©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning Of Human Paxillin, a Focal Adhesion Protein Phosphorylated by P210(*)

(Received for publication, October 31, 1994; and in revised form, December 12, 1994)

Ravi Salgia (1) Jian-Liang Li (2) Su Hao Lo (2) Beatrice Brunkhorst (2) Geoffrey S. Kansas (1) E. Sholeh Sobhany (3) Yaping Sun (2) Evan Pisick (1) Michael Hallek (1) Timothy Ernst (1) Ramana Tantravahi (3) Lan Bo Chen (2) James D. Griffin (1)(§)

From the  (1)Division of Hematologic Malignancies, the (2)Division of Cellular and Molecular Biology, and the (3)Division of Cytogenetics, Dana-Farber Cancer Institute, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Paxillin is a 68-kDa focal adhesion protein that is phosphorylated on tyrosine residues in fibroblasts in response to transformation by v-src, treatment with platelet-derived growth factor, or cross-linking of integrins. Paxillin has been shown to have binding sites for the SH3 domain of Src and the SH2 domain of Crk in vitro and to coprecipitate with two other focal adhesion proteins, vinculin and focal adhesion kinase (p125). After preliminary studies showed that paxillin was a substrate for the hematopoietic oncogene p210, we investigated the role of this protein in hematopoietic cell transformation and signal transduction. A full-length cDNA encoding human paxillin was cloned, revealing multiple protein domains, including four tandem LIM domains, a proline-rich domain containing a consensus SH3 binding site, and three potential Crk-SH2 binding sites. The paxillin gene was localized to chromosome 12q24 by fluorescence in situ hybridization analysis. A chicken paxillin cDNA was also cloned and is predicted to encode a protein approximately 90% identical to human paxil-lin. Paxillin coprecipitated with p210 and mul-tiple other cellular proteins in myeloid cell lines, suggesting the formation of multimeric complexes. In normal hematopoietic cells and myeloid cell lines, tyrosine phosphorylation of paxillin and coprecipitation with other cellular proteins was rapidly and transiently induced by interleukin-3 and several other hematopoietic growth factors. The predicted structure of paxillin implicates this molecule in protein-protein interactions involved in signal transduction from growth factor receptors and the BCR/ABL oncogene fusion protein to the cytoskeleton.


INTRODUCTION

Paxillin is a 68-kDa cytoskeletal protein found in specialized structures, termed focal adhesions, that occur at sites where cells adhere to the extracellular matrix(1, 2) . It is primarily within focal adhesions that transmembrane integrin molecules connect the actin cytoskeleton to the extracellular matrix. The formation of focal adhesions and the interaction of adhesion molecules with the cytoskeleton are dynamic processes that can be regulated by cytokines in normal cells, including epidermal growth factor and platelet-derived growth factor. Also, several oncogenes, such as v-src, are known to disrupt focal adhesions(3, 4) . The signaling pathways that regulate these events are not well understood.

A number of observations suggest that paxillin is involved in transducing signals from growth factor receptors to focal adhesions. In addition to epidermal growth factor and platelet-derived growth factor, paxillin is transiently tyrosine phosphorylated in response to several small peptide growth factors such as bombesin, endothelin, and vasopressin(5, 6) . Paxillin is also tyrosine phosphorylated in response to integrin-mediated cell adhesion (7) and during embryonic development(8) . The tyrosine kinase(s) that phosphorylate paxillin are unknown, but in vitro paxillin can be phosphorylated by the focal adhesion tyrosine kinase p125(9) . Further evidence linking paxillin to signal transduction pathways comes from the findings that paxillin binds to the SH3 domain of p60(10) and to the SH2 domain of v-Crk (11) in vitro, as well as to the carboxyl terminus of vinculin in vitro and in vivo(12) . Finally, paxillin is also prominently tyrosine phosphorylated in Rous sarcoma virus-transformed chick embryo fibroblasts(1) . Interestingly, paxillin is also prominently tyrosine phosphorylated during transformation by v-crk(11) .

In preliminary studies, we have found that p210 colocalized with paxillin in punctate, membrane-associated structures that also contained vinculin. P210 is known to be localized in part in the cytoskeleton, and this localization is believed to be critical for transformation of myeloid cells, perhaps by allowing access to critical substrates(14, 15, 16) . We found that tyrosine and serine phosphorylation of paxillin is strikingly and constitutively increased in myeloid cell lines transformed by BCR/ABL. In an effort to understand the structure and function of paxillin in hematopoietic cells, we cloned a full-length paxillin cDNA and investigated its interactions with other cellular proteins. The results indicate that paxillin has multiple protein-protein interaction domains, consistent with its potential role as a signal transduction molecule in the cytoskeleton. In contrast to its constitutive phosphorylation in BCR/ABL-transformed cells, paxillin phosphorylation in normal cells is regulated by numerous hematopoietic growth factors, including IL-3. (^1)


