WO1999047676A1 - Human leupaxin polypeptide and dna encoding it, their uses - Google Patents

Abstract

Disclosed are novel leupaxin polypeptides, polynucleotides encoding the polypeptides, expression constructs comprising the polynucleotides, host cell transformed with the polynucleotides, methods to produce the polypeptides, antibodies and binding partners specific for the polypeptides, methods to identify modulators of the polypeptides, and methods to identify modulators of polypeptide expression.

Description

HUMAN LEUPAXIN POLYPEPTIDE AND DNA ENCODING IT, THEIR USES

BACKGROUND OF THE INVENTION

Cell adhesion, spreading, and migration are mediated by integrin interactions with extracellular and cell surface ligands [Gumbiner, Cell, 84:345-357

(1996); Hynes, etal, Cell, 68:303-322 (1992)]. In adherent cell types such as epithelial cells and fibroblasts, readily identifiable complexes of cytoplasmic proteins localize at sites of integrin-dependent close cell contact with substratum. These complexes are designated focal adhesions/contacts and have been implicated in the regulation of cell locomotion, survival and proliferation [Lee, etal, Trends Cell Biol, 3:366-370 (1993);

Ruoslahti, et al, Cell, 77:477-478 (1994)].

Focal adhesions are rich in tyrosine phosphorylated proteins which suggests a role for tyrosine kinases in integrin signaling [Parsons, et al, Curr. Opin. Cell. Biol. 9:187-192 (1997); Miyamoto, et al, J. Cell. Biol. 131:791-805 (1995)]. Protein tyrosine kinases that are found in focal adhesions includes focal adhesion kinase (FAK), src, and src-family kinases. A FAK-related protein, PYK2, has also been identified and designated by various groups as CAKβ [Sasaki, etal, J. Biol. Chem. 270:21206-21219 (1995)], RAFK [Avraham, etal, J. Biol. Chem. 270:27742-27751 (1995)] and CADTK [Yu, etal, J. Biol. Chem., 77:29993-29998 (1996)]. PYK2 and FAK are closely related in overall structure and both are phosphorylated on tyrosine in response to integrin engagement, T cell receptor engagement, or chemokine stimulation. These stimuli all modulate integrin dependent adhesion. PYK2 and FAK both associate with paxillin, pl30cas, and src [Ganju, et al, J. Exp. Med., 185:1055-1063 (1997); Astier, et al, J. Biol. Chem., 272:228-232 (1997)]. Although PYK2 possesses a focal adhesion targeting domain that is highly homologous to the corresponding region in FAK, PYK2 displays a more diffuse cytoplasmic distribution than FAK, with only a small percentage of the protein found in focal adhesions [Matsuya, et al, J. Biol. Chem., 273: 1003-1014 (1998)]. These observations suggest that FAK and PYK2 have both overlapping and distinct functions. *%.

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In addition to FAK and paxillin, tyrosine phosphoproteins present in focal adhesions include vinculin,. zyxin, and the paxillin-like protein Hic-5 [Matsuya, et al, J. Biol. Chem., 273:1003-1014 (1998)]. Phosphorylation of tyrosine on these proteins can regulate interaction with proteins that possess src homology 2 domains (SH2) and phosphotyrosine binding (PTB) domains suggesting that tyrosine kinase activity plays a role in protein-protein interactions in these dynamic focal adhesion complexes [Miyamoto, et al, J. Cell. Biol. 131:791-805 (1995) Van der Gear, et al, Trends Biochem. Sci., 20:277-280 (1995)].

In addition, several focal adhesion proteins possess LIM domains that can mediate interactions with other proteins [Schmeichel, et al, Cell, 79:211-219 (1994)].

LIM domains are approximately 50 residues in length and contain conserved cysteine, histidine, and aspartate residues that form zinc binding modules [Perez- Alvarado, et al., Nat. Struct. Biol. 1:388-398 (1994); Kosa, et al, Biochemistry, 33:468-477 (1994); Michelsen, et al, J. Biol Chem. 269:11108-11113. (1994)]. Paxillin, Hic-5, zyxin, and cysteine-rich protein (CRP) contain a tandem array of three or four LIM domains in the carboxyl-terminal regions. Individual LIM domains demonstrate specificity for binding different proteins or protein motifs. For example, the zyxin LIMl domain supports a binding interaction with CRP [Schmeichel and Beckerle, Cell 79:211-219 (1994)] and paxillin LIM3 has been shown to participate in localization of paxillin to focal adhesions [Brown, et al, J. Cell. Biol. 135: 1109-1123 (1996)]. LIM domains in Enigma, a protein that interacts with the insulin receptor and the receptor tyrosine kinase Ret, bind to specific tyrosine-containing tight-turn motifs [Wu, et al., J. Biol. Chem. 271: 15934-15941 (1996)].

