WO2004072110A1 - Methods for inhibiting signal transduction - Google Patents

Abstract

The present invention provides polynucleotides encoding a SPRED polypeptide or an EVE polypeptide. Also provided are polypeptides encoded by the polynucleotides as well as antibodies to the polypeptides and antagonists and agonists of polypeptide function.

Description

METHODS FOR INHIBITING SIGNAL TRANSDUCTION

FIELD OF THE INVENTION.

The present invention relates generally to methods for inhibiting signal transduction. The invention also relates to a novel gene referred to herein as EVE3, polypeptides encoded by the gene, the production of such polypeptides, to their use in diagnosis and therapy, and to their use in identifying agonists and antagonists that are potentially useful in therapy for conditions such as hepatocellular carcinoma and hepatitis.

BACKGROUND TO THE INVENTION.

The process of normal development in an organism requires a highly coordinated and regulated control of cellular growth and differentiation. Positive signals for growth and differentiation are relayed by growth factors and their cognate receptors, most commonly receptor tyrosine kinases (RTKs), on cell membranes and amplified intracellularly by protein signal transduction networks. Negative control of signaling can be achieved by withdrawal of the initiator growth factor itself and/or by signal inhibitory proteins that serve to antagonize intracellular propagation of the initial stimulus. A paradigm of developmental control is that positive signals induce negative regulatory proteins that eventually inhibit these same signals, in a classical autoregulatory feedback loop.

One class of negative regulatory proteins that inhibit mitogenic signaling downstream of some RTKs is the Sprouty family. Sprouty (spry) was initially identified by genetic screens in Drosophila as an antagonist of Breathless fibroblast growth factor (FGF) receptor signaling during tracheal branching. Spry mutations induced excessive branching of the tracheal network whilst forced expression of spry blocked tracheal branching. Subsequently, it was shown in the Drosophila system that spry action was not limited to FGF activity but acted downstream of a range of RTKs including Torso and Sevenless. Unlike Drosophila where there is a single spry gene, four homologous mammalian spry genes have been identified. Mammalian spry genes exhibit a dynamic expression pattern throughout embryonic development and their expression can be rapidly upregulated by certain growth factors. Like Drosophila Sprouty, vertebrate Sprouty proteins appear to act as key regulators of developmental processes such as limb formation, lung branching morphogenesis and angiogenesis. A distinctive feature of vertebrate Sprouty proteins is their selective antagonism of only a subset of growth factors, with Sproutyl and Sprouty2 inhibiting FGF and vascular endothelial growth factor (VEGF) induced signaling but not epidermal growth factor (EGF) nor chemical (phorbol 12- myristate 13-acetate) activation of signaling.

The evidence is accumulating that unlike Drosophila Sprouty, vertebrate Sprouty proteins do not have pleiotropic inhibitory effects downstream of RTKs and hence much interest has focused on the molecular mechanisms by which they selectively block a ubiquitous intracellular growth signaling pathway, the

Ras/Raf/MAP kinase pathway. The initial genetic studies in Drosophila placed the inhibition of mitogen-activated protein kinase (MAPK) upstream of Ras whilst in the context of wing development, inhibition of MAPK occurred downstream of

Ras. Vertebrate Sprouty proteins have also been reported to act both upstream and downstream of Ras signaling, prompting the view that Sprouty proteins might act at multiple sites in the Ras/Raf/MAPK pathway by interacting with various signaling molecules. Thus, the present state of knowledge about regulation within signal transduction pathways is incomplete. Where and how the Sprouty proteins function remains unknown.

Recently, a family of two molecules, which are related to the Sprouty proteins was discovered in mouse, the Spredl and Spred2 proteins. These proteins have a C-terminal Sprouty domain, an N-terminal EVH1 (Ena Vasp homology 1 ) domain and a central c-kit binding domain (KBD). The Spreds were determined to be suppressors of Ras/MAPK signaling in response to a variety of extracellular stimuli. Deletion mutants indicated that the suppression of signaling required both the EVH1 and Sprouty domains.

Since control of cell proliferation and differentiation is coordinated by proteins of various signal transduction pathways and regulation of such signal transduction may provide a means of regulating cell growth and differentiation, there exists a need to provide molecules and methods which can interact with and regulate the activities of molecules within signal transduction pathways.

Regulation of signal transduction pathways may then permit regulation of cell, tissue or organ growth.

A particular problem relating to growth regulation in mammals is that of the liver. This organ has the unique capacity to control its growth and mass following injury. However, it is not known how the regeneration process is regulated. Therefore the provision of new molecules that can regulate molecules within signal transduction pathways raises the intriguing possibility that the regenerative capacity of this organ can be modulated. Of particular interest may be the ability to control and/or stimulate liver regeneration following treatment and resection of liver, especially following diseases such as hepatocellular carcinoma, hepatitis or cirrhosis; damage induced by drugs, infections, alcohol or toxins; congenital disorders; or surgery.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

SUMMARY OF THE INVENTION.

In one aspect the present invention provides an isolated polynucleotide encoding a SPRED3 polypeptide or an EVE3 polypeptide.

In another aspect the present invention provides an isolated polynucleotide selected from the group including a SPRED3 cDNA and an EVE3 cDNA.

In a further aspect the present invention provides an isolated polynucleotide encoding a SPRED1 polypeptide or a SPRED2 polypeptide. Preferably the polynucleotide encodes a mammalian SPRED1 polypeptide or a mammalian SPRED2 polypeptide. Most preferably the polynucleotide encodes a human SPRED1 polypeptide or a human SPRED2 polypeptide.

In yet a further aspect the present invention further provides an isolated polynucleotide selected from the group including a SPRED1 cDNA and an SPRED2 cDNA. Preferably the SPRED1 cDNA and SPRED2 cDNA are mammalian. More preferably the SPRED1 cDNA and SPRED2 cDNA are human.

In another aspect the present invention provides an isolated polypeptide which is a SPRED3 polypeptide, an EVE3 polypeptide, or a fragment or variant thereof.

In yet another aspect the present invention provides an isolated polypeptide which is a SPRED1 polypeptide, a SPRED2 polypeptide, or a fragment or variant thereof.

In a further aspect the present invention provides a first polypeptide sequence capable of destabilising a second polypeptide to which it is linked. Preferably, the sequence of the first polypeptide is at least 63%, 70%, 81 % or 90%, identical to the 11 amino acids encoded by exon 5 of an EVE3 gene.

In another aspect the present invention provides a host cell genetically engineered to contain a polynucleotide of the present invention. Preferably the host cell is selected from the group including a mammalian cell, an insect cell, a yeast cell, a fungal cell and a bacterium.

In yet another aspect the present invention provides a host cell engineered to produce a polypeptide of the present invention.

In still another aspect the present invention provides a method for identifying an agent capable of modulating the activity of a SPRED3 or EVE3 polypeptide or a fragment or variant thereof. Preferably, the agent is an agonist or antagonist of the SPRED3 or EVE3 polypeptide or fragment or variant thereof. In yet another aspect the present invention provides a method for identifying an agent capable of modulating the activity of a SPRED1 or SPRED2 polypeptide or a fragment or variant thereof. Preferably, the agent is an agonist or antagonist of the SPRED1 or SPRED2 polypeptide or fragment or variant thereof.

In a further aspect the present invention provides a method of using a SPRED3 or EVE3 polypeptide, or a fragment, variant, agonist or antagonist thereof, to modulate a cell function.

In another aspect the present invention provides a method of using a SPRED1 or SPRED2 polypeptide, or a fragment, variant, agonist or antagonist thereof, to modulate a cell function.

Yet a further aspect of the present invention provides an antagonist of a polypeptide of the present invention wherein the antagonist inhibits protein-protein interaction through the KBD of a polypeptide of the present invention.

In a further aspect the present invention provides a method of modulating tumour growth including administering to a mammal in need thereof an effective amount of KBD antagonist.

In another aspect the present invention provides a method of enhancing wound healing including administering to a mammal in need thereof an effective amount of a polypeptide, an agent or a composition as described herein.

In yet another aspect the present invention provides a method of modulating cell motility or migration including administering to a mammal in need thereof an effective amount of a polypeptide, an agent or a composition as described herein.

In another aspect the present invention provides a method of treating or preventing a liver condition including administering to a mammal in need thereof an effective amount of a polypeptide, an agent or a composition as described herein. A further aspect of the present invention provides a method of modulating a biological function including administering to a mammal in need thereof an effective amount of a polypeptide, an agent or a composition as described herein.

In a further aspect the present invention provides a method of treating or preventing a neural condition including administering to a mammal in need thereof an effective amount of a polypeptide, an agent or a composition as described herein.

In yet another aspect the present invention provides a method of modulating liver growth and/or regrowth by modulating the Ras/MAPK pathway. Preferably, the pathway is modulated using a polypeptide of the invention, or an agonist or antagonist thereof.

In another aspect the present invention provides antibodies specific for any one or more of the polypeptides of the present invention.

BRIEF DESCRIPTION OF THE FIGURES. Fig. 1 shows EVE3/SPRED3 characterization and liver restricted expression. (A) Domain structure of EVE3 and SPRED3. The EVH1 domain is shaded in red, the KBD domain in green and the Sprouty domain in yellow. The unique C- terminus of EVE3 is in blue. (B) The deduced human and mouse EVE3/SPRED3 amino acid sequences are aligned with identical residues shown in red and unique residues in black or blue. (C) Structure of the human EVE3 gene. Exon 5 encodes a unique C-terminal segment of EVE3, alternative splicing of exon 5 leads to a SPRED3 transcript. Human EVE3 is localized at position 19q13.2. (D, E) Northern blot analysis of EVE3/SPRED3 expression in 7 adult rat tissues, D and in 19 rat tissues and the human hepatoma cell line HepG2, E. Poly A+ RNA (2μg) was separated by denaturing agarose electrophoresis and transferred to Hybond XL gel. Membranes were probed with a human EVE3 cDNA probe at high stringency. The Ethidium Bromide (EthBr) staining (lower panels) indicates the RNA loading. RNA size markers in kb are indicated. Fig. 2 shows localization and movement of EVE3/SPRED3. (A) Subcellular localization of FLAG tagged EVE3, SPRED3 and C-terminal truncation mutants of SPRED2 (SPRED2ΔC) and SPRED1 (SPRED1ΔC) in NIH-3T3 cells. FLAG tagged protein detected with FLAG antibody are indicated by the red signal and DAPI staining of cell nuclei is depicted by the blue signal. (B) Localization of EVE3, SPRED3 and an N-terminal truncated mutant of SPRED3 (SPRED3ΔN, residues 196-409) in NIH-3T3 cells under serum starved (-), 10% serum (20 minutes) and PDGF stimulation (20 ng/ml, 20 minutes). Staining as in panel A.

Fig. 3 shows that EVE3 differentially blocks Ras/MAPK activation downstream of some growth factors but not other MAPK pathways. (A) HEK 293 cells were transfected with 0.2 μg of GFP-ERK2 construct and 2 μg of either empty vector (v: pEFBOS) or FLAG tagged human SPRED3 (S3) or EVE3 (E3). 24 hours post-transfection the cells were serum starved for 12 hours then restimulated for 20 mins with either 10% serum, PDBu (1 μg/ml), PDGF-BB (20 ng/ml), EGF (200 ng/ml), bFGF (100 ng/ml), HGF (40 ng/ml) or NGF-β (100 ng/ml) then lysed in RIPA buffer. Equal total protein amounts for each lysate were loaded on a SDS- PAGE gel and the immunoblot probed with the indicated primary antibodies. The left most lane of each blot represents the basal level of ERK2 activation (A) or of JNK and p38 (C) in serum starved cells. Note that in (A) the bottom FLAG panel detecting EVE3 expression was exposed for 1 hour whilst the upper FLAG panel detecting SPRED3 expression was exposed for 1 minute. (B) FLAG tagged EVE3/SPRED3 constructs were transfected into HEK 293 cells with Elk-1 reporter plasmids and stimulated with (+) or without (-) 10% serum for 6 hours then lysed and analyzed by the CAT ELISA assay. The results show the mean ± SD for three independent experiments. (C) HEK 293 cells were transfected as in A also with a FLAG tagged SPRED3 N-terminal deletion mutant, S3ΔN (2 μg; residues 196-409) and with (+) FLAG tagged JNK (0.2 μg) or HA tagged p38 (0.2 μg). 24 hours posttransfection the cells were treated for 10 mins with (+) or without (-) 0.5M sorbitol then lysed and probed with the indicated primary antibodies. (D) NIH-3T3 cells were transfected with either FLAG tagged EVE3 or SPRED3 cDNAs then fixed and stained with anti-FLAG (red signal) and either phospho-ERK, phospho-JNK or phospho-p38 antibodies (green signals). DAPI staining (blue signal) indicates position of cell nuclei. Fig. 4 shows that EVE3 inhibits NGF but not FGF mediated PC12 differentiation. (A, C) PC12 cells were transfected with the indicated FLAG tagged constructs then stimulated with (A) NGF (100 ng/ml) or (C) bFGF (100 ng/ml) for 24 hours to induce neurite formation. Cells were then fixed and stained with FLAG antibody (red signal) and anti-βlll-tubulin to detect mature neurons (green signal). Arrows represent FLAG positive cells. DAPI staining (blue signal) indicates cell nuclei. (B, D) Transfected, differentiated PC12 cells assessed as having neurites 1.5x longer than the cell body were scored and the means ± range compiled from the results of two independent experiments.