MATERIALS AND METHODS

Cell Lines and Cell Culture

The 32Dcl3 cell line was obtained from Dr. Joel Greenberger (University of Pittsburgh) and was cultured in RPMI 1640 containing 10% fetal calf serum and 15% WEHI-condition media (as a source of IL-3). 32Dcl3 expressing p210 clones were generated using previously described methods. One clone, 26.32Dp210BCR/ABL, was used for all experiments(17, 18) . HL-60, K562, U266, U937, FDCP-1, NIH3T3, A2058, 1262, Mo7e, Raji, and NSF-60 cells were obtained from the ATCC and were cultured in RPMI 1640 with 10% fetal calf serum. NIH3T3 fibroblasts transfected with a chimeric alpha/beta GM-CSF receptor were obtained as previously described(19) . BaF-3 cells transfected with erythropoietin receptor were obtained from Dr. A. D'Andrea (Dana-Farber Cancer Institute). FDCP-1 and BaF-3 cells are IL-3-dependent myeloid cells and were cultured as for 32Dcl3. Mo7e cells were cultured with GM-CSF as described(20) . Chick embryo fibroblasts were isolated as previously described(21) . Human neutrophils, monocytes, and T lymphocytes were isolated from blood from volunteer donors using standard protocols (22) . In some experiments, cells were stimulated with cytokines as described below. Human G-CSF, GM-CSF, IL-3, oncostatin-M, M-CSF, IL-6, IL-12, and murine IL-3 were obtained from Dr. Steven Clark (Genetics Institute, Cambridge, MA). Murine Steel factor and human erythropoietin were obtained from Amgen (Thousand Oaks, CA). Human IL-2 was obtained from Dr. Jerome Ritz (Dana-Farber Cancer Institute).

Immunoblotting, Immunoprecipitation, and Phosphoamino Acid Analysis

Logarithmically growing 32Dcl3 cells were starved either for 8 h in 0.5% bovine serum albumin or overnight in RPMI 1640 medium containing 10% fetal calf serum. Cells were then stimulated with murine IL-3 at a concentration of 10 ng/ml. The cells were lysed as previously described(17) . Other growth factors and cells were similarly stimulated and lysed(20, 22) . Immunoblots using anti-paxillin antibody (1:5000) were performed using previously published methods(18, 23) . Antiphosphotyrosine immunoprecipitations were performed with 50 µl protein-A-Sepharose beads coupled to 4G10 antibody at 2 mg/ml(17) . Phosphoamino acid analysis of in vivoP-labeled paxillin was performed as described (23) .

Cloning of Human and Chicken Paxillin cDNA

Monoclonal anti-paxillin antibody (Zymed) was used to screen a K562 gt11 cDNA library (Clontech), yielding a single 2.1-kb cDNA (Pax-0 in Fig. 1A)(24) . This 2.1-kb cDNA was then used to screen a cDNA library from human fibroblasts, WI38 (in gt11), identifying 2 subclones (Pax-1 and Pax-2), human fibroblast 1262 cell line (in ZapII), identifying 3 subclones (Pax-3, -4, -5), and PCR screen human A2058 library (in gt11, Pax-0.2). The length of the subclones varied from 0.2 to 3.7 kb. Each of these subclones was sequenced on both strands. For identification of the 5`-end, Pax-6 and Pax-7 were isolated from K562 and A2058 cell lines, respectively, using a 5`-RACE protocol (Clontech) with provided oligonucleotide primers for PCR. For identification of the 3`-end, Pax-8 was isolated from A2058 cell line using 3`-RACE protocol (Clontech) for PCR. A 1.7-kb partial cDNA encoding chicken paxillin was isolated by reverse transcriptase-PCR using oligonucleotide primers from Pax-0 and Pax-4 human cDNA (cPax-1, Fig. 1A). For identification of the 5`-end of chicken paxillin cDNA, RACE was performed (cPax-4), and an independent clone was isolated from chicken embryo fibroblast library (21) in gtll (cPax-5) using cPax-1 as a probe.


Figure 1: cDNA sequencing strategy and full-length cDNA structure of human and chicken paxillin. A, diagram of individual human and chicken paxillin cDNAs. The shadedbox represents the translation product from the deduced open reading frame. Pax-0 through Pax-8 represent individual cDNAs isolated from human libraries. B, BamHI; E, EcoRI. Pax-6 and Pax-7 were isolated by 5`-RACE from K562 and A2058 cell lines. Pax-8 was isolated by 3`-RACE from A2058 cell line. cPax-1, -4, and -5 are individual cDNAs isolated from chicken libraries. B, full-length cDNA structure of human paxillin. Shown are the nucleic acid base pairs with an open reading frame of 557 amino acids. Also shown is the starting Kozak sequence and the poly(A) adenylation sequence for the human paxillin cDNA. The potential v-Crk binding site(s) and potential SH3 binding site are identified. Noted are the four tandem LIM domains. C, comparison of predicted amino acid structures for human and chicken paxillin. The homology between human and chicken predicted amino acids is approximately 90%.