Other short sequences designated leucine-aspartate (LD) motifs in the a ino terminal region of paxillin participate in the binding to FAK and to vinculin [Brown, et al, J. Cell. Biol. 135:1109-1123 (1996)]. Domains of this type invariably include a signature leucine-aspartate dipeptide sequence at the amino terminus and were first characterized in paxillin as thirteen amino acid motifs that participate in specific protein binding. Paxillin regions LD2 and LD3 have been implicated in binding to FAK to localize the protein at focal adhesions, and domain LD2 is believed to mediate paxillin binding to vinculin. FAK and vinculin regions designated PBS participate in binding with paxillin LD - 3 - motifs [Tachibana, J. Exp. Med. 182:1089-1099 (1995)]. In paxillin, the LD regions appear to participate in localization of FAK at focal adhesions. Thus focal adhesion proteins such as paxillin contain multiple binding domains and likely serve as scaffolds to localize and regulate specific effector molecules to a subcellular site. Thus there exists a need in the art to identify proteins which mediate integrin binding and, in turn, modulate cell adhesion, spreading, and migration.

BRIEF SUMMARY OF THE INVENTION

In brief, the present invention provides polypeptides and underlying polynucleotides for a novel family of proteins designated leupaxins. The invention includes both naturally occurring and non-naturally occurring leupaxin polynucleotides and polypeptide products thereof. Naturally occurring leupaxin products include distinct gene and polypeptide species within the leupaxin family; these species include those which are expressed within cells of the same animal as well as corresponding species homologs expressed in cells of other animals. Within each leupaxin species, the invention further provides splice variants encoded by the same polynucleotide but which arise from distinct mRNA transcripts. Non-naturally occurring leupaxin products include variants of the naturally occurring products such as analogs (i.e., wherein one or more amino acids are added, substituted, or deleted) and those leupaxin products which include covalent modifications (i.e., fusion proteins, glycosylation variants, Met^'Meupaxin,

Met^-Lys^' eupaxin, Gly^' eupaxin and the like). The leupaxin family of proteins is distinguished from previously known localization families of proteins in that leupaxins include distinct amino acid sequences which suggest interaction with unique ligands as well as distinct modes of regulation. In a preferred embodiment, the invention provides a polynucleotide comprising the sequence set forth in SEQ ID NO: 1. The invention also embraces polynucleotides encoding the amino acid sequence set out in SEQ ID NO: 2. A presently preferred polypeptide of the invention comprises the amino acid sequence set out in SEQ ID NO: 2.

The present invention provides novel purified and isolated polynucleotides (e.g., double stranded and single stranded DNA sequences and RNA transcripts, both sense and complementary antisense strands, including splice variants thereof) encoding the - 4 - human leupaxins. DNA sequences of the invention include genomic and cDNA sequences (double stranded and single stranded sequences) as well as wholly or partially chemically synthesized DNA sequences. "Chemically synthesized," as used herein and is understood in the art, refers to polynucleotides produced by purely chemical, as opposed to enzymatic, techniques. "Wholly" synthesized DNA sequences are therefore produced entirely by chemical means, and "partially" synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means. A preferred DNA sequence encoding a human leupaxin polypeptide is set out in SEQ ID NO: 1. The worker of skill in the art will readily appreciate that the preferred DNA of the invention comprises a double stranded molecule, for example the molecule having the sequence set forth in SEQ ID NO: 1 along with the complementary molecule (the "non-coding strand" or "complement") having a sequence deducible from the sequence of SEQ ID NO: 1 according to Watson-Crick base paring rules for DNA. Also preferred are polynucleotides encoding the leupaxin polypeptide of SEQ ID NO: 2. The invention further embraces species, preferably mammalian, homologs of the preferred human leupaxin DNA.

The invention also embraces DNA sequences encoding leupaxin species which hybridize under moderately stringent conditions to the complete non-coding strand (complement) or distinct regions thereof, of the polynucleotide in SEQ ID NO: 1. DNA sequences encoding leupaxin polypeptides which would hybridize thereto but for the redundancy of the genetic code are contemplated by the invention. Exemplary moderate hybridization conditions are as follows: hybridization at 60°C in 5X SSC, and washing at 60°C in IX SSC. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described in Ausebel, et al. (Eds.), Protocols in Molecular Biology. John Wiley & Sons (1994), pp.

6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, etal, (Eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51. *_*.

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Autonomously replicating recombinant expression constructions such as plasmid and viral DNA vectors incorporating leupaxin sequences are also provided. Expression constructs wherein leupaxin-encoding polynucleotides are operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided.

According to another aspect of the invention, host cells are provided, including procaryotic and eukaryotic cells, either stably or transiently transformed or transfected with DNA sequences of the invention in a manner and under conditions which permits expression of leupaxin polypeptides of the invention. Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with leupaxin. Host cells of the invention are also conspicuously useful in methods for large scale production of leupaxin polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown by, for example, immuno affinity purification, gel permeation chromatography, ion exchange chromatography, gel electrophoresis, Western blotting, immunoprecipitation, or any of a number of other purification techniques well known and routinely practiced in the art. Purification techniques for isolating leupaxin polypeptides of the invention can be employed alone or in combination. Knowledge of leupaxin DNA sequences allows for modification of cells to modulate, increase or decrease, expression of endogenous leupaxin in host cell which naturally include polynucleotides that encode leupaxin. Cells can be modified (e.g., by homologous recombination) to provide modified leupaxin expression by replacing, in whole or in part, the naturally occurring leupaxin promoter with all or part of a heterologous promoter so that the cellular expression of leupaxin occurs at higher or lower levels. The heterologous promoter is inserted in such a manner that it is operatively-linked to leupaxin encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. 91/09955. The invention also contemplates that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate - 6 - transcarbamylase, and dihydroorotase) and or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the leupaxin coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the expression product of the leupaxin coding sequences in the cells. The DNA sequence information provided by the present invention also makes possible the development through, e.g. homologous recombination or "knock-out" strategies [Capecchi, Science 244: 1288-1292 (1989)], of animals that fail to express functional leupaxin or that express a variant of leupaxin. Such animals are useful as models for studying the in vivo activities of leupaxin and modulators of leupaxin. The invention also provides purified and isolated mammalian leupaxin polypeptides. Presently preferred is a leupaxin polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2. Leupaxin polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications

(e.g., glycosylation, truncation, lipidation, ubiquitination, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Leupaxin products of the invention may be full length polypeptides, biologically active fragments, or variants thereof which retain specific leupaxin biological or immunological activity. Variants may comprise leupaxin polypeptide analogs wherein one or more of the specified (t.e., naturally encoded) amino acids is deleted or replaced or wherein one or more non-specified amino acids are added: (1) without loss of one or more of the biological activities or immunological characteristics specific for leupaxin; or (2) with specific disablement of a particular biological activity of leupaxin. Fragment leupaxin polypeptides of the invention include specific protein binding domains including regions that participate in cytoplasmic localization. Presently preferred polypeptide fragments include regions comprising LD and LIM domains of the polypeptide set out in SEQ ID NO:2. LD domain fragments of leupaxin are exemplified by polypeptides comprising amino acid residues 4 through 15, 40 through 51, 93 through 104, and 128 through 139 as set out in SEQ ID NO: 2, as well as corresponding LD regions in other leupaxin polypeptides embraced by the invention. LIM domain fragments of leupaxin are - 7 - exemplified by polypeptides comprising amino acid residues 152 through 202, 211 through 261, 270 through 320, and 329 through 379 as set out in SEQ ID NO: 2, as well as corresponding LIM regions in other leupaxin polypeptides embraced by the invention.

Variant products of the invention include mature leupaxin products as well as leupaxin products including additional amino terminal residues. Leupaxin products having an additional methionine residue at position -1 (Met^"1 -leupaxin) are contemplated, as are leupaxin products having additional methionine and lysine residues at positions -2 and -1 (Met^-Lys^' eupaxin). Also contemplated are leupaxin products having multiple Met-Lys additional residues, in addition to other additional sequences which permit enhanced expression and/or recovery of leupaxin products of the invention. Variants of these types are particularly useful for recombinant protein production in bacterial cell types.

The invention also embraces leupaxin variants having additional amino acid residues which result from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide such as a glutathione-S-transferase (GST) fusion product provide the desired polypeptide having an additional glycine residue at position -1 as a result of cleavage of the GST component from the desired polypeptide. Variants which result from expression in other vector systems are also contemplated. The invention further embraces leupaxin products modified to include one or more water soluble polymer attachments. Particularly preferred are leupaxin products covalently modified with polyethylene glycol (PEG) subunits. Water soluble polymers may be bonded at specific positions, for example at the amino terminus of the leupaxin products, or randomly attached to one or more side chains of the polypeptide. Also comprehended by the present invention are antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, and the like) and other binding proteins specific for leupaxin products or fragments thereof. The term "specific for" indicates that the variable regions of the antibodies of the invention recognize and bind leupaxin polypeptides exclusively (i.e., able to distinguish specific leupaxin polypeptides from the family of leupaxin polypeptides despite sequence identity, homology, or similarity found - 8 - in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual: Cold Spring Harbor Laboratory; Cold Spring Harbor , NY (1988), Chapter 14. Antibodies that recognize and bind fragments of the leupaxin polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, leupaxin polypeptides. As with antibodies that are specific for full length leupaxin polypeptides, antibodies of the invention that specifically recognize leupaxin fragments are those which can distinguish specific leupaxin polypeptides from the family of leupaxin polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins. Specific binding proteins can be developed using isolated or recombinant leupaxin products, leupaxin variants, or cells expressing a modified leupaxin product such that the antigenic leupaxin product is expressed on the cells surface. Binding proteins are useful for purifying leupaxin products and detection or quantification of leupaxin products in fluid and tissue samples using known immunological procedures. Binding proteins are also manifestly useful in modulating (t^'.e., blocking, inhibiting, or stimulating) biological activities of leupaxin, especially those activities involved in signal transduction. Anti-idiotypic antibodies specific for anti-leupaxin antibodies are also contemplated.

The scientific value of the information contributed through the disclosures of DNA and amino acid sequences of the present invention is manifest. As one series of examples, knowledge of the sequence of a cDNA for leupaxin makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding leupaxin and leupaxin expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like. DNA DNA hybridization procedures carried out with DNA sequences of the invention under moderately stringent conditions are likewise expected to allow the isolation of DNAs encoding allelic variants of leupaxin; allelic variants are known in the art to include - 9 - structurally related proteins sharing one or more of the biochemical and/or immunological properties specific to leupaxin. Similarly, non-human species genes encoding proteins homologous to leupaxin can also be identified by Southern and/or PCR analysis and useful in animal models for leupaxin-related disorders. As an alternative, complementation studies can be useful for identifying other human leupaxin products as well as non-human proteins, and DNAs encoding the proteins, sharing one or more biological properties of leupaxin.

Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express leupaxin. Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in a leupaxin locus that underlies a disease state or states.