Fig. 5 shows that EVE3 blocks S-phase progression of synchronized NIH-3T3 cells. (A) NIH-3T3 cells (upper 4 panels) were serum starved for 24 hours then treated with (+) or without (-) 10% serum for 16 hours, then fixed and processed for BrdU incorporation into dividing S-phase positive cells (green nuclear signal). Cells were transfected with FLAG tagged EVE3 or SPRED3 (lower 2 panels) then starved and stimulated with 10% serum and processed for BrdU incorporation as above. FLAG positive cells are indicated by the red signal. (B) Compilation of BrdU positive cells coexpressing FLAG tagged EVE3 or SPRED3. Cells were either untransfected (UT) or transfected with empty vector (pEFBOS) or EVE3 or SPRED3 then starved for 24 hours then stimulated with (+) or without (-) 10% serum for 16 hours then processed for BrdU incorporation. Figures represent the mean ± range compiled from the results of two independent experiments. (C, D) The EVE3/SPRED3 combination enhances suppression of ERK activation. HEK 293 cells were transfected with GFP-ERK2 (0.2 μg) and 2 μg of either: empty vector (v), FLAG tagged constructs, SPRED3 (S3), EVE3 (E3), combined (S3 E3), or (D) EVE3 CAAX (E3 CAAX), and the lysates probed with the indicated (IB) antibodies. Note that in (C) the lower FLAG panel was exposed for 1 hour whilst the upper FLAG panel was exposed for 1 minute. (D) EVE3 CAAX is unable to block ERK activation. 0.2 μg and 0.5 μg of GFP ERK2 respectively were transfected in the two E3 CAAX lanes. (E, F) EVE3 and SPRED3 do not form heterodimers. HEK 293 cells were transfected with FLAG tagged EVE3, SPRED3 and/or HA tagged EVE3, SPRED3 as indicated and lysates (L) or immunoprecipitates using the FLAG antibody (IP F) probed with the indicated antibodies. In (F) lysates were made in either RIPA or A buffer.

Fig. 6 shows the human SPRED1 and SPRED2 amino acid sequences and gene structures. (A) The deduced human SPRED1 and SPRED2 amino acid sequences are as depicted with identical residues in red. The EVH1 domain is shaded in yellow, the KBD domain in green and the Sprouty domain in blue. (B) Gene structure of the human SPRED genes depicting their conserved seven coding exon structure. Human SPRED1 is localized at position 15q14 and human SPRED2 at position 2p13-14.

Fig. 7 shows that SPRED1 and SPRED2 expression is brain restricted and induced by Ras/MAPK pathway activation. (A,B) Northern blot analysis of SPRED1 and SPRED2 mRNA expression in rat tissues (A) and rat brain structures (B). 2 μg poly A+ RNA was isolated for each sample and loaded on the agarose gel. Northern blots were probed with the indicated human cDNA probes at low stringency. Predominant expression of both SPRED genes was observed in brain (A) then cerebral hemisphere, cerebellum and olfactory bulb (B). Ethidium bromide (EthBr) staining of mRNA and glyceraldehyde-3- phosphate dehydrogenase (GAPDH) reprobing is depicted as a loading control. RNA size markers in kb are indicated. (C) Western blot of SPRED1 and SPRED2 rat tissue protein expression. Rat tissue lysates in 1 % Triton X100, 20 mM Tris pH7.5, 150 mM NaCI, 2 mM EDTA and 10% glycerol buffer were immunoprecipitated using 25 μl rabbit SPRED1 or SPRED2 polyclonal serum then the blots probed with either SPRED primary antibody as indicated and secondarily probed using an anti-rabbit-HRP (lower panels) or protein G-HRP (upper panel). Both SPRED proteins were obscured or partially obscured by the primary antibody heavy chain (lower panel) necessitating secondary probing with protein G-HRP. SPRED proteins are indicated with the arrows. (D, E) Northern blot analysis of Spred mRNA induction in (D) NIH-3T3 1. 10% serum, 2. no serum, 3. PDGF 2 hrs, 4. PDGF 3 hrs, 5. PD 098059 (40 μM) 1 hr, PDGF 2 hrs,

6. PDBu 3 hrs, 7. PD 098059 (40 μM) 3 hrs, and HEK 293T cells conditions 8-14, duplicate conditions 1-7. (E) NIH-3T3 cells following the indicated treatments for the time points shown. 2 μg poly A+ RNA was loaded and probed with the indicated human SPRED cDNAs. GAPDH and EthBr panels serve as loading controls. (F) Western blot analysis of SPRED1 protein expression and phospho- ERK expression in O/N starved NIH-3T3 cells restimulated with 10% serum for the indicated time points. A nonspecific band served as a loading control.

Fig. 8 shows that the SPRED proteins are differentially expressed in brain tumours. Equivalent amounts (200 μg) of 8 independent primary human brain tumour lysates in RIPA buffer were run on 8% SDSPAGE gels and probed with either SPRED1 or SPRED2 rabbit polyclonal serum (upper panels) or with the matching preimmune (PI) serum (lower panels). S1 and S2 lanes represent human SPRED1 and SPRED2 cDNA transfected 293T cell lysates as controls.

Fig. 9 shows that SPRED proteins block activation of the RAS/MAPK pathway induced by a wide range of growth factors and stimuli. (A, B) HEK 293 cells were transfected with 0.2 μg of GFP-ERK2 construct and 2 μg of either empty vector (v: pEFBOS) or FLAG tagged human SPRED1 or SPRED2 (S1 , S2) or FLAG tagged C-terminal sprouty domain deletion mutants of human SPRED1 or SPRED2, residues 1-285 (S1ΔC) or residues 1-257 (S2ΔC). 24 hours post- transfection the cells were serum starved for 12 hours then restimulated for 20 mins with either 10% serum, PDBu 1 μg/ml, PDGF 20 ng/ml, EGF 200 ng/ml, bFGF 100 ng/ml, HGF 40 ng/ml, NGF 100 ng/ml then lysed in RIPA buffer. Equal total protein amounts for each lysate were loaded on a SDS-PAGE gel and the immunoblot probed with the indicated primary antibodies. The left most lane of each blot (A, B) represents the basal level of ERK2 activation in serum starved cells. (C) HEK 293 cells were transfected with either empty vector (v: 1 μg) or with indicated human SPRED cDNAs (1 μg) and with (where indicated) a mVEGF2 receptor cDNA (1 μg), 0.2 μg GFP-ERK2 and stimulated with mVEGF (100 ng/ml) for 20 minutes then lysed in RIPA buffer and equal lysate volumes run on a SDS-PAGE gel and the immunoblot probed with the indicated primary antibodies. The positions of phosphorylated GFP-ERK2 and endogenous phospho-ERK proteins is indicated by the arrows. (D) HEK 293 cells were transfected with either empty vector (v: 2 μg) or with indicated human SPRED2 cDNAs, including a FLAG tagged N-terminal EVH1 domain deletion mutant of

SPRED2 (S2ΔN) residues 201-418 (2 μg) and stimulated with 10% serum for 20 minutes then lysed in RIPA buffer and equal lysate volumes run on a SDS-PAGE gel and the immunoblot probed with the indicated primary antibodies. Both the phospho and pan ERK panels indicate endogenous ERK proteins. (E) FLAG tagged SPRED constructs were transfected into HEK 293 cells with Elk-1 reporter plasmids and stimulated with (+) or without (-) 10% serum for 6 hours then lysed and analyzed by the CAT ELISA assay. The results show the means + S.D. for three independent experiments.

Fig. 10 shows that SPRED proteins are mobilized to the plasma membrane following growth factor stimulation. (A, B) NIH-3T3 cells were starved O/N in serum free media then either fixed or stimulated for 20 minutes with (A) EGF (200 ng/ml) or (B) 10% serum then stained with rabbit polyclonal SPRED1 and SPRED2 serum (green signal) (A) or Flag Ab (green signal) (B). Arrows indicate enrichment of endogenous SPRED proteins at the plasma membrane. DAPI staining indicates cellular nuclei. (C) NIH-3T3 were transfected with indicated Flag tagged SPRED constructs then starved O/N in serum free media and treated for 20 minutes with 10% serum then stained with Flag Ab (red signal) and anti-phospho-ERK Ab (green nuclear signal).

Fig. 11 shows that the ERK activation block induced by SPRED proteins is sustained and not transitory. HEK 293 cells were transfected with 0.2 μg of GFP- ERK2 construct and 2 μg of either empty vector (v: pEFBOS) or FLAG tagged human SPRED2 (S2) or a FLAG tagged C-terminal sprouty domain deletion mutant of human SPRED2 residues 1-257 (S2ΔC). 24 hours post-transfection the cells were serum starved O/N then restimulated with 10% serum for the indicated time points (synchronized cells) then lysed in RIPA buffer or simply lysed at the indicated time points post-transfection and not serum starved (asynchronous). Equal lysate volumes were loaded on a SDS-PAGE gel and the immunoblot probed with the indicated primary antibodies.

Fig. 12 shows that SPRED 1 and SPRED2 proteins specifically block the RAS/MAPK pathway and not other related pathways. (A, B) HEK 293 cells were transfected with 2 μg of either vector alone (v: pEFBOS) or FLAG tagged human SPRED1 or SPRED2 (S1 , S2) or FLAG tagged C-terminal sprouty domain deletion mutant of human SPRED1 or SPRED2, (S1ΔC, S2ΔC) and with (+) FLAG tagged JNK (A) or HA tagged p38 (B). 24 hours post-transfection the cells were treated for 10 mins with (+) or without (-) 0.5M sorbitol then lysed in RIPA buffer. Equal lysate volumes were loaded on a SDS-PAGE gel and the immunoblot was probed with the indicated primary antibodies. (C) NIH-3T3 cells were transfected with either FLAG tagged SPRED1 or SPRED2 cDNAs then fixed and stained with anti-FLAG (red signal) and either phospho-ERK, phospho- JNK or phospho-p38 antibodies (green signals). (D) FLAG tagged SPRED1 and SPRED2 constructs were transfected into PC12 cells with CREB reporter plasmids and stimulated with (+) or without (-) forskolin (10 mM) for 6 hours then lysed and analyzed by the CAT ELISA assay. The results are for two independent experiments. (E) HEK 293 cells were transfected with (+) or without (-) FLAG tagged human SPRED2. 24 hours post-transfection the cells were serum starved O/N and then treated for 20 mins with (+) or without (-) bFGF (100 ng/ml) or (+), (-) 10% serum, then lysed in RIPA buffer. Equal lysate volumes were loaded on a SDS-PAGE gel and the immunoblot probed with the indicated primary antibodies.

Fig. 13 shows that SPRED1 and SPRED2 proteins inhibit NGF mediated PC12 differentiation. (A) PC12 cells were transfected with the indicated FLAG tagged SPRED constructs then stimulated with NGF (100 ng/ml) for 36 hours to induce neurite formation. Cells were then fixed and stained with FLAG antibody (red signal), βlll-tubulin to detect mature neurons (green signal) and DAPI staining of nuclei (purple signal). Arrows represent SPRED positive cells. (B) Transfected, differentiated PC12 cells assessed as having neurites 1.5x longer than the cell body were scored and compiled from the results of three independent experiments.