Fluorescence in Situ Hybridization and Southern Hybridization

A human P1 plasmid clone 2642 was obtained (Genome Systems, Inc., St. Louis, MO) from human foreskin fibroblast P1 library using the forward primer (5`-ACAGCCCTTGACCGGACGTGGCACC-3`) and reverse primer (5`-CGTGAAGCATTCCCGGCACACAAAGCA-3`) using PCR-based strategy(25) . The resulting clone was verified as to its authenticity by Southern hybridization of EcoRI-HindIII digest of P1 plasmid and utilizing P randomly primed Pax-4 cDNA as probe. For fluorescence in situ hybridization (FISH) studies, the P1 plasmid was biotinylated using a nick translation kit (Life Technologies, Inc.) and purified over a Sephadex G-50 spin column. Biotinylated P1 plasmid DNA (100 ng) was coprecipitated with 1-3 µg of human C(0)t-1 DNA (Life Technologies, Inc.) and 10 µg of salmon sperm DNA. The biotinylated probe was hybridized to human metaphase chromosome spreads and processed as previously outlined(26) . The resulting signal was detected with fluorescein avidin (Vector Laboratories), and chromosomes were counterstained with 4,6-diamidine-2-phenylindole and photographed using a Leitz Aristoplan microscope and Kodak Gold 100 ASA film.

Chromosome somatic cell hybrid panel blot was purchased from Oncor, Pax-4 cDNA probe was labeled with P random priming, and Southern hybridization was performed as described(24) .

DNA Sequence Analysis

DNA sequencing of double-stranded plasmid DNAs, on both strands, was done either with an Applied Biosystems 373A DNA sequencer using standard protocols and dye-labeled dideoxy nucleoside triphosphates as terminators or a Sequenase kit (U. S. Biochemical Corp.)(24) . DNA sequence alignments and groups of overlapping clones were constructed using Sequencer 2.0.10 (Gene Codes Corp., Ann Arbor, MI). Data base (NCBI-GenBank release 78, PIR release 37, and SwissProt release 26) searches were performed at the National Center for Biotechnology Information using the basic local alignment search tool (BLAST) network service. DNA and protein sequence homology alignments were performed using DNAStar MegAlign using the CLUSTAL method(27) .

Northern Analysis

RNA was isolated from various cells by extraction with guanidine isothiocyanate as previously described(24) . Samples of 15 µg total RNA were subjected to Northern analysis. Pax-0 and Pax-4 cDNA were used as probes.

Production of Paxillin Fusion Proteins and Antibody Production

A series of paxillin cDNAs were constructed and expressed as fusion proteins with glutathione S-transferase (GST) using pGEX vectors (Pharmacia Biotech Inc.)(28) . The 2.1-kb (Pax-0) EcoRI fragment, 2.5-kb (Pax-1) BamHI-EcoRI fragment, constructed BamHI-EcoRI fragments of LIM domain 1, LIM domains 1-4, and pre-LIM domain site were put into pGEX-3X plasmid. The fusion proteins were isolated as described(28) . Anti-paxillin antisera were prepared by repeated injection of 100-150 µg of fusion protein from 2.1 kb (Pax-0) into a New Zealand White female rabbit (24) .


RESULTS

Cloning of a Paxillin cDNA

The structure of human paxillin was deduced from a series of overlapping cDNA clones, as described under ``Materials and Methods'' (Fig. 1A). A heavily GC-rich region in the 5`-untranslated region of 83 nucleotides precedes a concensus initiator methionine with conserved Kozak sequences, followed by a single open reading frame of 1671 base pairs predicted to encode a protein of 557 amino acids with a predicted molecular mass of 61 kDa (Fig. 1B). The difference between this size and the observed molecular mass of 68 kDa may be due to anomalous migration secondary to the high proline content (10%) of paxillin or to post-translational modifications. A 3`-untranslated region of 1887 base pairs includes a polyadenylation signal (AATAAA) at position 3594. The predicted amino acid sequence of chicken paxillin cloned from chicken embryo fibroblasts is highly homologous, with 90% amino acid identity to human and only occasional nonconservative amino acid changes (Fig. 1, B and C).

Comparison of the structure of paxillin with other proteins revealed an array of discrete protein domains (Fig. 2A). Within the carboxyl-terminal third of paxillin are four tandem cysteine- and histidine-rich sequences termed LIM domains(29) . Although LIM domains are found in a number of proteins, including the cytoskeletal proteins zyxin and cysteine-rich protein, paxillin is the first protein reported to have four LIM domains. The homology of the LIM domains in paxillin with LIM domains from other proteins is represented in Fig. 2B.