Also made available by the invention are anti-sense polynucleotides which hybridize to polynucleotides encoding leupaxin. Full length and fragment anti-sense polynucleotides are provided. The worker of ordinary skill will appreciate that fragment anti-sense molecules of the invention include (i) those which specifically recognize and hybridize to leupaxin DNA (as determined by sequence comparison of DNAs encoding leupaxin to DNA encoding other known molecules) as well as (ii) those which recognize and hybridize to DNA encoding other members of the leupaxin family of proteins. Antisense polynucleotides that hybridize to multiple DNA encoding other members of the leupaxin family of proteins are also identifiable through sequence comparison to identify characteristic, or signature, sequences for the leupaxin family of molecules. Anti-sense polynucleotides are particularly relevant to regulating expression of leupaxin by those cells expressing leupaxin mRNA.

The DNA and amino acid sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of leupaxins. DNA and amino acid sequence information for leupaxin also permits identification of binding partner compounds with which a leupaxin polypeptide or polynucleotide will interact. Agents that modulate (i.e., increase, decrease, or block) leupaxin activity or expression may be identified by incubating a putative modulator with a leupaxin polypeptide or polynucleotide and determining the effect of the putative modulator on leupaxin activity or expression. The selectivity of a compound that - 10 - modulates the activity of the leupaxin can be evaluated by comparing its binding and/or modulating, activity on leupaxin to its binding and/or modulating activity on other proteins. Cell based methods, such as di-hybrid assays to identify DNAs encoding binding compounds and split hybrid or reverse di-hybrid assays to identify inhibitors of leupaxin polypeptide interaction with a known binding polypeptide, as well as in vitro methods to identify both known and heretofore unknown binding ligands, including assays wherein a leupaxin polypeptide, leupaxin polynucleotide, or a binding partner thereof is immobilized, and solution assays are contemplated by the invention.

Selective modulators may include, for example, antibodies and other proteins or peptides which specifically bind to a leupaxin polypeptide or a leupaxin-encoding nucleic acid, oligonucleotides which specifically bind to a leupaxin polypeptide or a leupaxin-encoding gene sequence, and other non-peptide compounds (e.g., isolated or synthetic organic and inorganic molecules) which specifically interact with a leupaxin polypeptide or underlying nucleic acid. Mutant leupaxin polypeptides which affect the enzymatic activity or cellular localization of the wild-type leupaxin polypeptides are also contemplated by the invention. Presently preferred targets for the development of selective modulators include, for example: (1) regions of the leupaxin polypeptide which contact other proteins and/or localize the leupaxin polypeptide within a cell, (2) regions of the leupaxin polypeptide which bind substrate, (3) allosteric regulatory binding sites of the leupaxin polypeptide, (4) phosphorylation site(s) of the leupaxin polypeptide and (5) regions of the leupaxin polypeptide which are involved in multimerization of leupaxin subunits. Still other selective modulators include those that recognize specific leupaxin-encoding and regulatory polynucleotide sequences. Modulators of leupaxin activity may be therapeutically useful in treatment of a wide range of diseases and physiological conditions in which leupaxin biological activity is known to be involved. - 11 -

DETAILED DESCRIPTION OF THE INVENTION

The present invention is illustrated by the following examples. Example 1 describes isolation of a cDNA encoding leupaxin and the encoded leupaxin polypeptide encoded is characterized in Example 2. Example 3 describes Northen analysis of leupaxin expression. Example 4 characterizes subcellular localization of leupaxin in different cell types. Example 5 demonstrates that leupaxin is a tyrosine kinase substrate. Example 6 relates to identification of a putative binding partner of leupaxin using immunoprecipitation. Example 7 relates to leupaxin participation in chemotaxis.

Example 1

Isolation of Leupaxin cDNA

In order to identify novel proteins expressed by macrophages, a random sequencing screen of a human macrophage cDNA library was carried out. Initially, a unique 1.2 kb clone cDNA was identified encoding an incomplete coding region homologous to the 3' region of paxillin. The sequence of the 5' terminus for the 1.2 kb clone is set out in SEQ ID NO: 3, and the sequence for the 3 ' terminus is set out in SEQ ID NO: 8. The In an attempt to identify the full length clone, the 1.2 cDNA was used as a probe to screen a spleen cDNA library as follows.

The 1.2 kb insert was labeled with ³²P using a Random Primed Labeling Kit (Boehringer Mannheim) and used as a hybridization probe to screen oligo(dT)-primed double-stranded cDNA prepared from poly(A)⁺ RNA isolated from normal human spleen. The library was constructed by adding BstXl linkers to isolated cDNA and cloning the resulting cDNA into the vector pcDNAl/Amp (InVitrogen). Labeled probe was added to colony replicas prepared by standard techniques in hybridization buffer (5X SSC, 5X Denhardt's, 1% SDS and 45% formamide) and hybridization was carried out overnight at 42°C. The final wash in buffer containing 0.5X SSC and 0.1% SDS was carried out at 50 °C.

Two 1.9 kb cDNAs were isolated, sequenced, and found to be identical over both complete coding regions. Each clone contained an open reading frame encoding 385 amino acid residues and a 5' translational start codon in the context of a consensus KOZAK sequence. In a BLASTP search of the National Center for Biotechnology Information (NCBI) database, the deduced amino acid sequence for the - 12 - clone was found to be most homologous to paxillin. Because the protein sequence appeared to be related to paxillin and expressed preferentially in leukocytes (discussed below), it was designated leupaxin. The polynucleotide and amino acid sequences of leupaxin are set out in SEQ ID NOs: 1 and 2, respectively.

