Far-UV CD spectroscopy is one of the commonly used methods to monitor changes in secondary structure of proteins, and we applied this technique to investigate if binding of Erk2, p38α and Jnkl peptides to WW domain was accompanied by any structural changes in the WWdomain. The peptides containing 14 amino acids in CD domains of Erk2, p38α and Jnkl were synthesized [sequences shown in Materials and Methods and in Fig. 7E(i)]. To analyze the changes in the structure of WW domain upon binding of corresponding peptides, the differences in the Far-UV CD spectrum of ArhGAP9 WW domain solutions in the presence and absence of peptides were monitored. The changes in secondary structure contents were analyzed using CDNN program, version 2.1. The analysis showed that the recombinant ArhGAP9 WW domain folded properly and possessed mostly beta sheets and random coils which are similar to the known WW domains [Fig. 7F(i)]. In the presence of the Erk2 peptide at equimolar concentration, ArhGAP9 WW domain showed significant difference in the CD spectra particularly from 205-190 nm [Fig. 7F(iv)]. Also in the presence of p38α peptide, ArhGAP9 WW domain showed significant difference in the CD spectra in the same region [Fig. 7F(Ui)]. In contrast, the addition of Jnkl peptide did not alter the CD spectra significantly although there were notable changes are present at 220-235 nm [Fig. 7F(U)] . These conformational changes monitored by the Far-UV CD were in agreement with the other known crystal and solution structures of WW proteins with their peptide targets. This result further supported the conclusion from both in vitro and in vivo experiments that both Erk2 and p38α interacted with ArhGAP9-WW domain but Jnkl did not. However exact conformational changes and the mechanism of binding can be studied only by solving the crystal or solution structures of arhGAP9-WW domain with its binding partners.

[00221] To obtain insights into the structural basis of specificity of the WW domain of ArhGAP9 binding to Erk2, p38α and Jnkl, we compared the three- dimensional structures of CD domains of the three proteins. It could be seen from the comparison that although the overall structures of Erk2 (PDB id: IERK), p38α (PDB id: 1P38) and Jnkl (PDB id: IJNK) are very similar, the CD domain of p38α shared greater homology than that of Jnkl to Erk2. Furthermore while Erk2 and p38α are significantly super- imposable with RMSD of 0.68 A, Erk2 and Jnkl CD domains are relatively distinct with RMSD of 2.18 A. The high sequence and structural similarity between Erk2 and p38α provided some insights into the structural basis for the specificity on the WW domain of ArhGAP9 for Erk2 and p38α but not Jnkl, as observed in the binding studies in vitro and in vivo. Taken together, these results strongly supported the hypothesis that the binding between the WW domain of ArhGAP9 and Erk2 or p38α was mediated by complementarily charged residues in WW and CD domains, respectively. The point mutation of these charged residues in the context of the full-length proteins was sufficient to result in significant reduction of binding. We therefore proposed that human ArhGAP9 is a novel MAP kinase docking protein.

[00222] Coexpression of ME K 2 and MKK6 reduced binding of Erk2 and p38α to WW domain of ArhGAP9, repectively. The CD domains of MAP kinases had been established as the binding sites for their upstream activating kinases, namely MEKl and 2 for Erkl and 2, and MKK3, 4 and 6 for p38α29. These upstream kinases all contained positively charged residues that formed the D domain for binding with the CD domain of MAP kinases. From our data which showed that upon EGFR stimulation the binding of ArhGAP9 to Erk2 and p38α was reduced, we postulated that the binding of MEK to Erk and MKK to p38α may compete with the binding o ArhGAP9 to these MAP kinases. To investigate if this was indeed the case, the binding of Erk2 or p38α to ArhGAP9 in the absence or presence of overexpressed MEK2 or MKK, respectively was compared. Lysates from 293T cells were transfected with p38α-Flag alone or p38α-Flag with MKK6-HA were incubated with immobilized ArhGAP9- WW-GST. The protein complexes washed and eluted for SDS-PAGE, followed by western blotting with anti-Flag. As shown in Fig. 8A(i), the amount of p38α that precipitated with the WW domain ArhGAP9 was significantly reduced in the presence of MKK6, suggesting that the binding of MKK6 to p38α could inhibit the binding of the WW domain ArhGAP9. The coexpression of MKK6 strongly induced phosphorylation and activation of p38α [Fig. 8A(Hi)], suggesting that MKK6 did associate with p38α. The control of GST alone showed no binding [Fig. 8A(ii)] and the expression levels of p38α and MKK6 in whole cell lysates were shown by western blotting with anti-Flag and anti-HA, respectively [Fig. 8A(iv,v)]. We had observed that a mutant ofp38α with the basic residues in the docking domain responsible for binding to the CD domain of MKK6 deleted failed to inhibit p38α binding to the WW domain ArhGAP9 (data not shown).