Figure 2: Schematic diagram of human paxillin protein and comparison of LIM domains with other known proteins containing LIM domains. A, various domains of paxillin with homologies to other proteins are depicted. There are four tandem LIM domains. There are three YXXP motifs that may be binding sites for v-Crk SH2. The proline-rich domain with potential Src-SH3 binding site is also identified. From precipitations of lysates with various constructs of GST-paxillin fusion proteins, a talin binding site is identified. B, comparison of LIM domains of paxillin with previously described proteins containing LIM domains. Consensus sequence for LIM domains is cysteine/histidine-rich with conserved repeats. The degreeofshading represents the degree of homology to each of the proteins. Lin-11, Isl-1, and Mec-3 are classic LIM homeodomain-containing proteins. RBTN-1 (also called rhombotin; Ttg-1) and RBTN-2 (Ttg-2) are newly described members of the LIM family of proteins. Zyxin (which contains three LIM domains, and for representation sake only two domains are shown) and human cysteine-rich protein (hCRP) are cytoskeletal associated proteins described to contain the LIM domains. Each LIM domain is designated 1, 2, 3, or 4 depending on proximity to the NH(2) terminus of the protein. Note that the LIM domain 3 of paxillin is a perfect match with other LIM domains, whereas there is similarity to domain 1, 2, and 4 of paxillin LIM domains. Each of the known sequences is obtained from GenBank.



In addition to LIM domains, paxillin has a proline-rich domain (amino acids 46-55) that contains the sequence PPPVPPPPSS, which may function as an SH3 domain binding site(30) . There are three YXXP motifs (Tyr-31, Tyr-118, and Tyr-181), each of which is a potential binding site for the SH2 domain of v-Crk(11) .

To confirm the authenticity of the human paxillin cDNA, a GST fusion protein was constructed using the open reading frame of a 2.1-kb (Pax-0) fragment and used to produce a rabbit polyclonal antisera, which was then shown to recognize authentic paxillin by immunoblot (Fig. 3) and to stain focal adhesions of fibroblasts (data not shown). A partial chicken cDNA (approximately half of the full-length cDNA reported in this paper) was also reported during preparation of this manuscript that is contained within the chicken cDNA reported here(31) .


Figure 3: Confirmation that the cloned cDNA is paxillin. A New Zealand White female rabbit was immunized with GST-fusion protein constructed from Pax-0 (Fig. 1A), and third immunization serum was used to identify that the antibody generated reacted with the 68-kDa paxillin protein (Western blot, 1:2000 dilution). Represented are whole cell lysates (lanes1 and 3) and commercial paxillin monoclonal antibody immunoprecipitates of NIH3T3 lysates (lanes2 and 4). Lanes1 and 2 are blotted with rabbit polyclonal antibody, whereas lanes3 and 4 are blotted with commercial paxillin monoclonal antibody. Arrow shows p68 paxillin.



Northern Analysis

Northern analysis of paxillin expression revealed a major 3.7-kb message in all tissues examined with the lowest levels in brain (Fig. 4A). Similarly, a 3.7-kb message was found in all of the cell lines examined. In some of the tissues and cell lines, there are other sizes of message detected for which the explanation is not clear. The 3.7-kb message size corresponds well to the size of the full-length cDNA (Fig. 4B).


Figure 4: Northern analysis of various tissue samples and cell lines using human paxillin cDNA as probe. A, human tissue RNA is as follows: lane1, heart; lane2, brain; lane3, placenta; lane4, testis; lane5, ovary; lane6, small intestine; lane7, colon; lane8, peripheral blood leukocyte. B, cell line RNA is as follows: lane 1, chick embryo fibroblasts; lane2, MRC-5; lane3, FS-2; lane4, A498; lane5, A2058; lane6, Hela; lane7, CV-1; lane8, CCL223; lane9, NIH3T3; lane10, NIH3T3 containing tensin-transfected cDNA. Also shown is the actin hybridization of RNA for standard controls.



FISH Localizes Paxillin Gene To Chromosome 12q24

Human P1 plasmid containing the paxillin gene was obtained by PCR-based strategy and used as a probe for FISH of metaphase chromosomes prepared from normal bone marrow cells. Of the 215 cells scored, 200 cells had two signals on chromosome 12 (93%, Fig. 5A), and 15 cells had one signal on chromosome 12 (7%). Chromosome 12 was identified by banding, and the localization of paxillin signal is further characterized as being on chromosome 12q24 (Fig. 5B). The localization of the paxillin gene on chromosome 12 was independently verified by Southern blotting on hybrid panel blot using full-length human paxillin cDNA (data not shown).


Figure 5: Chromosomal localization for paxillin gene. A, a normal human metaphase spread from phytohemagglutinin-stimulated peripheral blood was denatured and hybridized with paxillin P1 plasmid. Arrows point to the signals observed at the long arm end of a pair of C group chromosomes. B, same metaphase banded with DAPI localizing the signal in A to 12q24.