Example 2 Characterization of Leupaxin

The overall amino acid sequence identity between leupaxin and paxillin was determined to be 37%, however, the carboxy terminal regions of the proteins, leupaxin residues 151-385, showed 70% identity and 80% similarity. The conserved region common to leupaxin and paxillin was found to contain four LIM domains; homology between the four domains ranged from 67% to 76% identity. Leupaxin LIM domains contain two zinc finger motifs with a consensus sequence. As discussed supra, LIM domains have been implicated in protein binding and/or localization and in paxillin, LIM3 has been shown to mediate localization to focal adhesions and LIM2 appears to cooperate in this localization [Brown, et al., J. Cell Biol. 135:1109-1123 (1996)]though the focal adhesion ligand for paxillin LIM3 has not yet been identified. In view of the sequence similarity between leupaxin and paxillin, leupaxin LIM domains may also function in localization to focal contacts. The amino-terminal region of leupaxin is shorter than the corresponding region in paxillin and exhibits low sequence homology except for three short regions of approximately thirteen amino acids. Residues 1-15, 85-102 and 127-149 of leupaxin as defined in SEQ ID NO: 2 share 53%, 56% and 63% identity with the corresponding regions in paxillin. When conservative substitutions are taken into consideration, similarity between the sequences is 90%, 72% and 75%. These regions in paxillin are designated LD sequences because each contains the characteristic leucine and aspartate dipeptide pair near the amino terminus [Brown, et al., J. Cell Biol. 135:1109-1123 (1996)]. The leupaxin LD sequences align with regions in paxillin designated LD1, LD3 and LD4. The same three leupaxin LD regions can also be aligned with the LD regions identified in Hic-5, which like paxillin, has four identifiable LD domain. Leupaxin also includes a potential fourth LD motif at residues 39-51 that contains three invariable residues found in the LD2 domains of both paxillin and Hic-5. This additional potential - 13 -

LD domain is more closely related to the Hic-5 sequence, showing little homology to paxillin LD2. In the absence of a paxillin LD2 motif, leupaxin binding to either FAK or vinculin would therefore be predicted to differ from that of paxillin. Consistent with this prediction, preliminary results have failed to identify FAK in leupaxin immunoprecipitates from lymphoid cells. It is likely that leupaxin may interact with one or more other cytoplasmic proteins, possibly including other paxillin ligands such as src [Glenney, et al., J. Cell Biol. 108:2401-2408 (1989)], Csk [Sabe, et al., Proc. Natl Acad. Sci. USA. 91:3984-3988 (1994)], Lyn [Minoguchi, et al., Mol Immunol. 31:519-529 (1994)], crk [Birge, et al., Mol. Cell. Biol. 13:4648-4656 (1993)] and or talin, [Turner, et al, J. Cell. Biol. 111:1059-1068 (1990)].

In view of the known interactions between paxillin and other cytoplasmic proteins through LIM and LD domain sequences, and the similar sequences found in leupaxin, it is likely that leupaxin may also serve an adapter function and localize cytoplasmic molecules to specific subcellular locations. Example 3

Northern Analysis of Leupaxin Expression

In order to determine the range of cell types and tissues that express leupaxin, leupaxin cDNA was used to probe blots of mRNA isolated from various sources. A probe labeled by random priming containing the entire leupaxin coding region was used to hybridize to Human Cancer Cell Line, Human Immune System Northern and Human Multiple Tissue Northern blots (Clontech). Northern blots were hybridized at 68 °C for 1 hour in ExpressHyb Solution (Clontech) and blots were washed to a final stringency of 0.1X SSC / 0.1% SDS at 50°C. Consistent with the size of the leupaxin cDNA, the probe hybridized to a

2.4 kb mRNA present in lymphoid tissues including spleen, lymph node, thymus, and appendix. Markedly less leupaxin mRNA was detected in lung, bone marrow, fetal liver, and pancreas, and virtually none detected in heart, brain, placenta, adult liver, skeletal muscle, and kidney. Leupaxin mRNA was also detected in peripheral blood lymphocytes and the hematopoietic cell lines HL60, Molt4, and Raji cells, and to a lesser extent in

K562 cells. Leupaxin mRNA in four different epithelial cell lines was detected at levels similar to that observed in K652 cells. **_*.

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Leupaxin mRNA levels therefore appeared to be markedly higher in lymphoid tissues and certain hematopoietic cell lines relative to non-hematopoietic cell types. In addition, bone marrow cells appeared to contain low levels of leupaxin mRNA relative to lymphocytes and lymphoid tissues suggesting that leupaxin expression in hematopoietic cells may increase with differentiation.

Example 4 Expression and Subcellular Localization of Leupaxin

In order to further characterize leupaxin, and more particularly to determine subcellular localization of the protein, leupaxin was expressed as an enhanced green fluorescent protein (EGFP) chimeric protein in a lymphoblastoid line, JY8, and CHO fibroblast cells.