[00223] Consistent with the above in vitro data, we observed that the binding of full-length ArhGAP9 to p38α, in vivo was reduced in the presence of MKK6. 293T cells were transfected with p38α-Flag alone, p38α-Flag with ArhGAP9, p38α-Flag with MKK6-HA or all the three plasmids together. p38α-Flag was immunoprecipitated from the lysates with anti-Flag and the binding of ArhGAP9 was detected by western blotting with anti-ArhGAP9. It was observed that the amount of ArhGAP9 that coimmunoprecipitated with p38α from lysates of transfected 293T cells was reduced when MKK6 was cotransfected to cause the phosphorylation and activation of p38α [Fig. 8B(i)]. The amount of p38α that were immunoprecipitated was shown to be equivalent by western blotting with anti-Flag [Fig. 8B(Ui)] . The activation of p38α by coexpression of MKK6 was confirmed by western blotting with phospho-specific p38α antibodies [Fig. 8C(Ui)]. The expression levels of ArhGAP9, p38α and MKK6 in whole cell lysates were shown by western blotting with anti-ArhGAP9, Flag and HA, respectively [Fig. 8C(Hi, iv, v)]. Similarly, with the cotransfection of MEK2, the amount of Erk2 that precipitated with the ArhGAP9 both in vitro and in vivo was significantly reduced (data not shown).

[00224] Taken together, these results indicated that upstream activating kinases of Erk2 and p38α, namely MEK2 and MKK6, respectively were able to abrogate the binding of the MAP kinases to the WW domain of ArhGAP9, most likely due to their binding to the CD domains and thereby preventing the binding of ArhGAP9.

[00225] Binding of ArhGAP9 inhibited Erk2 and p38α activation by upstream signals. Since both ArhGAP9 and the MAP kinases have catalytic activity, we proceeded to investigate whether their interaction could mutually influence their activities. We and Furukawa et al (8,24) had shown that ArhGAP9 functioned as an active GTPase activating protein towards cdc42 and Racl but not RhoA (data not shown). To determine whether MAP kinase binding had any effect on the RhoGAP activity of ArhGAP9, the activation status of cdc42 in the lysates of cells transfected with ArhGAP9 in the presence or absence of Erk2 was compared. Recombinant PAK- binding domain (PBD) GST fusion protein which would bind preferentially to active cdc42 over the inactive form immobilized on glutathione Sepharose beads was used as a probe. Lysates from 293T cells transfected with myc-tagged cdc42, with and without ArhGAP9 and Flag-tagged p38α in different combinations shown in Fig. 8D were prepared and incubated with the immobilized PBD-GST. The GAP-inactive mutant of ArhGAP9 (R578K) was used as a negative control for the assay. The protein complexes were washed and eluted for SDS-PAGE, followed by western blotting with antimyc to ascertain the relative amount of active cdc42 in the lysate. As shown in Fig. 8D(i), there was no significant effect in the activity with or without the presence of p38α. The expression levels of cdc42, ArhGAP9 or p38α were shown by western blotting with anti-myc, ArhGAP9 or Flag, respectively [Fig. 8D(ii, iii and iv)]. Similar results were obtained which indicated that Erk2 binding to ArhGAP9 did not influence the RhoGAP activity of the latter (data not shown).

[00226] We next investigated whether the binding of ArhGAP9 to the Erk2 and p38α had an effect on the MAP kinase activities. 293T cells were transfected with p38α alone, or p38α with EGFR to induce the activation of p38α, or p38α with EGFR as well as ArhGAP9 to anlayse whether the activation of p38α was affected by the presence of ArhGAP9 or various ArhGAP9 mutants. The ArhGAP9 mutants tested included the R578K GAP-inactive mutant, the W219K and W242K mutants which caused the structure of the WW domain to be disrupted and unable to bind to its targets, the R339A,K340A and R342A,K343A mutants which disrupted the ability of the PH domain to bind to its phospholipid targets and the W181,182K mutant which would render the SH3 domain defective in targeting its proline-containing targets. The mutants transfected in each sample were as indicated in the figure legend of Fig. 4b.