Phosphorylation of Paxillin and Interaction with Other Proteins in Response to Growth Factors and p210in Hematopoietic Cells

The structure of paxillin suggested that this protein could interact with several other cellular proteins. We therefore analyzed the effects of growth factor stimulation and transformation by BCR/ABL on phosphorylation of paxillin and coprecipitation with cellular proteins. Factor-deprived 32Dcl3 cells, an IL-3-dependent murine myeloid cell line, exhibited a major 68-kDa band and a doublet of 44/46 kDa (Fig. 6) by Western blotting using paxillin monoclonal antibody. The relationship between the 68-kDa form and the 44/46 forms is unclear, but these lower forms are not detected in all cells, indicating that they may be proteolytic breakdown products of the major 68-kDa form, alternatively spliced forms of paxillin, or other proteins immunologically related to paxillin. The 44/46-kDa paxillin-related proteins we observed may be identical to previously reported proteins of the same molecular weights variably detected in NIH3T3 cells(2) . In hematopoietic cells, these 44/46-kDa proteins were readily detected by anti-paxillin immunoblotting in 32Dcl3, FDCP-1, K562, HeLa, neutrophils, T-cells, NK-cells, and monocytes but not in U266 and HL-60 cell lines. Little or no paxillin was detected in Mo7e, Raji, or NSF-60 cells.


Figure 6: IL-3 and P210 induces paxillin migration in 32Dcl3 myeloid cells. Paxillin is a 68-kDa protein that is recognized by the mouse monoclonal antibody. IL-3 stimulation time course is represented (lanes1-6: 0, 1, 5, 15, 30, and 60 min, respectively). Four individual 32Dcl3.P210 subclones were also evaluated for paxillin immunoblotting. Proteins were separated by 7.5% SDS-PAGE and immunoblotted using anti-paxillin monoclonal antibody. There is a migration of the paxillin band with IL-3 stimulation and constitutively in P210 cells. The antibody also recognizes a doublet at 44/46 kDa.



IL-3 stimulation of factor-deprived 32Dcl3 cells led to a rapid (1-5 min) shift to multiple slower migrating isoforms up to 80 kDa (Fig. 6). These slower migrating forms of paxillin have increased phosphorylation of both serine and tyrosine residues (Fig. 7), and similar results were observed with other hematopoietic cell lines and growth factors. For example, there is also hyperphosphorylation in FDCP-1 and BAF-3 cells in response to IL-3, human neutrophils and NIH3T3 alpha/beta1 fibroblasts in response to GM-CSF, FDCP-1 in response to Steel factor, human neutrophils in response to G-CSF, HL-60 in response to trans-retinoic acid, human T-cells in response to phytohemagglutinin, human macrophages in response to M-CSF, and BAF-3 cells in response to erythropoietin. These shifts are due to principally or exclusively increased phosphorylation because treatment of paxillin immunoprecipitates and whole cell lysates with potato acid phosphatase reduced this complex set of bands to a single 68-kDa band (data not shown). A similar pattern of paxillin phosphorylation was observed in 32Dcl3 cells transformed to factor independence by expression of p210 ( Fig. 6and Fig. 7), except that this phosphorylation pattern is constitutive. Finally, we did not observe a change in phosphorylation status of paxillin with IL-2 in T-cells, IL-6 in U266 cells, IL-12 in NK cells, and oncostatin-M in U266 cells.


Figure 7: Phosphoamino acid analysis of in vivoP-labeled paxillin. After immunoprecipitation of P-labeled paxillin protein with the monoclonal antibody, cell lysates were applied to a 7.5% SDS-PAGE gel and electrophoresed. The appropriate paxillin bands were cut, and phosphoamino acids were analyzed. Lane1, 32Dcl3, unstimulated; lane2, 32Dcl3, IL-3 stimulated for 15 min; lane3: clone 26.32D.P210BCR/ABL.



Given the structure of paxillin and the observation of increased phosphorylation following growth factor stimulation or oncogenic transformation, we asked if growth factor or p210-induced phosphorylation was associated with changes in the interaction of paxillin with other cellular proteins. Lysates of factor-deprived, IL-3-stimulated, or p210-transformed 32Dcl3 cells were immunoprecipitated with antibody to paxillin and then blotted with antibody to phosphotyrosine. Proteins of 210, 116, 94, 60, 55, and 44 kDa were detected in lysates from factor-stimulated or p210-transformed cells but not from resting cells (Fig. 8A). Western blotting of these immunoprecipitates identified the 210 kDa as p210 in oncogene-transformed cells and the 116-kDa protein as vinculin (data not shown).