Fig. 14 shows that SPRED1 and SPRED2 proteins block S-phase entry of synchronized NIH-3T3 cells. NIH-3T3 cells were transfected with the indicated SPRED constructs, serum starved for 12 hours, treated with (+) or without (-) 10% serum for 16 hours, then fixed and processed for BrdU incorporation into dividing S-phase positive cells. (A) Non-transfected cells with or without serum, processed for BrdU incorporation (green nuclear signal). (B) FLAG tagged SPRED positive cells (red signal) with BrdU nuclear incorporation (green signal). (C) Compilation of BrdU positive cells expressing SPRED constructs or NF2 following serum stimulation for 16 hours. Figures represent the results of at least two independent experiments. (D) HEK 293 cells were transfected with vector alone (pEFBOS) or the FLAG tagged SPRED2 or the C-terminal SPRED2 mutant and lysates probed with the indicated cell cycle related antibodies.

Fig. 15 shows that SPRED1 and SPRED2 proteins heterodimerize via their Sprouty domain. (A, B) HEK 293 cells were transfected with the indicated Flag tagged SPRED constructs including SPREDΔC CAAX vectors comprising the SPREDΔC constructs fused with the C-terminal 17 residues of Ras. Cells were serum starved O/N then treated with 10% serum for 20 mins and lysates probed with the indicated antibodies. (C) HEK 293T cells were transfected with Flag tagged SPRED2 and HA tagged SPRED1 constructs and lysates (L) or immunoprecipitates (IP) performed with the anti-SPRED2 Ab (S2) run out on the gel and probed with the indicated antibodies.

DETAILED DESCRIPTION OF THE INVENTION.

Control of cell proliferation and differentiation is coordinated by proteins of various signal transduction pathways. A typical example of these pathways is the Ras/MAPK (mitogen-activated pjotein kinase) pathway as discussed supra.

The inventors have identified novel genes which encode polypeptides that can potently block signal transduction by the Ras/MAPK pathway and can potentially suppress JNK kinase activation. The latter is a related MAPK pathway in the cell involved in, amongst other functions, cell survival/apoptosis.

In one aspect the present invention provides an isolated polynucleotide encoding a SPRED3 polypeptide or an EVE3 polypeptide. Preferably the polynucleotide encodes a mammalian SPRED3 polypeptide or a mammalian EVE3 polypeptide. Most preferably the polynucleotide encodes a human SPRED3 polypeptide or a human EVE3 polypeptide. In another aspect the present invention also provides an isolated polynucleotide encoding a SPRED1 polypeptide or a SPRED2 polypeptide. Preferably the polynucleotide encodes a mammalian SPRED1 polypeptide or a mammalian SPRED2 polypeptide. Most preferably the polynucleotide encodes a human SPRED1 polypeptide or a human SPRED2 polypeptide.

As used herein, the term "polynucleotide" is intended to include DNA, RNA and PNA, single and double stranded nucleic acids, naturally produced and synthetic nucleic acids.

As used herein, the term "polypeptide" is intended to include full length protein and fragments of the protein. Thus "protein", "polypeptide" and "peptide" can be used interchangeably and include all polypeptides of more than approximately 6 consecutive amino acids of a sequence.

As used herein, the term "SPRED3" is intended to include all forms of SPRED3, fragments of SPRED3, molecules having SPRED3 function and variants of SPRED3 resulting from alternate splicing of the primary transcript from the EVE3 gene. SPRED3 function includes a function as described herein including but not limited to: inhibition of Ras/MAPK pathway, inhibition of c-Jun N-terminal kinase pathway, blocking cell cycle phase progression and preventing cell differentiation in response to growth factors.

As used herein, the term "EVE3" is intended to include all forms of EVE3, fragments of EVE3, molecules having EVE3 function and variants of EVE3 resulting from alternate splicing of the primary transcript from the EVE3 gene. EVE3 function includes a function as described herein including but not limited to: inhibition of Ras/MAPK pathway, inhibition of c-Jun N-terminal kinase pathway, blocking cell cycle phase progression and preventing cell differentiation in response to growth factors.

As used herein, the term "SPRED1" is intended to include all forms of SPRED1 , fragments of SPRED1 , molecules having SPRED1 function and variants of SPRED1 resulting from alternate splicing of the primary transcript from the SPRED1 gene. SPRED1 function includes a function as described herein including but not limited to: inhibition of Ras/MAPK pathway, blocking cell cycle phase progression and preventing cell differentiation in response to growth factors.

As used herein, the term "SPRED2" is intended to include all forms of SPRED2, fragments of SPRED2, molecules having SPRED2 function and variants of SPRED2 resulting from alternate splicing of the primary transcript from the SPRED2 gene. SPRED2 function includes a function as described herein including but not limited to: inhibition of Ras/MAPK pathway, blocking cell cycle phase progression and preventing cell differentiation in response to growth factors.

SPRED1 and SPRED2 include an N-terminal EVH1 protein interaction domain, a central KBD region (so named because a mouse Spredl polypeptide can bind c- kit through this region) and a C-terminal cysteine-rich Sprouty domain which may be involved in membrane translocation and localisation of SPRED polypeptides. The Sprouty domain may also mediate dimerisation to form homodimers or heterodimers of SPRED polypeptides.

SPRED3 and EVE3 are two distinct polypeptides encoded by the same gene (EVE3), with the larger SPRED3 polypeptide produced by translation of an alternatively spliced transcript. SPRED3 shares a similar domain structure to SPREDs 1 and 2, but the KBD is only weakly conserved (28% identity). EVE3, however, may include an N-terminal EVH1 domain and a unique C-terminal region encoded by exon 5, which possesses an in-frame stop codon.

The present invention therefore provides an isolated polynucleotide encoding a functional domain of a SPRED3 polypeptide or an EVE3 polypeptide. Preferably the functional domain is selected from the group including an EVH1 domain, a KBD and a Sprouty domain.

The present invention further provides an isolated polynucleotide including the genomic sequence of an EVE3 gene. Preferably the EVE3 gene is a mammalian EVE3 gene. More preferably the EVE3 gene is a human EVE3 gene or a mouse EVE3 gene. Most preferably the EVE3 gene is selected from the group of EVE3 genes depicted in SEQ ID NO:1 and SEQ ID NO:2.

The present invention yet further provides an isolated polynucleotide including the genomic sequence of a SPRED gene. Preferably the SPRED gene is a mammalian SPRED gene. More preferably the SPRED gene is a human SPRED1 gene or a human SPRED2 gene. Most preferably the SPRED gene is selected from the group of SPRED genes depicted in SEQ ID NO:9 and SEQ ID NO:10.

A genomic sequence may be transcribed into RNA and subsequently processed to produce a messenger RNA (mRNA). An mRNA sequence may be represented by its corresponding complementary DNA (cDNA) sequence.

In one aspect the present invention provides an isolated polynucleotide selected from the group including a SPRED3 cDNA and an EVE3 cDNA. Preferably the SPRED3 cDNA and EVE3 cDNA are mammalian. More preferably the SPRED3 cDNA and EVE3 cDNA are human. Even more preferably the SPRED3 cDNA and EVE3 cDNA are derived from the genomic EVE3 sequences depicted in either SEQ ID NO:1 or SEQ ID NO:2. Yet more preferably the SPRED3 cDNA and EVE3 cDNA encode polypeptides depicted in SEQ ID NOS:5 and 6 respectively. Most preferably the SPRED3 cDNA and EVE3 cDNA are those depicted in SEQ ID NOS: 3 and 4 respectively.

In a further aspect the present invention provides an isolated polynucleotide selected from the group including a SPRED1 cDNA and an SPRED2 cDNA. Preferably the SPRED1 cDNA and SPRED2 cDNA are mammalian. More preferably the SPRED1 cDNA and SPRED2 cDNA are human. Even more preferably the SPRED1 cDNA and SPRED2 cDNA encode polypeptides depicted in SEQ ID NOS:13 and 14 respectively. Most preferably the SPRED1 cDNA and SPRED2 cDNA are those depicted in SEQ ID NOS:11 and 12 respectively. The present invention provides variants of the cDNAs depicted in SEQ ID NOS:3 and 4, wherein the variants will hybridise to either of SEQ ID NOS:3 and 4 under conditions of moderate or high stringency, as understood by a person skilled in the art. Preferably, the nucleotide sequence of a variant is at least 60%, 70%, 80%, 90%, 95% or 98% identical to the sequence depicted by either of SEQ ID NO:3 or SEQ ID NO:4.

Alternatively, due to degeneracy of the genetic code, a variant may encode a polypeptide which has an amino acid sequence at least 60%, 70%, 80%, 90%, 95% or 98% identical to a SPRED3 or an EVE3 polypeptide sequence. Preferably, a variant encodes a polypeptide which has a sequence selected from the group including SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:8, or a fragment thereof.

As used herein, the phrase " conditions of moderate or high stringency " refers to conditions under which a probe, primer or oligonucleotide will hybridise to its target sequence, but to no other sequences. These conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridise to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide. Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 60%, 70%, 80%, 90%, 95%, 98%, or 100% homologous to each other typically remain hybridized to each other. A non- limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6χSSC, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2χSSC, 0.01% BSA at 50°C.

The present invention also provides variants of the cDNAs depicted in SEQ ID NOS:11 and 12, wherein the variants will hybridise to either of SEQ ID NOS:11 and 12 under conditions of moderate or high stringency, as understood by a person skilled in the art. Preferably, the nucleotide sequence of a variant is at least 60%, 70%, 80%, 90%, 95% or 98% identical to the sequence depicted by either of SEQ ID NO:11 or SEQ ID NO:12.

In addition, due to degeneracy of the genetic code, a variant may encode a polypeptide which has an animo acid sequence at least 60%, 70%, 80%, 90%, 95% or 98% identical to a SPRED1 or an SPRED2 polypeptide sequence. Preferably, a variant encodes a polypeptide which has a sequence selected from the group including SEQ ID NO:13 and SEQ ID NO:14, or a fragment thereof.

Oligonucleotides of the present invention include sequences provided by the present invention and have at least 15 nucleotides and preferably at least about 20 nucleotides.

Northern analyses indicated that in vivo expression of EVE3 transcripts may be limited to particular organs. Preferably the organ is liver.

Northern analyses indicated that in vivo expression of SPRED1 and SPRED2 transcripts may be limited to particular organs. Preferably the organ is brain. The polypeptides of the invention include, but are not limited to, SPRED3 and EVE3, and fragments and variants thereof. Also encompassed by the term "polypeptides of the invention" are human SPRED1 and human SPRED2, and fragments and variants thereof.

The isolated polypeptides of the present invention include, but are not limited to, an isolated polypeptide encoded by an EVE3 gene. One such polypeptide is encoded by the cDNA represented in SEQ ID NO:4. The sequence of that polypeptide is shown in SEQ ID NO:6 and is herein referred to as EVE3 polypeptide or protein.

Surprisingly, the EVE3 primary transcript may undergo alternative splicing (SEQ ID NO:3, as discussed supra) and this results in a different, longer polypeptide being produced. This polypeptide is also provided by the present invention and has been named SPRED3 (SEQ ID NO:5). Most surprisingly, the polypeptide resulting from exon skipping (SPRED3) is related to two other polypeptides, SPRED1 and SPRED2. The sequences of human SPRED3, SPRED1 and SPRED2 are shown in SEQ ID NOS:5, 13 and 14 respectively. The sequence of the mouse SPRED3 homologue has been determined and is shown in SEQ ID NO:8.

EVE3 (EVH1 enhanced) is the shorter polypeptide and contains only an N- terminal Ena Vasp Homology 1 (EVH1 ) domain and a unique C-terminal segment. The second, larger protein, produced by alternative splicing of exon 5 has been named SPRED3; it encompasses the EVH1 domain, a central domain with some homology to a c-kit binding domain (KBD) and a C-terminal Sprouty domain, similar to the SPRED1 and SPRED 2 proteins (Fig. 1A-C).

The EVH1 domain is a protein-protein interaction domain that binds with high specificity to poly-proline motifs present in target molecules suggesting that the prospective targets of EVE3 action may contain this distinctive proline motif.

The KBD domain of SPRED3 has approximately 28% identity to a similar region SPRED1 and SPRED2. The central domain of mouse Spredl has been shown to facilitate binding to c-kit receptor tyrosine kinase. Thus this domain most likely facilitates protein-protein interactions with as yet unidentified target proteins.