[00227] Equal amounts of the whole cell lysates prepared from these transfected cells were resolved by SDS-PAGE followed by western blotting with anti- phosphotyrosine which predominantly detected the activated EGFR that was the major tyrosine phosphorylated protein in these lysates (Fig. 4b(i)). It could be concluded that the expression and activation of EGFR in each sample were equivalent. The expression levels of wildtype and the different ArhGAP mutants were shown to be comparable by anti-ArhGAP9 western blotting (Fig. 4b(ii)). The expression Flag-tagged p38α was confirmed to be comparable by anti-Flag western blotting (Fig. 4b(iii)). The activation status of p38α in each of the samples was analysed by anti-phospho-p38 western blotting (Fig. 4b(iv)). From Fig. 4b(iv), it could be seen that p38α that was transfected alone showed a slight detectable level of activation (lane 2) which increased with the coexpression of the EGFR (lane 3). With the expression of wildtype ArhGAP9 (lane 4), we observed a reduction of p38α activation by EGFR, indicating that ArhGAP9 could suppress the activation of p38α downstream of EGFR. With the coexpression of all the ArhGAP9 mutants (lanes 5-10), there was also reduction of p38α activation by EGFR albeit to varying extents. Most interestingly, we observed that the W219K and W242K mutants (lanes 6 and 7) which would cause the structure of the WW domain to be disrupted and therefore bound MAP kinases poorly, were able to allow for activation of p38α to a level higher than wildtype ArhGAP9 (lane 3). The ability of the WW domain mutants to allow partial restoration of p38α activation by EGFR compared to wildtype ArhGAP9 strongly indicated that the binding of ArhGAP9 to MAP kinases would prevent the activation of the latter by their upstream kinases. It was observed that the R342A,K343A (lane 9) and W181,182K (lane 10) mutations in the PH and SH3 domains, respectively might also have a slight ability to restore EGFR-induced p38α activation. The contribution of the PH and SH3 domains of ArhGAP9 in modulating MAP kinase activation will be investigated outside the context of this report.

[00228] Taken together, the above data suggests that the binding of ArhGAP9 through its WW domain to the MAP kinase p38α, did not have a significant effect on the Rho GAP activity of ArhGAP9, but caused the inhibition of p38α activation by upstream signals.

[00229] ArhGAP9 binding prevented MAP kinase-induced loss of actin stress fibres. Rho GTPase and MAP kinase signaling have both been implicated in the control of the actin cytoskeleton. Specifically it was reported that activation of the MAP kinases Erkl, 2 and 5 pathways caused disruption of the actin cytoskeleton28. To investigate if the interaction of ArhGAP9 and MAP kinases had an effect on the actin cytoskeleton, wild type or the R246,247A mutant of ArhGAP9 which was defective in binding to Erk2 and p38α were expressed in Swiss 3T3 cells by microinjection of the respective cDNAs together with GFPactin, followed by imaging of the actin structure. As shown in Fig. 9(i) in the presence of wild type ArhGAP9, the cells maintained distinct bundles of actin stress fibres as in the control cells where GFP-actin alone was expressed. However, in the presence of the R246,247A ArhGAP9 mutant, the stress fibres dissolved and showed a diffuse pattern of actin. The average number of stress fibres in cells expressing wild type ArhGAP9 was comparable to the control cells whereas the average number of stress fibres in cells expressing mutant ArhGAP9 was reduced significantly [Fig. 9(U)] . These results supported the hypothesis that binding of wild type ArhGAP9 to MAP kinases sequestered them in the inactive forms, preventing the activation of the MAP kinases and therefore allowing the formation and maintenance of actin stress fibres in fibroblasts. Conversely, the R246,247A mutant being unable to bind the MAP kinases, would potentially allow activation of the MAP kinases to result in the disassembly of the actin stress fibres.

[00230] Figure 10 depicts a proposed mechanism of negative regulation of MAP kinase by ArhGAP9. a. ArhGAP9 contains a WW domain which possesses a basic di- Arginine motif while MAP kinase (MAPK) contains a Common Docking (CD) domain that contains conserved acidic residues, b. hi quiescent state, ArhGAP9 interacts with MAPK through electrostatic interaction between the complementary basic and acidic residues in the WW and CD domains, blocking the access of MAPK by other docking proteins therefore negatively regulating MAPK activation, c, d. In the induced state, the presumable increase in local concentration of active upstream MAPK kinase (MAPKK) displaces ArhGAP9 by docking onto CD domain of MAPK, causing the diminished binding of ArhGAP9 to MAPK. The interaction between MAPK and MAPKK results in the phosphorylation of MAPK in the kinase activation loop to activate the latter.