Figure 8: Identification of multiple proteins that associate with paxillin. A, association of paxillin with other phosphotyrosine proteins as determined by paxillin immunoprecipitation and antiphosphotyrosine immunoblot. After immunoprecipitation of paxillin protein with the monoclonal antibody, isolated proteins were applied to a 7.5% SDS-PAGE gel and electrophoresed, and phosphotyrosine-containing proteins were visualized by immunoblots. Lane1, 32Dcl3, unstimulated; lane2, 32Dcl3, IL-3 stimulated for 15`; lane3, clone 26.32D.P210BCR/ABL; lanes4-6 are 32Dcl3, unstimulated, 32Dcl3, IL-3 stimulated for 15`, and clone 26.32D.P210BCR/ABL cell lysates, respectively, immunoprecipitated with control antibody 3c11c8 (-interferon). B, association of GST-paxillin fusion protein with talin. Various GST fusion proteins of paxillin domains were constructed as described under ``Materials and Methods,'' and the expressed fusion proteins were used to precipitate lysates from clone 26.32D.P210BCR/ABL cell line. GST protein alone and GST-SH2 domain of tensin were used as controls. Thereafter, isolated proteins were applied to a 7.5% SDS-PAGE gel and electophoresed, and talin, vinculin, and p125 proteins were visualized by immunoblots. Lane1, GST-protein alone; lane2, GST-SH2-tensin; lane3, Pax-0-GST-paxillin (amino acids 100-557); lane4, Pax-1-GST-paxillin (amino acids 227-557); lane5, LIM domains 1 through 4-GST-paxillin (amino acids 325-557); lane6, LIM domain 1-GST-paxillin (amino acids 325-374); lane7, pre-LIM domain-GST-paxillin (amino acids 303-327); lane8, whole cell lysate.



Using various constructed GST-fusion proteins of paxillin (Fig. 8B), it is seen that talin is precipitated with Pax-0 GST-fusion protein (amino acids 100-557) but not Pax-1 (amino acids 227-557), pre-LIM domain, LIM 1 domain, or LIM 1-4 domain GST-fusion proteins. None of these fusion proteins precipitate vinculin or p125. As a control, a tensin-SH2 GST-fusion protein did not precipitate talin, vinculin, or p125.


DISCUSSION

The cytoskeleton is essential for many cellular functions, including regulation of cell shape, flexibility, mobility, and adhesive properties(32) . The focal adhesion is a specialized structure of the cytoskeleton and plasma membrane that forms at areas of contact between the cell and extracellular matrix proteins such as fibronectin(33) . Integrins serve as receptors for extracellular matrix proteins, localize in focal adhesions, and their intracellular domains interact directly with focal adhesion proteins such as talin and alpha-actinin and indirectly with actin(34) . The expression and function of adhesion receptors and the formation of focal adhesions can be modulated by various events, including exposure to cytokines and growth factors, although the mechanisms are generally not understood. Phosphorylation of proteins within focal adhesions is closely associated with changes in the structure of the actin cytoskeleton(7, 35, 36, 37, 38) . Focal adhesions have very high concentrations of proteins phosphorylated on tyrosine residues, and a tyrosine kinase unique to this structure, p125, has been described(39, 40) . Further evidence implicating tyrosine phosphorylation in the regulation of focal adhesion functions comes from the observation that cross-linking of integrins induces tyrosine phosphorylation of focal adhesion plaque proteins(41) . Thus, the focal adhesion appears to be a structure that receives signals from both sides of the membrane in a complex manner.

Paxillin is a 68-kDa focal contact protein initially identified by making antibodies to phosphotyrosine-containing proteins in Rous sarcoma virus-transformed chick embryo fibroblasts(1) . The pI of paxillin ranges from pH 6.31-6.86, with increased acidic forms (consistent with multiple phosphorylation sites) migrating more slowly on SDS-polyacrylamide gels(2) . The antibody also recognizes two lower forms of 46 and 44 kDa, with pIs of pH 6.9. Up to 20-30% of paxillin is phosphorylated on tyrosine in Rous sarcoma virus-transformed cells and in embryonal tissues(1, 2, 8, 9) . The significance of tyrosine phosphorylation is unknown, although the phosphorylation of this protein appears to be a consequence of signal transduction from membrane receptors in normal fibroblasts. Paxillin is one of the major tyrosine phosphoproteins in cells transformed by v-src or v-crk(11) . Further, paxillin can interact directly with both oncogenes through the SH3 domain of Src (10) and the SH2 domain of Crk(11, 42) , based on in vitro binding studies. Binding of v-Src and v-Crk to paxillin may be a major determinant in concentrating both of these oncogenes in focal adhesions and therefore could be important in the altered cytoskeletal structure and adhesive properties that accompany transformation by both viral oncogenes.