As discussed supra, the EVE3 polypeptide is structurally similar to SPRED3 but lacks KBD and Sprouty domains. SPRED3 is structurally similar to SPRED1 and SPRED2 (discussed infra). It has been found that Sprouty domain deletion mutants of mouse SPRED1 and SPRED2, similar in domain structure to EVE3, were able to augment cell differentiation and growth factor induced activation of ERK2 suggesting that these constructs have dominant negative capabilities, that is, they are unable to inhibit signal transduction.

Surprisingly, and in contrast to what might be expected in light of the mouse Spred studies, the Applicants have found EVE3 to be a potent inhibitor of Ras/MAPK signaling induced by certain growth factors and chemical stimuli. As such EVE3 represents a new class of Ras/MAPK inhibitory protein.

The inhibitory activity of EVE3 appears to be restricted to that of the Ras/MAPK pathway with no observed effect on the two related intracellular MAPK pathways of JNK and p38 kinase. This is in contrast to that of SPRED3 which appears to interfere with activation of the JNK pathway and block ERK activation induced by a growth factor that EVE3 is unable to block, such as FGF. In this regard it is possible that EVE3 and SPRED3 may act separately or in concert to regulate an expansive repertoire of MAPK functions in a cell, a point supported by the observed enhanced inhibitory activity of EVE3 and SPRED3 when coexpressed.

So while, EVE3 and SPRED3 polypeptides are potent inhibitors of the Ras/MAPK pathway and SPRED3 can also inhibit the c-Jun amino-terminal kinase (JNK) pathway; the two polypeptides differ in their effects on blocking of cellular differentiation, but both can block cell cycle progression.

Human SPRED1 and SPRED2 appear to be broad regulators of the Ras/MAPK pathway and are able to block a wide range of activators of this pathway, including both natural and chemical inducers. This is in contrast to EVE3 and SPRED3 which appear to be more specific in their inhibitory activity. A further difference between SPREDs 1 and 2 and EVE3/SPRED3 is that SPREDs 1 and 2 are not able to effect related MAPK pathways such as the JNK pathway.

In another aspect the present invention provides an isolated polypeptide which is a SPRED3 polypeptide, an EVE3 polypeptide, or a fragment or variant thereof. Preferably, the polypeptide is capable of blocking cell cycle progression. Preferably, the polypeptide is capable of binding to a component of a signal transduction pathway. More preferably, the polypeptide is capable of modulating a signal transduction pathway. Yet more preferably, the polypeptide is capable of modulating a cellular response to an extracellular signal. Even more preferably, the polypeptide is capable of modulating cellular growth and/or differentiation. More preferably still, the polypeptide is capable of modulating signal transduction through a MAPK pathway. Most preferably, the polypeptide is capable of modulating signal transduction through a Ras/MAPK pathway.

The isolated SPRED3 and EVE3 polypeptides, or fragments or variants thereof, of the present invention may be a mammalian polypeptide. Preferably, the polypeptide is either a mouse polypeptide or a human polypeptide. More preferably, the polypeptide is selected from the group depicted in SEQ ID NOS:5, 6 and 8.

In yet another aspect the present invention provides an isolated polypeptide which is a SPRED1 polypeptide, a SPRED2 polypeptide, or a fragment or variant thereof. Preferably, the polypeptide is capable of blocking cell cycle progression. Preferably, the polypeptide is capable of binding to a component of a signal transduction pathway. More preferably, the polypeptide is capable of modulating a signal transduction pathway. Yet more preferably, the polypeptide is capable of modulating a cellular response to an extracellular signal. Even more preferably, the polypeptide is capable of modulating cellular growth or differentiation. More preferably still, the polypeptide is capable of modulating signal transduction through a MAPK pathway. Most preferably, the polypeptide is capable of modulating signal transduction through a Ras/MAPK pathway. The isolated SPRED1 and SPRED2 polypeptides, or fragments or variants thereof, of the present invention may be mammalian polypeptides. Preferably, a polypeptide is a human polypeptide. More preferably, a polypeptide is selected from the group depicted in SEQ ID NOS:13 and 14.

Thus, the polypeptides of the invention include, but are not limited to, SPRED3 and EVE3, and fragments and variants thereof. Also encompassed by the term "polypeptides of the invention" are human SPRED1 and human SPRED2, and fragments and variants thereof.

Polypeptides falling within the scope of the present invention are not limited to those shown in the sequence listing, but also include variants and fragments of such polypeptides. Preferably, variants are at least 60%, 70%, 80%, 90%, 95% or 98% identical to the sequences provided by any one of SEQ ID NOS:5, 6 and 8. Variants also include polypeptides in which conservative amino acid changes have been made. Conservative amino acid changes are well known to those of skill in the art and involve replacing an amino acid with another amino acid that is similar in at least one physicochemical property; for example aspartate and glutamate are both negatively charged at neutral pH and similar in size.

Unexpectedly, the 11 amino acids encoded by exon 5 of the EVE3 gene (LSQYFRHMLCP) appear to confer instability on the EVE3 protein. This sequence seems to be restricted to humans since the mouse and rat exon 5 homologues do not appear to encode such a sequence. A putative exon approximately corresponding to human exon 5 encodes a predicted 21 amino acid sequence, but whether this is actually present in any mature protein is as yet unknown. Without wishing to be limited by theory it appears that the 11 amino acids direct ubiquitination of the polypeptide thus leading to its degradation by the proteosome, although there is no consensus ubiquitination signal within the sequence. Interestingly, bacteria employ an 11 amino acid C-terminal extension to direct degradation of incomplete polypeptides that result from ribosome stalling during translation. However, the mechanism behind this process is unrelated to ubiquitiniation. Furthermore, the bacterial sequence is highly hydrophobic and is recognised by a specific protease. In still another aspect the present invention provides a first polypeptide sequence capable of destabilising a second polypeptide to which it is linked. Preferably, the sequence of the first polypeptide is at least 63%, 70%, 81% or 90%, identical to the 11 amino acids encoded by exon 5 of the EVE3 gene. Most preferably the first polypeptide sequence is LSQYFRHMLCP. The present invention also provides the use of the destabilising polypeptide sequence to regulate the amount of a protein. For example, a nucleotide sequence encoding the first polypeptide is ligated in-frame to a nucleotide sequence encoding the protein whose amount is to be regulated, the resulting sequence thereby encoding a fusion protein wherein the first polypeptide sequence destabilises the fusion protein.

Surprisingly, it was found that EVE3 is a potent inhibitor of ERK activation induced by phorbol 12, 13-dibutyrate (PDBu), PDGF, hepatocyte growth factor (HGF) and nerve growth factor (NGF) and could repress serum mediated activation (Fig. 3A). However, EVE3 could not block ERK activation induced by epidermal growth factor (EGF) or fibroblast growth factor (FGF) (Fig. 3A), suggesting specificity in the inhibitory activity of EVE3. SPRED3 also has an ability to inhibit ERK activation, in particular that induced by serum, FGF and HGF (Fig. 3A). SPRED3 further demonstrated selectivity in its inhibitory properties with no observable effect on NGF, EGF, PDGF or PDBu induced activation of the Ras/MAPK pathway as assessed by ERK2 activation (Fig. 3A).

Another aspect of the present invention provides a method for identifying an agent capable of modulating the activity of a SPRED3 or EVE3 polypeptide or a fragment or variant thereof. Preferably, the agent is an agonist or antagonist of the SPRED3 or EVE3 polypeptide or fragment or variant thereof. Most preferably the method includes the steps of: (i) contacting the SPRED3 or EVE3 polypeptide or fragment or variant thereof, with the agent under conditions permitting binding between the polypeptide and the agent; (ii) detecting specific binding of the agent to the polypeptide; and (iii) determining whether the agent binds to the polypeptide. Yet another aspect of the present invention provides a method for identifying an agent capable of modulating the activity of a SPRED1 or SPRED2 polypeptide or a fragment or variant thereof. Preferably, the agent is an agonist or antagonist of the SPRED1 or SPRED2 polypeptide or fragment or variant thereof. Most preferably the method includes the steps of: (i) contacting the SPRED1 or SPRED2 polypeptide or fragment or variant thereof, with the agent under conditions permitting binding between the polypeptide and the agent; (ii) detecting specific binding of the agent to the polypeptide; and (iii) determining whether the agent binds to the polypeptide.

As used herein, the term "modulators" of activity of a polypeptide of the present invention include, but are not limited to polypeptides, small molecules, inhibitory RNA molecules (si RNA or iRNA), any other molecule that can interact with a nucleic acid encoding a polypeptide of the present invention such that translation is enhanced or is not effected, or a molecule that can interact with a polypeptide of the present invention to enhance its activity or reduce its activity.

The invention encompasses agonists and antagonists of the polypeptides of the present invention, including small molecules, large molecules, or fragments or variants of polypeptides of the present invention, that compete with the polypeptides of the present invention, and antibodies, as well as nucleotide sequences that can be used to inhibit the expression of the polypeptides of the present invention (e.g., siRNA, iRNA, antisense and ribozyme molecules, and open reading frame or regulatory sequence replacement constructs) or to enhance the expression of the polypeptides of the present invention (e.g., expression constructs that place the described polynucleotide under the control of a strong promoter system), and transgenic animals that express a polypeptide of the present invention sequence, or "knock-outs" (which can be conditional) that do not express a functional polypeptide of the present invention. Knock-out mice can be produced in several ways, one of which involves the use of mouse embryonic stem cell ("ES cell") lines that contain gene trap mutations in a murine homologue of at least one of the polypeptides of the present invention. The polypeptides of the present invention, as well as fragments, mutated, truncated, or deleted forms of the polypeptides, and/or fusion proteins can be prepared for a variety of uses. These uses include, but are not limited to, the generation of antibodies, as reagents in diagnostic assays, for the identification of other cellular gene products related to a polypeptide of the present invention, and as reagents in assays for screening for compounds that can be used as pharmaceutical reagents useful in the therapeutic treatment of mental, biological, or medical disorders and diseases. Given the similarity information and expression data, the described polypeptides of the present invention can be targeted (by drugs, oligos, antibodies, etc.) in order to treat disease, or to therapeutically augment the efficacy of therapeutic agents.

The use of peptides as therapeutic or pharmaceutical agents in accordance with the present invention includes the use of peptides modified to improve their efficacy. Such improvements include, but are not limited to, addition of short carbohydrate groups or carrier groups to permit entry into a cell.

The present invention further provides a composition including an agent as herein defined and a pharmaceutically effective carrier. The present invention yet further provides the use the composition to modulate the activity of a SPRED3 or EVE3 polypeptide or fragment or variant thereof.

The present invention further provides a composition including an agent as herein defined and a pharmaceutically effective carrier. The present invention yet further provides the use the composition to modulate the activity of a SPRED1 or SPRED2 polypeptide or fragment or variant thereof.

A further aspect of the present invention provides a method of using a SPRED3 or EVE3 polypeptide, or a fragment, variant, agonist or antagonist thereof, to modulate a cell function. Preferably the cell function is cell proliferation and/or differentiation. Alternatively, the cell function is signal transduction through the Ras/MAPK pathway. A further aspect of the present invention provides a method of using a SPRED1 or SPRED2 polypeptide, or a fragment, variant, agonist or antagonist thereof, to modulate a cell function. Preferably the cell function is cell proliferation and/or differentiation. Alternatively, the cell function is signal transduction through the Ras/MAPK pathway.

Yet a further aspect of the present invention provides an antagonist of a polypeptide of the present invention wherein the antagonist inhibits protein-protein interaction through the KBD of a polypeptide of the present invention.

In yet a further aspect the present invention provides a method of modulating tumour growth including administering to a mammal in need thereof an effective amount of KBD antagonist. Preferably the tumour is a stromal tumour. Most preferably the tumour has a mutated KIT gene.

In wound healing it is desirable to prevent excessive fibrosis while still allowing healing. Without wishing to be limited by theory, the surprising ability of SPRED3 and EVE3 to inhibit signal transduction activated by specific growth factors, but not affect signalling activated by EGF, suggests that these polypeptides may be useful in enhancing wound healing while preventing excessive fibrosis.