[00231] As can be understood by one skilled in the art, many modifications to the exemplary embodiments described herein are possible. The invention, rather, is intended to encompass all such modification within its scope, as defined by the claims.

[00232] All documents referred to herein are fully incorporated by reference.

[00233] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of this invention, unless defined otherwise.

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Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting activation of a MAP kinase comprising contacting the MAP kinase with a WW domain.

2. The method of claim 1 wherein the WW domain has two basic amino acids at the C-terminus.

3. The method of claim 1 wherein the WW domain is the WW domain of human ArhGAP9.

4. The method of claim 1 wherein the WW domain comprises the sequence as set forth in SEQ ED NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10.

5. The method of claim 2 wherein each of the two basic amino acids are lysine, arginine or histidine.

6. The method of claim 5 wherein both of the two basic amino acids are arginine.

7. A method of inhibiting a cell having increased activation of a MAP kinase comprising administering to the cell an effective amount of a molecule that increases cellular levels of a polypeptide comprising at least a WW domain.

8. The method of claim 7, wherein the WW domain has two basic amino acids at the C-terminus.

9. The method of claim 7 wherein the molecule is the polypeptide comprising at least a WW domain.

10. The method of claim 7 wherein the molecule is a peptidomimetic or a small molecule.

11. The method of claim 9 wherein the molecule is a nucleic acid molecule encoding the polypeptide for expression in the cell.

12. The method of claim 7 wherein the polypeptide is human ArhGAP9.

13. The method of claim 12 wherein the polypeptide comprises the sequence as set forth in SEQ ID NO: 11.

14. The method of claim 7 wherein the WW domain is the WW domain of human ArhGAP9.

15. The method of claim 7 wherein the WW domain comprises the sequence as set forth in SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10.

16. The method of claim 8 wherein each of the two basic amino acids are lysine, arginine or histidine.

17. The method of claim 16 wherein both of the two basic amino acids are arginine.

18. A method of treating a disease state characterized by the presence of cells that have increased activation of a MAP kinase, in a patient in need of such treatment, comprising administering to the patient an effective amount of a polypeptide comprising at least a WW domain.

19. The method of claim 18 wherein the WW domain has two basic amino acids at the C-terminus.

20. The method of claim 18 wherein the disease state is cancer or rheumatoid arthritis.

21. The method of claim 18 wherein the administering comprises administering a nucleic acid molecule encoding the polypeptide for expression in the cell.

22. The method of claim 18 wherein the polypeptide is human ArhGAP9.

23. The method of claim 21 wherein the polypeptide comprises the sequence as set forth in SEQ ID NO: 11.

24. The method of claim 18 wherein the WW domain is the WW domain of human ArhGAP9.

25. The method of claim 18 wherein the WW domain comprises the sequence as set forth in SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10.

26. The method of claim 19 wherein each of the two basic amino acids are lysine, arginine or histidine.

27. The method of claim 26 wherein both of the two basic amino acids are arginine.

28. A composition comprising a polypeptide comprising at least a WW domain or comprising a nucleic acid encoding the polypeptide.

29. The composition of claim 28 wherein the WW domain has two basic amino acids at the C terminus.

30. The composition of claim 28 wherein the polypeptide is human ArhGAP9.

31. The composition of claim 30 wherein the polypeptide comprises the sequence as set forth in SEQ ID NO: 11.

32. The composition of claim 31 wherein the polypeptide is the WW domain of human ArhGAP9.

33. The composition of claim 28 wherein the WW domain comprises the sequence as set forth in SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10.

34. The composition of claim 29 wherein each of the two basic amino acids are lysine, arginine or histidine.

35. The composition of claim 34 wherein both of the two basic amino acids are arginine.

36. A kit comprising a polypeptide comprising at least a WW domain, the WW domain having two basic amino acids at the C-terminus, or a nucleic acid molecule encoding the polypeptide, and instructions for inhibiting a cell having increased activation of a MAP kinase or for treating a disease state characterized by the presence of cells that have increased activation of a MAP kinase.