To identify any role paxillin might play in regulating cytoskeletal structure in either transformed or normal hematopoietic cells, we cloned a full-length cDNA encoding human paxillin by expression and authenticated the clone by demonstrating that an antibody raised against the amino terminus of the protein precipitated paxillin. The cDNA has an open reading frame of 1671 base pairs, which encodes a predicted protein of 557 amino acids and molecular mass of 61 kDa. The expected molecular mass of paxillin is 68 kDa, and the difference between the expected and predicted molecular weights could be due to the high proline content of this protein or alternative splicing. The formal proof that paxillin described here shares identical amino acid sequences with native paxillin is still lacking since we have not purified the native paxillin and analyzed individual amino acid composition. We have also cloned chicken paxillin, using human paxillin cDNAs as probes to screen a chicken embryo fibroblast library. While this manuscript was in preparation, a partial cDNA of chicken paxillin (approximately half of the full-length cDNA reported here), identified initially by peptide sequencing of paxillin protein and thereafter using degenerate oligonucleotide primers to screen a chicken fibroblast library, was reported by Turner and Miller(31) . This published partial sequence is contained within the chicken paxillin sequence reported here, which predicts a protein 90% identical to human paxillin, further supporting the authenticity of our human cDNA. There is a major human RNA message on Northern blot of 3.7 kb, expressed in most tissues, although low in brain.

Comparison of the predicted sequence with known proteins reveals several interesting domains. There is a series of 4 motifs identical or very closely related to LIM domains, with the general consensus sequence CX2CX16-23(W/Y/F)HX2CX2CX16-21CX2-3(C/H/D) (29) . The LIM motif is named for three homeodomain proteins, each containing two LIM domains, lin-11, a Caenorhabditis elegans cell lineage gene(43) ; isl-1, preferentially expressed in pancreatic islet cells(44) ; and mec-3, involved in neuron differentiation in C. elegans(43) . A number of other LIM domain proteins have been identified, including rhombotin 1 and 2(29, 45, 46, 47, 48, 49, 50, 51) , identified in chromosomal translocations in T-cell acute leukemias and two cytoskeletal proteins, zyxin and cysteine-rich protein(52, 53, 54) . LIM domains are predicted to bind two molecules of Zn and could form two zinc finger-like structures. Theoretically, therefore, one molecule of paxillin could bind up to eight molecules of zinc and form eight zinc fingers. One of the three LIM domains of zyxin has been shown to bind cysteine-rich proteins(55) . In cysteine-rich protein, metal binds to the two sites within a single LIM domain is sequential(56) , with preferential occupancy of a site involving the 4 carboxyl-terminal cysteines(57) . Proteins containing LIM domains tend to be highly conserved among different species. For example, rat, hamster, and human Isl-1 amino acid sequences are identical(58) , while chicken and human cysteine-rich proteins are 91% identical(54) . The high degree of homology (90%) between chicken and human paxillin is therefore not surprising. It is noteworthy that many LIM proteins are nuclear and are involved in regulating development or differentiation. However, zyxin and cysteine-rich proteins are cytoskeletal proteins, and like paxillin, neither has been detected in the nucleus.

Analysis of paxillin structure reveals several other potential motifs of interest. Paxillin has been shown to bind to the SH2 domain of v-Crk in vitro, and recent studies indicate that phosphopeptides with pYXXP motifs will bind to Crk SH2 domains(11) . Human paxillin has three of these motifs, and it will be worthwhile to determine which interact with c-CRK and v-Crk. Cells transformed by v-crk have increased tyrosine phosphorylation, which is of interest because v-Crk does not contain a tyrosine kinase itself. Recent data from Feller et al.(59) indicate that c-Abl binds to the first c-Crk SH3 domain and can phosphorylate Tyr-221 in the spacer region between the two SH3 domains in c-Crk. This creates a high affinity binding motif for Crk-SH2, and possible intramolecular binding of Crk-SH2 to Tyr-221. This model of Crk function predicts that the interaction of c-Crk and paxillin would be controlled by the relative degrees of phosphorylation of Crk Tyr-221 and paxillin YXXP motifs, both potentially competing for c-Crk-SH2 binding. The transient phosphorylation of paxillin induced by growth factors, if it occurs on the appropriate tyrosine residues, could increase paxillin-Crk interaction, facilitating signal transduction to (or from) focal adhesions.

In BCR/ABL-transformed cells, paxillin is phosphorylated, and interaction of c-CRK and paxillin might be increased. However, CRK-L, a CRK-related protein, has been shown to be tyrosine phosphorylated in CML cells(60) , and the interactions of c-CRK, CRK-L, and paxillin may be complex. In the CML cell lines studied here, paxillin coprecipitates with p210, suggesting that a paxillin-CRK-L-P210 complex could be formed, although these studies are preliminary. The formation of such a complex may not only anchor p210 at the cell membrane, adjacent to other critical substrates, but also interrupt the normal signaling pathways that utilize paxillin and CRK-L. Alterated regulation of integrin molecule function would be a potential outcome. In v-Crk, Tyr-221 is deleted, and constitutive complexes of v-Abl and v-Crk exist. It would be predicted that these complexes could also contain paxillin, and this possibility is currently being tested. Overall, the available data suggest that the interaction between Crk or CRK-L and paxillin may be regulated by phosphorylation and could contribute to signaling in focal adhesions.