In another aspect the present invention provides a method of enhancing wound healing including administering to a mammal in need thereof an effective amount of a polypeptide, an agent or a composition as described herein. Preferably the polypeptide, agent or composition includes a SPRED3 or EVE3 polypeptide, or fragment or variant thereof.

Without wishing to be limited by theory, the surprising ability of SPRED3 and EVE3 to inhibit HGF signal transduction suggests a potential role for these polypeptides in the inhibition of cell motility since HGF has also been known as Scatter Factor. This may provide a tool for modulating for example, tumour metastasis or cell migration involved in proliferation or repair. In another aspect the present invention provides a method of modulating cell motility or migration including administering to a mammal in need thereof an effective amount of a polypeptide, an agent or a composition as described herein. Preferably the polypeptide, agent or composition includes a SPRED3 or EVE3 polypeptide, or fragment or variant thereof. Alternatively the polypeptide, agent or composition includes an inhibitor of SPRED3 or EVE3 function.

Given the growth inhibitory effect of EVE3-encoded polypeptides, and the apparently liver-restricted expression of EVE3 transcript, it is possible that EVE3 and/or SPRED3 may play a role in regulating liver growth or regeneration. In a preferred embodiment EV£3-encoded polypeptides are used to regulate liver growth and/or regeneration. Thus, in a particularly preferred embodiment EVE3 and/or SPRED3 polypeptides can be used to regulate liver growth.

In an analogous manner it may be possible to regulate brain or nerve cell growth or division since SPRED1 and SPRED2 are highly enriched in brain tissue and may therefore act to block the major mitogenic stimulatory pathway downstream of a broad range of RTKs. Overactivation of the EGFR has been implicated in a variety of human brain tumors, notably glioblastoma and meningioma. Mutation of intracellular members of the Ras signaling pathway has also been correlated to CNS neoplasm progression. Loss of SPRED protein expression or function might be a contributing factor to the development or disease progression of brain tumors with both of these proteins potentially acting as classical tumor suppressors.

Without wishing to be limited by theory, the surprising differential effects of SPRED3 and EVE3 on PDGF signal transduction imply a possible role in the regulation of fibrosis, angiogenesis, and sclerosis which suggests an important role in cell growth and differentiation, especially in association with conditions including, but not limited to, cancer and cirrhosis.

A further aspect of the present invention provides a method of modulating a biological function including administering to a mammal in need thereof an effective amount of a polypeptide, an agent or a composition as described herein. Preferably the polypeptide, agent or composition includes a SPRED3 or EVE3 polypeptide, or fragment or variant thereof. Alternatively the polypeptide, agent or composition includes an inhibitor of SPRED3 or EVE3 function. Most preferably the biological function is selected from the group including normal cell growth, fibrosis, angiogenesis and sclerosis.

In particular embodiments of the present invention molecules of the present invention may be used to treat medical conditions. Such use involves the administering, to a patient in need thereof, a therapeutically effective amount of a molecule of the present invention.

Another aspect of the present invention provides a method of treating or preventing a liver condition including administering to a mammal in need thereof an effective amount of a polypeptide, an agent or a composition as described herein. Preferably, the method includes the step of modulating a signal transduction pathway. Most preferably, the method includes modulating the signal transduction pathway in order to effect regeneration of the mammal's liver.

Also provided by the present invention is a method of treating or preventing a condition affecting a liver following or induced by surgery, trauma hepatitis caused by damage induced by drugs, infections (including viral) alcohol or toxins, a disease such as cirrhosis, hepatic fibrosis or a congenital disorder giving rise to liver abnormalities or storage diseases, including administering to a mammal in need thereof an effective amount of a polypeptide, an agent or a composition as described herein.

Yet another aspect of the present invention provides a method of treating or preventing a neural condition including administering to a mammal in need thereof an effective amount of a polypeptide, an agent or a composition as described herein. Preferably, the method includes the step of modulating a signal transduction pathway. Most preferably, the method includes modulating the signal transduction pathway in order to effect treatment of a nerve or neural cell of the mammal. Still another aspect of the present invention provides a method of modulating liver growth and/or regrowth by modulating the Ras/MAPK pathway. Preferably, the pathway is modulated using a polypeptide of the invention, or an agonist or antagonist thereof. More preferably, the pathway is modulated using a polypeptide selected from the group including SPRED3 and EVE3, or an agonist or antagonist thereof. Most preferably, the pathway is modulated using a polypeptide selected from the group including SEQ ID NO:5 and SEQ ID NO:6, or an agonist or antagonist thereof.

Yet another aspect of the present invention provides a host cell genetically engineered to contain a polynucleotide of the present invention. Preferably the host cell is selected from the group including a mammalian cell, an insect cell, a yeast cell, a fungal cell and a bacterium. In preferred embodiments of the present invention the host cell is Escherichia coli. Preferably the polynucleotide is in operative association with a regulatory sequence wherein the regulatory sequence is functional in the host cell and directs expression of the polynucleotide of the invention.

A variety of methodologies exist for making the polypeptides of the present invention, as well as fragments of the polypeptides. For example, a recombinant organism may be generated which directs expression of polypeptides or fragments thereof. These polypeptides or fragments may further include specific epitopes which facilitate purification. Polypeptides and fragments may also be synthesised by commercially available peptide synthesisers. This method is particularly useful for the generation of small peptides.

In another aspect of the present invention provides a host cell engineered to produce a polypeptide of the present invention. The polypeptide may be modified to include an epitope which does not naturally occur in that polypeptide, but is added in order to facilitate purification of the polypeptide. Such epitopes are well known to those of skill in the art. Preferably the epitope is selected from the group including 6xHis, maltose-binding protein (MBP), glutathione S- transferase (GST), myc, haemagglutinin (HA) and FLAG peptide (Asp-Tyr-Lys- Asp-Asp-Asp-Asp-Lys). Another aspect of the present invention provides antibodies specific for any one or more of the polypeptides of the present invention.

Methods for generating polyclonal and/or monoclonal antibodies are well known in the art. The present invention provides a variety of antibodies directed to different epitopes on the surface of each of the polypeptides of the present invention.

As used herein, the term "antibodies" includes, but is not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), phage display "antibodies", chimeric antibodies, human antibodies, "humanised" antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, domain antibodies, epitope-binding fragments of any of the above, and any other molecule that specifically binds a polypeptide of the present invention.

The present invention will now be more fully described with reference to the following non-limiting examples.

EXAMPLES

Example 1 : Cloning of human EVE3 gene and its expression in various tissues.

The novel human EVE3 and SPRED3 sequences were constructed by linking together information from a combination of two anonymous Expressed Sequence

Tag (EST) clones, GenBank accession numbers: BF983829 and BF409205 via the polymerase chain reaction (PCR) using a proof reading enzyme system

(Platinum Pfx polymerase: Invitrogen) according to the manufacturer's conditions.

However, the sequence was not complete and non-overlapping regions were filled in by PCR using sequence information from ESTs and the genomic EVE3 gene obtained from public databases. Sequences for the genomic gene,

SPRED3 cDNA and EVE3 cDNA are shown in SEQ ID NOS:1 , 3 and 4 respectively. The mouse homologue of SPRED3 was also obtained, its genomic sequence is shown in SEQ ID NO:2, the corresponding cDNA sequence is shown in SEQ ID NO:7 and its encoded protein is shown in SEQ ID NO:8.

Tissue distribution of EVE3 expression was determined using multi-tissue Northern blots. Poly A+ RNA was resolved on an agarose-formaldehyde gel. RNA was transferred to Hybond-XL membrane (Amersham) overnight in 20 x SSC via capillary action. The membrane was blocked in hybridization buffer Ultra-Hyb (Ambion) for 1 hour at 42°C. cDNA probes (25 ng) were labeled using the Megaprime DNA labeling system (Amersham Pharmacia Biotech) with α- [32P]-dATP (Amersham Pharmacia Biotech) and subsequently purified using oligonucleotide purification columns (Boehringer Mannheim) and then hybridized to the membrane overnight at 42°C. Filters were washed at high stringency (65°C, 0.1 x SSC for 1 hour). The results are shown in Figure 1 D.

Example 2: EVE3 polypeptide has novel activity

Sprouty and Spred proteins have been reported to translocate to the cell membrane following growth factor treatment, a step that is necessary for their inhibitory function. In order to determine the cellular localization of EVE3 and SPRED3 and whether they were similarly sensitive to growth factor treatment FLAG tagged constructs of EVE3 and SPRED3 were expressed in NIH-3T3 fibroblasts. A diffuse cytoplasmic staining for SPRED3 and a distinctive punctate, cytoplasmic pattern for EVE3 (Fig. 2A) were observed. Since the EVE3 polypeptide resembled a truncated SPRED polypeptide, the applicants sought to replicate EVE3 function with truncated SPRED1 and SPRED2. To determine whether this pattern was unique to EVE3, C-terminal deletion mutants of SPRED1 (SPRED1ΔC) and SPRED2 (SPRED2ΔC) were constructed, analogous to that of the EVE3 structure and observed that the EVE3-like truncations of these two proteins had no such punctate localization within the cell (Fig. 2A). After overnight serum deprivation the localization of the transfected proteins was unchanged (Fig. 2B). Upon treatment with serum for 20 minutes the position of EVE3 remained unchanged, however we detected an enrichment of some SPRED3 at the membrane in a proportion of transfected cells (Fig. 2B). Platelet derived growth factor (PDGF) treatment for 20 minutes similarly had no effect on the punctate expression pattern of EVE3 and there was no obvious movement of SPRED3 to the membrane (Fig. 2B). An N-terminal truncated mutant of SPRED3, lacking the EVH1 domain (SPRED3ΔN) was also unable to move to the membrane following PDGF treatment (Fig. 2B).

Example 3: EVE3 and SPRED3 polypeptides block Ras/MAPK signalling

To determine whether EVE3 and SPRED3 could regulate ERK activation following growth factor stimulation, HEK 293 cells (which respond to a wide range of growth factors) were transfected with FLAG tagged EVE3 or SPRED3 and a GFP-ERK2 construct. ERK2 activation was then detected with a phospho- specific antibody following treatment of serum starved cells with seven different stimuli able to induce activation of the Ras/MAPK pathway. Surprisingly, the results clearly demonstrated that EVE3 was a potent inhibitor of ERK activation induced by phorbol 12, 13-dibutyrate (PDBu), PDGF, hepatocyte growth factor (HGF) and nerve growth factor (NGF) and could repress serum mediated activation (Fig. 3A). However, there was no observable effect in blocking ERK activation induced by epidermal growth factor (EGF) or fibroblast growth factor (FGF) (Fig. 3A), suggesting specificity in the inhibitory activity of EVE3. SPRED3 also demonstrated an ability to inhibit ERK activation, in particular that induced by serum, FGF and HGF (Fig. 3A). SPRED3 also demonstrated selectivity in its inhibitory properties with no observable effect on NGF, EGF, PDGF or PDBu induced activation of the Ras/MAPK pathway as assessed by ERK2 activation (Fig. 3A). The inhibitory effects of EVE3 were observed in spite of the consistently lower levels of expressed EVE3 compared with those of SPRED3 (Fig 3A), which when quantitatively analyzed indicated that EVE3 is a factor of 4 to 6 more effective in inhibiting ERK activation compared with SPRED3. The same trend was observed in an Elk-1 transactivation assay, an in vivo readout of endogenous ERK activation (Fig 3B). The N-terminal EVH1 domain of SPRED3 (so that its structure resembled that of EVE3) was then deleted and the ability of this truncated protein to effect ERK activation in response to serum was examined. This mutant was unable to suppress serum-mediated activation of the Ras/MAPK pathway as assessed by Elk-1 mediated transcription of a Chloramphenicol acetyltransferase (CAT) reporter cassette (Fig. 3B). In fact this construct appeared to enhance serum mediated Elk-1 activation suggesting it may possess dominant negative properties. CAT reporter assays.

Phospho-Elk-1 activation assays (Stratagene) were performed using the CAT Elisa system (Roche Diagnostics). HEK 293 Cells were transfected with the following expression constructs; gal4-CAT plasmid (100 ng), pFA2-Elk-1 transactivator plasmid (50 ng) and pCMV-lacZ (50 ng). Twenty four hours after transfection cells were serum deprived for 24 hours and then stimulated with 10% serum for 6 hours. Cells were then lyzed and the lysates loaded in duplicate onto CAT antibody coated wells in 96 well microtitre plates. CAT Elisa absorbance recordings were standardized according to LacZ expression and protein concentration.