The entire leupaxin coding region was ligated in reading frame to the 3 ' terminus of the EGFP coding sequence previously isolated from the vector pEGFP-Cl (Clontech). The resulting EGFP-leupaxin chimeric DNA was inserted into vector pCEP4

(in Vitrogen, Carlsbad, CA) and the resulting plasmid, pEGFP-PX2-CEP4, transfected by electroporation into either a JY cell line previously transfected to stably express the IL-8 receptor or CHO cells. Transfectants were selected by culturing in media containing 0.5 mg/ml of Hygromycin B. EGFP-leupaxin JY8 transfectants were placed on coverslips coated with ICAM-1-Ig and allowed to adhere for 45 minutes at 37°C. Bound cells were fixed in 3% paraformaldehyde, washed with Dulbeccos PBS and then counterstained with rhodamine phalloidin (Molecular Probes) for 30 minutes at room temperature. Coverslips were washed with D-PBS and mounted in N-propyl-gallate (NPG). Cells were visualized with Deltavision using a CCD camera to detect images through a Zeiss Axiovert microscope. Image blur is corrected computationally using constrained iterative deconvolution algorithms. To facilitate determination of leupaxin localization in JY8 cells, the assay was carried out with cells that were adherent to, and spread on, an ICAM-1 coated substrate. ICAM-1 is a ligand for the only β₂ integrin α_Lβ₂ expressed in JY8 cells.

In JY8 cells, leupaxin was found to be diffusely distributed in the f-actin rich cortical cytoskeleton that stains with rhodamine-phalloidin and in a region adjacent to the f-actin rich cortical cytoplasm. Leupaxin was also detected in proximal regions of filipodia-like projections, but was excluded, or at least detected at a much lower level, in *_*.

- 15 - the distal tips of the projections. In general, GFP-leupaxin was diffusely distributed throughout the nucleus and cytoplasm. In addition, it was unclear if leupaxin was present in adherent cell focal adhesions. Because it has previously been suggested that focal adhesions in motile cells, such as leukocytes, are relatively diffuse, much smaller, greater in number, and more uniformly distributed than in adherent cells, association of leupaxin with focal adhesions cannot be ruled out on the basis of heterologous expression alone. In a second experiment, using a non-hematopoietic cell type, DG44 CHO cells (ATCC) were transfected with pEGFP-PX2-CEP4 by electroporation and stable expressing cells were selected in hygromycin B (700 μg/ml). Coverslips were coated overnight with 0.1 mg/ml human fibronectin (Sigma) at 4°C. Cells were trypsinized and plated in serum-free medium on coated coverslips. Sixteen hours later, cells were fixed in 3.7% paraformaldehyde in Dulbeccos phosphate-buffered saline (PBS) for 8 minutes, rinsed in PBS, and permeabilized for 2 minutes in CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 0.5 % Triton X-100, 10 mM Pipes pH 6.8). The coverslips were then incubated at room temperature with the anti-phosphotyrosine antibody py20 at 10 μg/ml (Transduction Labs) for 45 minutes, rinsed in PBS and incubated with lissamine- conjugated F(ab₂') goat-anti-mouse antibody at 10 μg/ml (Jackson Labs) for 45 minutes, rinsed in PBS, and visualized by confocal microscopy using a BioRad confocal microscope station. In CHO cells, EGFP-leupaxin localized to distinct foci, which, like focal adhesions, are enriched with vinculin and proteins phosphorylated on tyrosine residues.

Overall, these results indicate that in nonhematopoietic CHO fibroblasts, the leupaxin-GFP fusion protein localized to focal contacts, while in the lymphoid cells, protein localization was relatively diffuse. These observations are consistent with previous reports suggesting relatively diffuse adhesive sites in lymphoid cells and discrete focal contacts in non-hematopoietic cells [Kolega, et al., J. CellSci. 54:23-34 (1982)].

While it is unclear which sequences in leupaxin participate in localization, one LIM domain in leupaxin is approximately 70% identical to paxillin LIM3 at the amino acid level. Because LIM3 appears to be essential for paxillin localization to focal contacts [Brown, et al., J. Cell Biol. 135:1109-1123 (1996)], it is possible that leupaxin localization is mediated through the corresponding homologous sequence and possibly *_*.

- 16 - through interaction with the same, or a similar, ligand that mediates localization of paxillin. To date, no paxillin ligand that localizes the protein to focal contacts has been identified, but candidate ligands could include proteins having LIM-interacting motifs, proteins with other LIM domains, or integral membrane proteins with tyrosine containing tight turn motifs [Wu and Gill, J. Biol. Chem. 269:25085-25090 (1994)].

Example 5 Detection of Tyrosine Phosphorylated Leupaxin

In order to determine if leupaxin is a tyrosine kinase substrate, the EGFP- leupaxin chimera was expressed in JY8 cells, immunoprecipitated from cell lysate using anti-EGFP polyclonal antisera, and tested for reactivity with a phosphotyrosine specific monoclonal antibody. The rationale for this examination was based on the knowledge that (i) tyrosine phosphorylated proteins such as paxillin concentrate in focal adhesions and likely to mediate signaling following integrin engagement, (ii) leupaxin contains at least ten potential tyrosine phosphorylation sites, and (iii) leupaxin was shown above to localize with tyrosine phosphorylated proteins in focal adhesions in nonlymphoid CHO cells.