Paxillin has previously been shown to bind to the SH3 domain of Src, and analysis of the primary structure of paxillin reveals a proline-rich motif that could serve as an SH3 binding domain. The sequence PPPVPPPPSS (amino acids 46-55) is consistent with a consensus SH3 binding site, related, but not identical to, the SH3 binding sites previously identified in SOS, p59, 3BP1, and 3BP2(30) . It has been suggested that the colocalization of Src to focal adhesions may be the result of this interaction(10) . The precise function of SH3 domains is uncertain, but SH3 domains are found in a variety of cytoskeletal and membrane proteins and have been suggested to participate in protein interactions in these structures. In addition, some SH3 domains have recently been shown to increase enzyme activities of binding partners. Notably, the GTPase activity of dynamin is increased by SH3 domain binding(61) , and it will be of interest to determine if the binding of paxillin to Src-SH3 affects Src kinase activity.

Using FISH analysis, we have localized the gene for paxillin on chromosome 12q24. From known genomic data, there are several genes known to reside in this region. For example, the gene for Darier's disease (12q23-24.1), a rare autosomal dominant skin disorder in which there is abnormal adhesion between keratinocytes(62) , and autosomal dominant cerebellar ataxia (second locus, 12q23-24.1) (63) appear to be in the vicinity of the paxillin gene. At this time, the functional relationships to any or all of these genes is not known and is being investigated.

We have begun to generate fusion proteins containing different domains of paxillin fused to GST to map potential binding sites. Here, we show that GST-fusion proteins containing amino acids 100-557 bind to talin while amino acids 227-557 do not, suggesting that a talin binding site resides between amino acids 100-227. In similar studies, Turner and Miller (31) have shown that the non-LIM domains of chicken paxillin (amino acids 56-313 in our full-length predicted paxillin structure) bind vinculin and p125. Since a fusion protein containing amino acids 100-557 did not bind vinculin or p125, amino acids 56-100 could have critical residues for binding vinculin and/or p125, and this possibility is currently being tested.

Our studies show that paxillin is tyrosine phosphorylated in myeloid cell lines transformed by the human oncogene P210. This is of interest in its own right, as very few potential substrates of the p210 tyrosine kinase have been identified(18) . We also found that paxillin is transiently tyrosine phosphorylated in response to several hematopoietic cytokines. Hematopoietic cells adhere to extracellular matrix proteins through integrins, and these adhesive events are likely to be critically important for migration and homing properties of hematopoietic cells(13) . For example, GM-CSF has been shown to induce rapid polymerization of F-actin in neutrophils, up-regulation of integrin expression and function, and enhanced adhesion and chemotaxis of neutrophils(22) . We found that growth factors such as GM-CSF and IL-3 increase the tyrosine phosphorylation of proteins within these structures. The aberrent tyrosine phosphorylation of paxillin or other focal adhesion plaque proteins, caused by p210, could contribute to the known alteration in adhesive properties of CML cells(13) . P210 reduces ability to bind to a number of extracellular matrix proteins in the marrow microenvironment, and this is believed to contribute to the extreme leukocytosis and extramedullary hematopoiesis characteristic of this disorder.

In summary, we have shown that paxillin is a common target for a tyrosine kinase(s) in hematopoietic cells involved in IL-3 signal transduction and thus may be involved in mediating signals from various receptors to focal adhesion-like structures in this lineage as well as in others. The structure of paxillin suggests that it could interact with other proteins through many different motifs, including a series of LIM domains and SH2 and SH3 binding domains, and thus contribute to the changes in cytoskeletal structure that take place in response to cytokines, integrin cross-linking, and differentiation. It will now be possible to determine the nature of the interaction of vinculin, Crk, Src, and Fak with paxillin. Finally, the phosphorylation of paxillin by p210 and the reported interaction of p210 with CRK-L suggest that the role of paxillin in CML be examined.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants CA 60821, CA36167, and GM 3818. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U14588 [GenBank]and U14589[GenBank].

§
To whom correspondence should be addressed: Division of Hematologic Malignancies, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3360; Fax: 617-632-4388.

(^1)
The abbreviations used are: IL, interleukin; kb, kilobase(s); PCR, polymerase chain reaction; FISH, fluorescence in situ hybridization; GM-CSF, granulocyte-macrophage-colony stimulating factor; RACE, rapid amplification of cDNA ends; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; CML, chronic myelogenous leukemia.


ACKNOWLEDGEMENTS

We thank the Molecular core facility at the Dana-Farber Cancer Institute for help in sequence analysis. We especially thank Paul Morrison, Lori Wirth, and Christine Earabino. Thanks are also due to our students Karen Slattery, Toshiki Saito, and Paul Kim for tremendous efforts in the lab. We thank Dr. Uma Devi Tantravahi, Dept. of Pathology, Brown University Medical Center, for help with labeling the P1 plasmid probe and procedure of in situ hybridization.


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