Immunochemical analysis.

Immunoprecipitation, immunoblotting and immunofluorescence were performed with the following antibodies: anti-FLAG, anti-HA (Sigma-Aldrich), anti-GFP (Clontech), antiphosphotyrosine (Santa Cruz), anti-βlll tubulin, anti-phospho p38 kinase, anti-phospho MAP kinase (Erk1 , Erk2), anti-phospho JNK kinase (Promega), anti-phospho-p44/42 MAP Kinase antibody (Cell Signaling Technology).

Immunoprecipitation.

Cells were lyzed in A buffer: 1% Triton X100, 20 mM Tris pH 7.5, 150 mM NaCI, 2 mM EDTA and 10% glycerol buffer, 1 x protease inhibitor cocktail (Calbiochem), 1 x phosphatase inhibitor cocktail (Calbiochem) or RIPA buffer: 0.5 % NP-40, 20 mM Tris pH 7.5, 0.1 % SDS, 1% Triton X100, 0.15 M NaCI, 2 mM EDTA, 10% glycerol, 1 x protease inhibitor cocktail (Calbiochem), 1 x phosphatase inhibitor cocktail (Calbiochem) and lysates were precipitated with 3 μg of FLAG antibody for between one and twenty four hours at 4°C on a rotating wheel. Protein LA beads (Sigma, 20 μl 1 :1) were then added for a further hour at 4°C. Immunocomplexes were pelleted and washed 5 times in lysis buffer. 20 μl of the samples were resolved on 10% or 12% SDS-PAGE gels for analysis.

To determine if EVE3 and SPRED3 would be selective in their inhibitory activity, the effects of EVE3 and SPRED3 expression on activation of two related intracellular MAPK pathways were tested, namely those of c-Jun amino-terminal kinase (JNK) and the stress activated p38 MAPK. HEK 293 cells were transfected with FLAG tagged JNK and HA tagged p38 along with EVE3 or SPRED3 and the activation of these kinases was checked with phosphospecific antibodies. As shown in Fig. 3C, EVE3 was unable to interfere with activation of these kinases by sorbitol induced osmotic stress. However, SPRED3 was able to reduce JNK activation whilst having no effect on p38 MAPK activation (Fig. 3C). These results were confirmed in another cell line, where transfection of EVE3 in NIH-3T3 cells failed to repress the nuclear accumulation of endogenous activated JNK and activated p38 MAPK in these cells but did inhibit phosphorylation of endogenous ERK1 and ERK2 (Fig. 3D). In contrast, SPRED3 was able to block the nuclear accumulation of phosphorylated JNK in these cells as well as that of ERK1 and ERK2 but had no such effect on p38 MAPK (Fig.3D).

PC12 cell differentiation assay.

For immuno-Iabeling experiments cells were grown on glass coverslips in 12-well or 6-well cell culture dishes. PC12 rat phaeochromocytoma cells were plated onto laminin or poly-l-lysine pretreated coverslips in 24 well plates in DMEM supplemented with 10% horse serum and 5% foetal calf serum and subsequently transfected with various mammalian expression constructs using GenePORTER 2 transfection reagent (Gene Therapy Systems). After transfection the cells were deprived of serum and 100 ng/ml of nerve growth factor (NGF) or fibroblast growth factor (100 ng/ml) was added. The cells were then incubated for 48-72 hours before fixation. Differentiated cells were assessed as those cells that had developed neurite outgrowth greater than 1.5 times the diameter of the cell body.

Differentiation of the rat PC12 phaeochromocytoma cell line into sympathetic-like neurons characterized by the extension of neurite outgrowths is critically dependent on the sustained activation of the Ras/MAPK pathway (Marshall, 1995), with both NGF (Marshall, 1995) and FGF (Hadari et al., 1998) able to induce differentiation of these cells. Therefore, the Applicants wished to determine whether EVE3 and SPRED3 would be able to sustain a differentiation block of PC12 cells and whether the growth factor specific effects we had observed with these factors would be maintained in a differentiation assay. PC12 cells were transfected with EVE3, SPRED3 or SPRED3ΔN and then differentiation of the cells was stimulated with either NGF or FGF. As shown in Fig. 4, EVE3 was able to block NGF mediated differentiation of these cells whilst SPRED3 was unable to do so (Fig. 4A, B). Conversely, EVE3 was unable to interfere with FGF mediated differentiation of these cells whilst SPRED3 was able to block neurite outgrowth induced by this growth factor (Fig. 4C, D). The inhibitory activity of SPRED3 in this assay was dependent on the EVH1 domain with deletion of this region abolishing its ability to block FGF mediated neurite extension (Fig. 4C, D). The same SPRED3ΔN construct had also no capacity to interfere with NGF mediated neurite formation (Fig. 4A, B).

Example 4: EVE3 polypeptides can block cell cycle progression

Activation of the ERK proteins is essential for cell growth and is involved in the regulation of mitosis. Growth factor mediated progression of proliferating cells through the S-phase of the cell cycle can be sensitively monitored by detecting incorporation of the nucleotide analog 5-bromo-2'-deoxyuridine (BrdU) into newly synthesized DNA using anti-BrdU immunofluorescent staining.

EVE3 and SPRED3 were expressed in NIH-3T3 fibroblasts, cells were synchronized in the quiescent GO stage of the cell cycle by 24 hour serum starvation then mitogenically stimulated with serum for 16 hours and tested for BrdU incorporation in transfected cell nuclei. As can be seen in Fig. 5A and B, both EVE3 and SPRED 3 were potent inhibitors of the mitogenic effects of serum on synchronized NIH-3T3 fibroblasts, with EVE3 or SPRED3 expressing cells unable to progress through the G1-S restriction point.

S-phase progression assay.

The S-phase assays utilizing BrdU incorporation were performed using NIH-3T3 cells transfected with expression constructs as indicated and after a 12 hour period were placed in serum free media for 24 hours. Cells were then stimulated with 10% serum for 16 hours, and at this time BrdU (Roche) was added at a concentration of 10 μM. Cells were stained with anti-BrdU antibody (Roche) and other antibodies as indicated. The percentage of FLAG-positive cells that were also positive for BrdU incorporation was determined by counting. Example 5: Expression of human SPRED1 and SPRED2 genes in various tissues.

Northern blot analyses on a variety of tissue samples were used to ascertain the expression profiles of SPRED1 and SPRED2. Poly A+ RNA was isolated from seven different adult rat tissues and probed with SPRED1 and SPRED2 specific cDNA probes. The tissue expression patterns of the SPRED1 and SPRED2 mRNAs were highly correlated, with strong expression in brain for both genes and much weaker expression in testis, spleen and heart (Fig. 7A). A doublet of hybridizing bands was consistently observed with the SPRED1 cDNA probe, which may represent alternatively spliced transcripts for this gene (Fig. 7A). Probing of a rat brain poly A+RNA blot to different brain regions indicated highest SPRED expression in cerebral hemisphere with expression also detectable in cerebellum and olfactory bulb (Fig 7B). To determine the protein expression profiles of the SPRED genes, GST fusion proteins of human SPRED1 and SPRED2 were produced and specific sera to both proteins were obtained. Immunoprecipitation of 9 separate rat whole tissue lysates with SPRED1 and SPRED2 specific antibodies, strikingly, demonstrated detectable expression in only the brain tissue sample (Fig. 7C). To examine whether SPRED1 and SPRED2 expression could be induced by activators of Ras signal transduction NIH-3T3 and HEK 293T cells were serum starved overnight and then stimulated with PDGF or PDBu, with in some cases pretreatment with the MEK1 inhibitor PD 098059, for different time periods then harvested and RNA from the cells probed. Both SPRED1 and SPRED2 mRNA levels were markedly upregulated following stimulation in NIH-3T3 cells (Fig. 7D, lanes 3-6). However, this was not true of all cell types with HEK 293T cells exhibiting only weak induction of the SPRED mRNAs (Fig. 7D). The kinetics of SPRED mRNA induction were analysed with a time course experiment. Serum starved NIH-3T3 cells were stimulated with either PDBu or serum and RNA samples out to 24 hours post-stimulation were prepared. SPRED1 mRNA levels were strongly induced at 2-4 hours post- stimulation and then declined at 8 hours and appeared stable out to 24 hours (Fig. 7E). The kinetics of SPRED1 protein induction in response to serum in the same cells (Fig. 7F) was also examined. SPRED1 levels were first clearly detectable 2 hours post-induction and gradually increased at all time points out to 24 hours (Fig. 7F). This trend was inversely proportional to the kinetics of ERK activation in the same cells with strongest activation at 30 minutes and weakest activation at 24 hours (Fig. 7F). The expression profiles of SPRED1 and SPRED2 in a range of primary human brain tumor samples ranging from benign vestibular schwannoma to non-invasive meningioma to highly malignant glioblastoma multiforme, were examined. SPRED1 levels were uniform in the 8 different vestibular schwannoma samples, but low level and variable in the meningioma and in the glioblastoma samples (Fig 8). In contrast SPRED2 was not detected in the vestibular schwannoma samples, was present at variable levels in the meningioma samples and was virtually absent in all of the glioblastoma patient tissues analyzed (Fig. 8).

Example 6: SPRED1 and SPRED2 block ERK activation induced by a range of stimuli, but differ in the subdomains required to do so. To determine if SPRED1 or SPRED2 were selective in their ability to block Ras/Raf/MEK/ERK pathway signaling induced by certain stimuli, eight different stimuli to induce phosphorylation of transfected ERK2 in serum starved HEK 293 cells, were used. These were PDGF, EGF, basic (b) FGF, hepatocyte growth factor (HGF), NGF, VEGF, serum and PDBu. Full-length SPRED1 and SPRED2 were able to block ERK2 activation in these cells induced by all of these stimuli. SPRED1 and SPRED2 were particularly potent in blocking ERK2 activation downstream of serum, bFGF and HGF, while SPRED2 in addition efficiently blocked activation by EGF, NGF and VEGF (Fig. 9A, B and C). However, a very different result was obtained when the properties of C-terminal truncated mutants of SPRED1 (S1ΔC) and SPRED2 (S2ΔC) and a N-terminal truncation mutant of SPRED2 (S2ΔN) were analyzed. Truncated versions of both proteins lacking the C-terminal conserved Sprouty domain were constructed and tested for their ability to block ERK2 activation. Deletion of this domain destroyed the ability of SPRED2 to block ERK2 activation induced by all of the eight stimuli tested (SPRED2ΔC, Fig. 9B, C and D). In contrast, SPRED1ΔC was effective in blocking ERK2 activation induced by all of the stimuli tested (Fig. 9A and C). The same trend was observed in a Elk-1 transactivation assay, an in vivo readout of endogenous ERK activation (Fig. 9E). Example 7: Some SPRED1 and SPRED2 translocates to the plasma membrane following EGF activation.

NIH-3T3 cells were serum starved overnight then stimulated with EGF for 20 minutes and the localization of the endogenous SPRED1 and SPRED2 proteins in the cells was observed. A small percentage of the cellular SPRED proteins exhibited plasma membrane enrichment of these proteins following EGF treatment in comparison to untreated cells where the expression was predominantly cytoplasmic (Fig 10A). Surprisingly, the SPRED1ΔC mutant which retained the ability to block ERK activation also moved to the plasma membrane following serum stimulation (Fig. 10B). The SPRED2ΔC mutant was unable to translocate to the plasma membrane (data not shown). Following mitogenic stimulation there is a rapid intracellular redistribution of ERK proteins with an accumulation of phosphorylated ERK proteins in the nucleus of NIH-3T3 cells within 10 minutes of stimulation. Activation of the RAS-MAPK pathway is essential for this translocation as blocking MEK activity prevents ERK enrichment in the nucleus. The ability of SPRED proteins to block the nuclear enrichment of phosphorylated ERK was tested by transiently expressing SPRED1 and SPRED2 in NIH-3T3 cells and observing the nuclear accumulation of phosphorylated ERK following serum stimulation. As indicated in Fig. 10C, cells transfected with full- length SPRED proteins consistently failed to translocate endogenous phospho- ERK to the nucleus whilst surrounding non-transfected cells did. Removal of the C-terminal Sprouty domain of both proteins had very different effects on ERK translocation. SPRED2ΔC lost the ability to block ERK translocation, however SPRED1 ΔC retained the ability to inhibit the nuclear accumulation of endogenous ERK as effectively as full-length SPRED1 (Fig. 10C).