EGFP-leupaxin protein was immunoprecipitated from JY8 transfectants (expressed as described in Example 4) using EGFP polyclonal antisera (Clontech). Approximately 40 x 10⁶ cells were lysed in 1.5 ml 1% CHAPS lysis buffer containing 0.01 mg/ml each soybean trypsin inhibitor (SBTI), aprotinin, and leupeptin, 1 mM 4-(2- aminoethyl) benzenesulfonyl fluoride (AEBSF), and 2 mM Na₃VO₄. Immunoprecipitated

EGFP and EGFP-leupaxin were separated on a 12% Tris-glycine Novex gel and transferred to a PVDF membrane by standard techniques. Tyrosine phosphorylation was determined by Western blotting with the anti-phosphotyrosine monoclonal antibody RC20H (Transduction Labs). Phenylphosphate (0.5 mM) was preincubated with RC20H for 40 minutes at 4°C in inhibition studies. As a negative control, EGFP was analyzed in parallel.

The EGFP-leupaxin fusion migrated as a 78 kD protein whereas EGFP migrated at approximately 33 kD. The difference in observed molecular weight (45 D) was consistent with the predicted size of leupaxin. The EGFP-leupaxin band bound the anti-phosphotyrosine antibody and the binding was inhibited with phenylphosphate confirming specificity. No antibody binding was detected with EGFP alone, suggesting that leupaxin is a tyrosine kinase substrate. *_*

- 17 -

The results suggest that leupaxin function may be regulated by a tyrosine kinase activity which is consistent with the previous demonstration that paxillin is phosphorylated on tyrosine and that phosphorylation can be induced with cell adhesion or engagement of integrins with monoclonal antibodies [Turner and Miller, J. Cell Sci. 107:1583-1591 (1994); Burridge, et al., J. Cell Biol. 119:893-903 (1992); and Fuortes, et al., J. Cell Biol. 127:1477-1483 (1994)], cell transformation [Glenney and Zokas, J. Cell Biol. 108:2401-2408 (1989)], and in response to mitogens and signaling through G- protein coupled seven transmembrane receptors [Fuortes, et al., J. Cell Biol. 127:1477- 1483 (1994); Zachary, et al., J. Biol Chem. 268:22060-22065 (1993)]. Thus tyrosine phosphorylation of paxillin occurs in the induction and process of cell adhesion, motility, and growth.

Example 6 Co-immunoprecipitation of PYK2 and Leupaxin

In order to assess the possible association of leupaxin with PYK2, proteins in cell lysate from JY8 cells transformed as described above were immunoprecipitated using anti-PYK2 polyclonal antisera.

Briefly, JY8 GFP or GFP-leupaxin transfectants (20 x 10⁶ cells) were lysed in 1 ml 1% CHAPS lysis buffer containing protease inhibitors (complete-EDTA inhibitor tablet, Boehringer Mannheim) and 1 ml Na₃VO₄. Approximately 5 μg anti-PYK2 polyclonal antibody was added and bound proteins immunoprecipitated according to standard techniques. Proteins were also immunoprecipitated using anti-GFP polyclonal antisera (Clontech). Immunoprecipitated proteins were separated by electrophoresis using a 12% Tris-glycine (Novex) gel and following resolution, proteins were transferred to a PVDF membrane. Western blottting was carried out by standard techniques using an anti- GFP monoclonal antibody (Clontech) or an anti-PYK2 monoclonal antibody, P47120

(Transduction Labs) to detect precipitated proteins.

Immunoblots demonstrated that EGFP-leupaxin co-immunoprecipitated with PYK2 from the JY8 lysate using anti-PYK2 antibody. The association appeared to be specific for leupaxin as GFP did not co-immunoprecipitate with PYK2 although similar amounts of PYK2 were immunoprecipitated from both lysates. In the reciprocal experiment, PYK2 was found to co-immunoprecipitate with GFP-leupaxin using the anti- GFP monoclonal antibody. The association was specific for leupaxin as no PYK2 was - 18 - precipitated with GFP although equal amounts of GFP and GFP-leupaxin were immunoprecipitated from the two lysates. Control antibodies did not permit precipitation of either PYK2 or leupaxin.

The results suggest that leupaxin/PYK2 association may occur in leukocytes. PYK2 is in the FAK family of proteins and is expressed preferentially in leukocytes. Activity of PYK2 can be postulated to overlap those known for FAK, in particular, activities such as cell motility, spreading, and apoptosis. Thus, leupaxin may serve to localize PYK2 to various subcellular sites to support and modulate these biological activities. Leupaxin may therefore modulate PYK2 activity and serve a regulatory function in integrin-mediated or G protein coupled receptor transmembrane signaling.

Example 7 Leupaxin Participation in Chemotaxis In order to identify a role for leupaxin in chemotaxis, a leupaxin amino terminal fragment including LD motifs and a carboxy terminal fragment containing LIM domains were separately expressed in JY8 as GFP fusion proteins.

Briefly, leupaxin fragments were expressed in the pEGFP-CEP4 expression vector. DNA encoding the leupaxin domain from amino acid residue 2 to 150 (as set out in SEQ ID NO: 2) was amplified using PCR using the full length leupaxin clone as template DNA in a reaction with primers as set out in SEQ ID NOs: 4 and 5.