Example 8: The Spred induced block of the Ras signaling pathway is not transitory but sustained. To examine the effect of SPRED2 on Ras signaling in HEK 293 cells transfected with SPRED2 and GFP-ERK2, cells were synchronized by serum starvation overnight, then stimulated with serum for various times. SPRED2 was able to block ERK2 activation at all time points analyzed out to 36 hours post stimulation, with SPRED2ΔC unable to block (Fig. 11 ). A similar result was obtained with asynchronous cells, which were not serum starved post-transfection, with the SPRED2 mediated block observed at all time points out to 36 hours (Fig. 11 ).

Example 9: Spred proteins specifically block the Ras/MAPK pathway. To determine whether the Spred proteins could affect three closely related kinase pathways (the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 MAPKs) HEK 293 cells were transfected with FLAG tagged JNK and HA tagged p38 along with SPRED constructs. The activation of these kinases was checked with phospho-specific antibodies. As shown in Fig. 12A and B, neither SPRED1 nor SPRED2 or their C-terminal mutants were able to block activation of these kinases by sorbitol induced osmotic stress. This result was confirmed in another cell line, NIH-3T3 cells where transfection of the same SPRED constructs failed to repress the nuclear accumulation of endogenous activated JNK and activated p38 in these cells (Fig. 12C). SPRED proteins were similarly unable to block Forskolin mediated activation of the CREB transcription factor in PC12 cells (Fig. 12D) but SPRED2 was effective in blocking RSK90 activation in response to serum and bFGF (Fig. 12E).

Example 10: SPRED proteins block NGF mediated neurite formation in PC12 cells.

In order to determine whether SPRED1 , SPRED2 and in particular whether the C-terminal truncated mutant of SPRED1 could block PC12 cell differentiation, similar experiments to those described in Example 3 were performed. As demonstrated in Fig. 13 both wt SPRED proteins efficiently hindered NGF dependent neurite outgrowth in these cells. However in this assay the stark difference in the C-terminal mutants capacity to block Ras/MAPK pathways was confirmed with SPRED1ΔC able to repress neurite formation yet SPRED2ΔC was unable to do so (Fig 13A and B).

Example 11 : SPRED proteins can heterodimerize via their Sprouty domains.

To determine whether the SPRED proteins might interact co-expression of the SPRED 1 and SPRED2 proteins was performed. Co-expression of the SPRED proteins has no obvious modifying effect on their inhibitory activity. However the SPRED2ΔC mutant appeared to have a dominant negative effect on SPRED2 function, with the inhibitory activity of the protein slightly impaired in the presence of the truncated C-terminal mutant (Fig. 15B). Both SPRED1 and SPRED2 are able to form stable heterodimers when coexpressed in cells, with HA tagged SPRED1 able to coimmunoprecipitate with Flag tagged SPRED2 using an anti- SPRED2 specific antibody (Fig 15C). This interaction was mediated via the Sprouty domain with an N-terminal deletion mutant of SPRED2 still able to interact with SPRED1 but the C-terminal deletion mutant, lacking the Sprouty domain, unable to do so (Fig. 15C).

Finally, it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

Claims

I . An isolated polynucleotide encoding a SPRED3 polypeptide.

2. The polynucleotide of claim 1 , wherein the polynucleotide encodes a mammalian SPRED3 polypeptide.

3. The polynucleotide of claim 1 or 2, wherein the polynucleotide encodes a human SPRED3 polypeptide.

4. The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide has a sequence at least 60% identical to that shown in SEQ ID NO:3.

5. The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide has a sequence at least 70% identical to that shown in SEQ ID NO:3.

6. The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide has a sequence at least 80% identical to that shown in SEQ ID NO:3.

7. The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide has a sequence at least 90% identical to that shown in SEQ ID NO:3.

8. The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide has a sequence at least 95% identical to that shown in SEQ ID NO:3.

9. The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide has a sequence at least 98% identical to that shown in SEQ ID NO:3.

10. The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide has a sequence identical to that shown in SEQ ID NO:3.

I I . The polynucleotide of claim 1 or 2, wherein the polynucleotide encodes a mouse SPRED3 polypeptide.

12. The polynucleotide of any one of claims 1 , 2 to 11 , wherein the polynucleotide has a sequence at least 60% identical to that shown in SEQ ID NO:7.

13. The polynucleotide of any one of claims 1 , 2 to 11 , wherein the polynucleotide has a sequence at least 70% identical to that shown in SEQ ID NO:7.

14. The polynucleotide of any one of claims 1 , 2 to 11 , wherein the polynucleotide has a sequence at least 80% identical to that shown in SEQ

ID NO:7.

15. The polynucleotide of any one of claims 1 , 2 to 11 , wherein the polynucleotide has a sequence at least 90% identical to that shown in SEQ ID NO:7.

16. The polynucleotide of any one of claims 1 , 2 to 11 , wherein the polynucleotide has a sequence at least 95% identical to that shown in SEQ ID NO:7.

17. The polynucleotide of any one of claims 1 , 2 to 11 , wherein the polynucleotide has a sequence at least 98% identical to that shown in SEQ ID NO:7.

18. The polynucleotide of any one of claims 1 , 2 to 11 , wherein the polynucleotide has a sequence identical to that shown in SEQ ID NO:7.

19. An isolated polynucleotide encoding an EVE3 polypeptide.

20. The polynucleotide of claim 19, wherein the polynucleotide encodes a mammalian EVE3 polypeptide.

21. The polynucleotide of claim 19 or 20, wherein the polynucleotide encodes a human EVE3 polypeptide.

22. The polynucleotide of any one of claims 19 to 21 , wherein the polynucleotide has a sequence at least 60% identical to that shown in SEQ ID NO:4.

23. The polynucleotide of any one of claims 19 to 21 , wherein the polynucleotide has a sequence at least 70% identical to that shown in SEQ ID NO:4.

24. The polynucleotide of any one of claims 19 to 21 , wherein the polynucleotide has a sequence at least 80% identical to that shown in SEQ ID NO:4.

25. The polynucleotide of any one of claims 19 to 21 , wherein the polynucleotide has a sequence at least 90% identical to that shown in SEQ

ID NO:4.

26. The polynucleotide of any one of claims 19 to 21 , wherein the polynucleotide has a sequence at least 95% identical to that shown in SEQ ID NO:4.

27. The polynucleotide of any one of claims 19 to 21 , wherein the polynucleotide has a sequence at least 98% identical to that shown in SEQ ID NO:4.

28. The polynucleotide of any one of claims 19 to 21 , wherein the polynucleotide has a sequence identical to that shown in SEQ ID NO:4.

29. An isolated polynucleotide that will hybridise with the polynucleotide of any one of claims 1 to 28 under conditions of moderate or high stringency.

30. An isolated SPRED3 polypeptide selected from the group consisting of: i) a polypeptide having an amino acid sequence as shown in SEQ ID NO:5; ii) a polypeptide having an amino acid sequence as shown in SEQ ID

NO:8; iii) a polypeptide having an amino acid sequence at least 60% identical to i) or ii); iv) a polypeptide having an amino acid sequence at least 70% identical to i) or ii); v) a polypeptide having an amino acid sequence at least 80% identical to i) or ii); vi) a polypeptide having an amino acid sequence at least 90% identical to i) or ii); vii) a polypeptide having an amino acid sequence at least 95% identical to i) or ii); viii) a polypeptide having an amino acid sequence at least 98% identical to i) or ii); and ix) fragments and variants of i) to viii); wherein the polypeptide has a SPRED3 function.

31. A polypeptide according to claim 30, wherein the polypeptide is encoded by the polynucleotide of any one of claims 1 to 18.

32. An isolated EVE3 polypeptide selected from the group consisting of: i) a polypeptide having an amino acid sequence as shown in SEQ ID

NO:6; ii) a polypeptide having an amino acid sequence at least 60% identical to i); iii) a polypeptide having an amino acid sequence at least 70% identical to i); iv) a polypeptide having an amino acid sequence at least 80% identical to i): v) a polypeptide having an amino acid sequence at least 90% identical to i); vi) a polypeptide having an amino acid sequence at least 95% identical to i); vii) a polypeptide having an amino acid sequence at least 98% identical to i); and viii) fragments and variants of i) to vii); wherein the polypeptide has an EVE3 function.

33. A polypeptide according to claim 32, wherein the polypeptide is encoded by the polynucleotide of any one of claims 19 to 28.

34. Use of the polypeptide of any one of claims 30 to 33 to modulate a signal transduction pathway.

35. The use of claim 34, wherein the signal transduction pathway is a Ras/MAPK pathway or a c-Jun amino-terminal kinase pathway.

36. Use of the polypeptide of any one of claims 30 to 33 to inhibit cellular differentiation.

37. Use of the polypeptide of any one of claims 30 to 33 to inhibit cell cycle progression.

38. An isolated polypeptide consisting of a first polypeptide sequence and a second polypeptide sequence, wherein the first polypeptide sequence capable of destabilising the second polypeptide, and wherein the first polypeptide sequence is selected from the group consisting of: i) an amino acid sequence 63% identical to an amino acid sequence encoded by exon 5 of an EVE3 gene; ii) an amino acid sequence 70% identical to an amino acid sequence encoded by exon 5 of an EVE3 gene; iii) an amino acid sequence 81% identical to an amino acid sequence encoded by exon 5 of an EVE3 gene; iv) an amino acid sequence 90% identical to an amino acid sequence encoded by exon 5 of an EVE3 gene; v) an amino acid sequence identical to an amino acid sequence encoded by exon 5 of an EVE3 gene; and vi) LSQYFRHMLCP.

39. A host cell genetically engineered to contain a polynucleotide according to any one of claims 1 to 29.

40. A host cell according to claim 39, wherein the host cell is selected from the group consisting of: a mammalian cell, an insect cell, a yeast cell, a fungal cell and a bacterium.

41. A host cell according to claim 39 or 40, wherein the host cell is a bacterium.

42. A host cell according to claim 41 , wherein the bacterium is Escherichia coli.

43. A host cell genetically engineered to express a polypeptide according to any one of claims 30 to 33.

44. A host cell according to claim 43, wherein the host cell is selected from the group consisting of: a mammalian cell, an insect cell, a yeast cell, a fungal cell and a bacterium.

45. A host cell according to claim 43 or 44, wherein the host cell is a bacterium.

46. A host cell according to claim 45, wherein the bacterium is Escherichia coli.

47. A method of identifying an agent capable of modulating an activity of the polypeptide of any one of claims 30 to 33, the method comprising the steps of: contacting the agent and the polypeptide; detecting specific binding of the agent to the polypeptide; and - determining whether the agent binds to the polypeptide.

48. A method of identifying an agonist or an antagonist of the polypeptide of any one claims 30 to 33, the method comprising the steps of: exposing a candidate agonist or antagonist to the polypeptide; and determining whether the candidate agonist or antagonist modulates a biological activity and the polypeptide.

49. An antagonist identified by the method of claim 48.

50. An agonist identified according to the method of claim 48.

51. An isolated antibody which specifically binds to a polypeptide of any one of claims 30 to 33.

52. An isolated antibody according to claim 51 wherein the antibody is selected from the group consisting of: i) polyclonal antibodies; ii) monoclonal antibodies (mAbs); iii) phage display "antibodies"; iv) chimeric antibodies; v) human antibodies; vi) "humanised" antibodies; vii) single chain antibodies; viii) Fab fragments; ix) F(ab')₂ fragments; x) fragments produced by a Fab expression library; xi) anti-idiotypic (anti-Id) antibodies; xii) domain antibodies; and xiii) epitope-binding fragments of any of i) to xii).

53. A composition comprising a pharmaceutically acceptable carrier and any one or more of the following:

) a polypeptide according to any one of claims 30 to 33; i) an agent according to claim 47; ii) an antagonist according to claim 49; v) an agonist according to claim 50; v) an antibody according to claim 51 or 52.

54. Use of the composition of claim 53 to modulate a biological function.

55. The use according to claim 54, wherein the biological function is a cell function.

56. The use according to claim 55, wherein the cell function is selected from the group consisting of cell motility, cell migration, cell proliferation and cell differentiation.

57. The use according to claim 54, wherein the biological function involves a c-kit-binding domain of a polypeptide according to any one of claims 30 to 33.

58. Use of the composition of claim 53 to modulate growth of a tumour.

59. The use according to claim 58, wherein modulation of growth occurs by modulation of stroma.

60. The use according to claim 58, wherein modulation of growth occurs by modulation Ras function.

61. The use according to claim 60, wherein the tumour is a gastrointestinal tumour.

62. The use according to claim 60, wherein the tumour is a pancreatic tumour.

63. The use according to claim 60, wherein the tumour is a stromal tumour.

64. The use according to any one of claims 60 to 63, wherein the tumour has a mutated KIT gene.

65. Use of the composition of claim 53 to enhance wound healing.

66. Use of the composition of claim 53 to inhibit fibrosis.

67. Use of the composition of claim 53 to treat or prevent a liver condition.

68. Use of the composition of claim 53 to regulate liver growth.

69. Use of the composition of claim 53 to inhibit angiogenesis.

70. Use of the composition of claim 53 to treat or prevent a neural condition.

71. Use of the composition of claim 53 to modulate liver growth and/or regrowth by modulating a Ras/MAPK pathway.

72. A method of treating a tumour comprising the step of administering to a patient in need thereof, a therapeutically effective amount of a composition according to claim 53.

73. A method of treating a neural condition comprising the step of administering to a patient in need thereof, a therapeutically effective amount of a composition according to claim 53.

74. A method of treating a liver condition comprising the step of administering to a patient in need thereof, a therapeutically effective amount of a composition according to claim 53.

75. A method of treating a wound comprising the step of administering to a patient in need thereof, a therapeutically effective amount of a composition according to claim 53.

76. A method of regulating liver growth comprising the step of administering to a patient in need thereof, a therapeutically effective amount of a composition according to claim 53.

77. A method of diagnosing a condition in a patient, the method comprising the steps of: obtaining a sample from the patient; and detecting the level of a polypeptide according to any one of claims 30 to 33.

78. The method of claim 77, wherein the step of detecting uses an antibody according to claim 51 or 52.

79. A kit for detecting the presence of a polypeptide according to any one of claims 30 to 33, comprising an antibody according to claim 51 or 52.

80. An isolated oligonucleotide capable of hybridising with a polynucleotide of any one of claims 1 to 29, wherein the oligonucleotide is 15 to 25 nucleotides in length.

81. An oligonucleotide according to claim 80, wherein the oligonucleotide is 15 to 20 nucleotides in length.

82. An oligonucleotide according to claim 80 or 81 , wherein the oligonucleotide consists of deoxyribonucleotides, ribonucleotides or polyamide nucleotides.

83. A genetically modified non-human animal wherein expression of a SPRED3 polypeptide is reduced.

84. A genetically modified non-human animal wherein expression of a SPRED3 polypeptide is increased.

85. A genetically modified non-human animal wherein expression of an EVE3 polypeptide is reduced.

86. A genetically modified non-human animal wherein expression of an EVE3 polypeptide is increased.

87. The non-human animal of any one of claims 83 to 86, wherein the non-human animal is a mouse.

88. An isolated polynucleotide encoding a human SPRED1 polypeptide.

89. The polynucleotide of claim 88, wherein the polynucleotide has a sequence at least 60% identical to that shown in SEQ ID NO:11.

90. The polynucleotide of claim 88, wherein the polynucleotide has a sequence at least 70% identical to that shown in SEQ ID NO:11.

91. The polynucleotide of claim 88, wherein the polynucleotide has a sequence at least 80% identical to that shown in SEQ ID NO:11.

92. The polynucleotide of claim 88, wherein the polynucleotide has a sequence at least 90% identical to that shown in SEQ ID NO:11.

93. The polynucleotide of claim 88, wherein the polynucleotide has a sequence at least 95% identical to that shown in SEQ ID NO:11.

94. The polynucleotide of claim 88, wherein the polynucleotide has a sequence at least 98% identical to that shown in SEQ ID NO:11.

95. The polynucleotide of claim 88, wherein the polynucleotide has a sequence identical to that shown in SEQ ID NO:11.

96. An isolated polynucleotide capable of hybridising with the polynucleotide of any one of claims 88 to 95 under conditions of moderate or high stringency.

97. An isolated oligonucleotide that can specifically bind to a polynucleotide of any one of claims 88 to 95, wherein the oligonucleotide is 15 to 25 nucleotides in length.

98. An oligonucleotide according to claim 97, wherein the oligonucleotide is 15 to 20 nucleotides in length.

99. An oligonucleotide according to claim 97 or 98, wherein the oligonucleotide consists of deoxyribonucleotides, ribonucleotides or polyamide nucleotides.

100. An isolated polynucleotide encoding a human SPRED2 polypeptide.

101. The polynucleotide of claim 100, wherein the polynucleotide has a sequence at least 60% identical to that shown in SEQ ID NO:12.

102. The polynucleotide of claim 100, wherein the polynucleotide has a sequence at least 70% identical to that shown in SEQ ID NO: 12.

103. The polynucleotide of claim 100, wherein the polynucleotide has a sequence at least 80% identical to that shown in SEQ ID NO: 12.

104. The polynucleotide of claim 100, wherein the polynucleotide has a sequence at least 90% identical to that shown in SEQ ID NO: 12.

105. The polynucleotide of claim 100, wherein the polynucleotide has a sequence at least 95% identical to that shown in SEQ ID NO:12.

106. The polynucleotide of claim 100, wherein the polynucleotide has a sequence at least 98% identical to that shown in SEQ ID NO: 12.

107. The polynucleotide of claim 100, wherein the polynucleotide has a sequence identical to that shown in SEQ ID NO:12.

108. An isolated polynucleotide capable of hybridising with the polynucleotide of any one of claims 100 to 107 under conditions of moderate or high stringency.

109. An isolated oligonucleotide that can specifically bind to a polynucleotide of any one of claims 100 to 107, wherein the oligonucleotide is 15 to 25 nucleotides in length.

110. An oligonucleotide according to claim 109, wherein the oligonucleotide is 15 to 20 nucleotides in length.

111. An oligonucleotide according to claim 100 or 101 , wherein the oligonucleotide consists of deoxyribonucleotides, ribonucleotides or polyamide nucleotides.

112. An isolated SPRED1 polypeptide selected from the group consisting of: i) a polypeptide having an amino acid sequence as shown in SEQ ID NO:13; ii) a polypeptide having an amino acid sequence at least 60% identical to i); iii) a polypeptide having an amino acid sequence at least 70% identical to i): iv) a polypeptide having an amino acid sequence at least 80% identical to i); v) a polypeptide having an amino acid sequence at least 90% identical to i): vi) a polypeptide having an amino acid sequence at least 95% identical to i); vii) a polypeptide having an amino acid sequence at least 98% identical to i); and viii) fragments and variants of i) to vii); wherein the polypeptide has a SPRED1 function.

113. A polypeptide according to claim 112, wherein the polypeptide is encoded by the polynucleotide of any one of claims 88 to 95.

114. An isolated SPRED2 polypeptide selected from the group consisting of: i) a polypeptide having an amino acid sequence as shown in SEQ ID

NO:14; ii) a polypeptide having an amino acid sequence at least 60% identical to i); iii) a polypeptide having an amino acid sequence at least 70% identical to i); iv) a polypeptide having an amino acid sequence at least 80% identical to

0; v) a polypeptide having an amino acid sequence at least 90% identical to i); vi) a polypeptide having an amino acid sequence at least 95% identical to i): vii) a polypeptide having an amino acid sequence at least 98% identical to i); and viii) fragments and variants of i) to vii); wherein the polypeptide has a SPRED2 function.

115. A polypeptide according to claim 114, wherein the polypeptide is encoded by the polynucleotide of any one of claims 100 to 107.

116. Use of a polypeptide according to any one of claims 112 to 115 to inhibit cell cycle progression.

117. Use of a polypeptide according to any one of claims 112 to 115 to modulate a signal transduction pathway.

118. The use of claim 117, wherein the signal transduction pathway is a MAPK pathway or a Ras/MAPK pathway.

119. A host cell genetically engineered to contain a polynucleotide according to any one of claims 88 to 95 or claims 100 to 107.

120. A host cell according to claim 119, wherein the host cell is selected from the group consisting of: a mammalian cell, an insect cell, a yeast cell, a fungal cell and a bacterium.

121. A host cell according to claim 119 or 120, wherein the host cell is a bacterium.

122. A host cell according to claim 121 , wherein the bacterium is Escherichia coli.

123. A host cell genetically engineered to express a polypeptide according to any one of claims 112 to 115.

124. A host cell according to claim 123, wherein the host cell is selected from the group consisting of: a mammalian cell, an insect cell, a yeast cell, a fungal cell and a bacterium.

125. A host cell according to claim 123 or 124, wherein the host cell is a bacterium.

126. A host cell according to claim 125, wherein the bacterium is Escherichia coli.

127. A method of identifying an agent capable of modulating an activity of the polypeptide of any one of claims 112 to 115, the method comprising the steps of: contacting the agent and the polypeptide; detecting specific binding of the agent to the polypeptide; and determining whether the agent binds to the polypeptide.

128. A method of identifying an agonist or an antagonist of the polypeptide of any one claims 112 to 115, the method comprising the steps of: exposing a candidate agonist or antagonist to the polypeptide; and determining whether the candidate agonist or antagonist modulates a biological activity and the polypeptide.

129. An antagonist identified by the method of claim 128.

130. An agonist identified according to the method of claim 128.

131. An isolated antibody which specifically binds to a polypeptide of any one of claims 112 to 115.

132. An isolated antibody according to claim 131 wherein the antibody is selected from the group consisting of: i) polyclonal antibodies; ii) monoclonal antibodies (mAbs); iii) phage display "antibodies"; iv) chimeric antibodies; v) human antibodies; vi) "humanised" antibodies; vii) single chain antibodies; viii) Fab fragments; ix) F(ab')2 fragments; x) fragments produced by a Fab expression library; xi) anti-idiotypic (anti-Id) antibodies; xii) domain antibodies; and xiii) epitope-binding fragments of any of i) to xii).

133. A composition comprising a pharmaceutically acceptable carrier and any one or more of the following:

) polypeptide according to any one of claims 112 to 115; i) an agent according to claim 127; ii) an antagonist according to claim 129; iv) an agonist according to claim 130; v) an antibody according to claim 131 or 132.

134. Use of the composition of claim 133 to modulate a biological function.

135. The use according to claim 134, wherein the biological function is a cell function.

136. The use according to claim 135, wherein the cell function is selected from cell motility and cell migration.

137. Use of the composition of claim 133 to modulate growth of a tumour.

138. The use according to claim 137, wherein modulation of growth occurs by inhibition of stroma.

139. The use according to claim 137, wherein modulation of growth occurs by inhibition Ras function.

140. The use according to claim 139, wherein the tumour is a gastrointestinal tumour.

141. The use according to claim 139, wherein the tumour is a pancreatic tumour.

142. Use of the composition of claim 133 to enhance wound healing.

143. Use of the composition of claim 133 to treat or prevent a liver condition.

144. Use of the composition of claim 133 to treat or prevent a neural condition.

145. Use of the composition of claim 133 to modulate brain or nerve cell growth.

146. Use of the composition of claim 133 to modulate liver growth and/or regrowth by modulating a Ras/MAPK pathway.

147. A method of treating a tumour comprising the step of administering to a patient in need thereof, a therapeutically effective amount of a composition according to claim 133.

148. A method of treating a neural condition comprising the step of administering to a patient in need thereof, a therapeutically effective amount of a composition according to claim 133.

149. A method of modulating brain or nerve cell growth comprising the step of administering to a patient in need thereof, a therapeutically effective amount of a composition according to claim 133.

150. A method of treating a liver condition comprising the step of administering to a patient in need thereof, a therapeutically effective amount of a composition according to claim 133.

151. A method of treating a wound comprising the step of administering to a patient in need thereof, a therapeutically effective amount of a composition according to claim 133.

152. A method of diagnosing a condition in a patient comprising the step of detecting in a sample, a polypeptide according to any one of claims 112 to 115.

153. The method of claim 152, wherein the step of detecting uses an antibody according to claim 131 or 132.

154. A kit for detecting the presence of a polypeptide according to any one of claims 112 to 115, comprising an antibody according to claim 131 or 132.