ATATCTCGAGAAGAGTTAGATGCCTTATTGG SEQ ID NO: 4

ATATAAGCTTTCAGCCCTTGGGCACTGTGG SEQ ID NO: 5

PCR conditions included 30 cycles of denaturation at 92° C for 0.5 minutes, annealing at 42°C for 0.5 minutes, and extension at 72°C for 0.5 minutes. The resulting amplification product was digested with Xhόl and H dIII and inserted into the expression vector previously digested with the same enzymes to give plasmid pEGFP-LD-CEP4. DNA encoding the leupaxin LIM domain fragment from amino acid residue

145 to 386 was also amplified using PCR with the full length leupaxin cDNA as template and primer as set out in SEQ ID NOs: 6 and 7. - 19 -

ATATCTCGAGCCACAGTGCCCAAGGGCC SEQ ID NO: 6

ATATAAGCTTTTACAGTGGGAAGAGCTT SEQ ID NO: 7

Reactions conditions included 30 cycles of denaturation for 30 seconds at 92 °C, annealing for 30 seconds at 40 °C, and extension for 30 seconds at 72 °C. The amplification product was digested with Xhol and Hindlll and the resulting DNA inserted in the expression vector previously digested with the same enzymes. The plasmids, pEGFP-CEP4, pEGFP- PX2-CEP4, pEGFP-LD-CEP4 and pEGFP-LIM-CEP4 were separately transfected into JY8 cells and transformants selected using media containing 0.5 mg/ml hygromycin. Expression of the individual fusion proteins was confirmed using FACS analysis and

Western blotting using an anti-GFP monoclonal antibody (Boerhinger Mannheim). JY8 cells found to overexpress leupaxin or a leupaxin fragment were assayed for chemotactic movement toward IL-8 on surfaces coated with ICAM-1, VCAM-1 or Vitronectin.

In the results from three assays, JY8 transfectants expressing the amino terminal leupaxin fragment demonstrated approximately 50-300% greater migration on surfaces with immobilzed VCAM-1 compared to cells expressing GFP alone suggesting a role for leupaxin in chemotaxis.

While the present invention has been described in terms of specific embodiments, it is understood that variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the appended claims should be placed on the invention.

Claims

- 20 - What is claimed is:

1. A purified and isolated leupaxin polypeptide.

2 The polypeptide according to claim 1 comprising the leupaxin amino acid sequence set out in SEQ ID NO: 2.

3. A polynucleotide encoding the polypeptide according to claim 1 or 2.

4. The polynucleotide according to claim 3 comprising the sequence set forth in SEQ ID NO: 1.

5. A polynucleotide encoding a human leupaxin polypeptide selected from the group consisting of: a) the polynucleotide according to claim 2; and b) a DNA which hybridizes under moderately stringent conditions to the complement of the polynucleotide of (a).

6. The polynucleotide of claim 5 which is a DNA molecule.

7. The DNA of claim 6 which is a cDNA molecule.

8. The DNA of claim 6 which is a genomic DNA molecule.

9. The DNA of claim 6 which is a wholly or partially chemically synthesized DNA molecule.

10. An anti-sense polynucleotide which specifically hybridizes with the complement of the polynucleotide of claim 5. - 21 -

11. A expression construct comprising the polynucleotide according to claim 5.

12. A host cell transformed or transfected with the polynucleotide according to claim 11.

13. A method for producing a leupaxin polypeptide comprising the steps of: a) growing the host cell according to claim 12 under conditions appropriate for expression of the leupaxin polypeptide and b) isolating the leupaxin polypeptide from the host cell or the medium of its growth.

14. An antibody specifically immunoreactive with the polypeptide according to claim 1 or 2.

15. The antibody according to claim 14 which is a monoclonal antibody.

16. A hybridoma which secretes the antibody according to claim 15.

17. An anti-idiotype antibody specifically immunoreactive with the antibody according to claim 14.

- 22 -

18. A method to identify a specific binding partner compound of the leupaxin polypeptide according to claim 1 or 2 comprising the steps of: a) contacting the leupaxin polypeptide with a compound under conditions which permit binding between the compound and the leupaxin polypeptide; b) detecting binding of the compound to the leupaxin polypeptide; and c) identifying the compound as a specific binding partner of the leupaxin polypeptide.

19. The method according to claim 18 wherein the specific binding partner modulates activity of the leupaxin polypeptide.

20. The method according to claim 19 wherein the compound inhibits activity of the leupaxin polypeptide.

21. The method according to claim 19 wherein the compound enhances activity of the leupaxin polypeptide.

22. A compound identified by the method according to claim 18.

23. A method to identify a specific binding partner compound of the leupaxin polynucleotide according to claim 5 comprising the steps of: a) contacting the leupaxin polynucleotide with a compound under conditions which permit binding between the compound and the leupaxin polynucleotide; b) detecting binding of the compound to the leupaxin polynucleotide; and c) identifying the compound as a specific binding partner of the leupaxin polynucleotide. - 23 -

24. The method according to claim 23 wherein the specific binding partner modulates expression of a leupaxin polypeptide encoded by the leupaxin polynucleotide.

25. The method according to claim 24 wherein the compound inhibits expression of the leupaxin polypeptide.

26. The method according to claim 24 wherein the compound enhances expression of the leupaxin polypeptide.

27. A compound identified by the method according to claim 23.

28. A composition comprising the compound according to claim 27. pharmaceutically acceptable carrier