©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CRKL Links p210 with Paxillin in Chronic Myelogenous Leukemia Cells (*)

(Received for publication, September 6, 1995; and in revised form, September 25, 1995)

Ravi Salgia (1) Naoki Uemura (1) Keiko Okuda (1) Jian-Liang Li (1) Evan Pisick (1) Martin Sattler (1) Ron de Jong (3) Brian Druker (4) Nora Heisterkamp (3) Lan Bo Chen (2) John Groffen (3) James D. Griffin (1)(§)

From the  (1)Division of Hematologic Malignancies and (2)Cellular and Molecular Biology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115, the (3)Section of Molecular Carcinogenesis, Department of Pathology, Childrens Hospital, Los Angeles, California 90027, and the (4)Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, Oregon 97201-3098

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary neutrophils from patients with CML, the major novel tyrosine-phosphorylated protein is CRKL, an SH2-SH3-SH3 linker protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene product. Anti-CRKL immunoprecipitates from CML cells, but not normal cells, were found to contain p210 and c-ABL. Several other phosphoproteins were also detected in anti-CRKL immunoprecipitates, one of which has been identified as paxillin, a 68-kDa focal adhesion protein which we have previously shown to be phosphorylated by p210. Using GST-CRKL fusion proteins, the SH3 domains of CRKL were found to bind c-ABL and p210, while the SH2 domain of CRKL bound to paxillin, suggesting that CRKL could physically link p210 to paxillin. Paxillin contains three tyrosines in Tyr-X-X-Pro (Y-X-X-P) motifs consistent with amino acid sequences predicted to be optimal for binding to the CRKL-SH2 domain (at positions Tyr-31, Tyr-118, and Tyr-181). Each of these tyrosine residues was mutated to a phenylalanine residue, and in vitro binding assays indicated that paxillin tyrosines at positions 31 and 118, but not 181, are likely to be involved in CRKL-SH2 binding. These results suggest that the p210 oncogene may be physically linked to the focal adhesion-associated protein paxillin in hematopoietic cells by CRKL. This interaction could contribute to the known adhesive defects of CML cells.


INTRODUCTION

Chronic myelogenous leukemia (CML) (^1)is characterized by the production of an active tyrosine kinase fusion protein, p210. In cell lines and primary cells, p210 translocates to the cytoskeleton (1) and causes the tyrosine phosphorylation of several cellular proteins, including the SH2-SH3-SH3 adaptor protein CRKL(2, 3) . In cell lines either derived from patients with advanced phase CML or generated by transfecting the BCR/ABL oncogene, there are a number of cellular proteins which are constitutively tyrosine-phosphorylated, such as p120(4) , p120(5) , p52(6) , p93(7) , p95(8) , p68(9) , and p72(10) . In contrast, in the early (stable) phase of CML, there are only a few proteins which either interact with BCR/ABL or are phosphorylated by BCR/ABL(3) . In earlier studies, we and others identified a 39-kDa tyrosine phosphoprotein complexed with BCR/ABL in CML stable phase cells as CRKL (2, 3, 11) .

The CRKL protein has an overall homology of 60% to c-CRK, the human homologue of the v-crk oncogene(12) . v-crk is the oncogene in the CT10 avian retrovirus and has a deletion of the C-terminal SH3 domain(13, 14) . v-Crk, c-CRK, and CRKL each have one SH2 domain, and two SH3 domains, without other known functional motifs (13) . The functions of c-CRK and CRKL in normal signaling are unknown, although recent studies have linked c-CRK to signaling in normal T-cells(15) . Since several proteins in v-Crk-transformed cells are heavily phosphorylated on tyrosyl residues, it has been suggested that v-Crk and c-Crk may serve as regulatory subunits of a tyrosine kinase (16) . Recently, c-Abl has been identified as a possible Crk-associated tyrosine kinase(17) . c-Abl binds in vitro to the first Crk SH3 domain and phosphorylates Crk on tyrosine 221. The phosphorylation on Crk (Tyr-221) creates a binding site for the Crk SH2 domain, possibly inhibiting its binding to other proteins(13, 17) . It is not known if Crk and Abl interact in vivo. Interestingly, despite the structural similarities of c-CRK and CRKL and the apparent interaction of v-Crk and c-Abl, our preliminary studies indicated that CRKL is phosphorylated in CML cells, while c-CRK is not.

In an effort to determine if CRKL functions to link p210to other cellular proteins, we looked for proteins which coprecipitated with CRKL in p210-containing cell lines. A 68-kDa protein precipitating with CRKL was identified as paxillin, a focal adhesion protein we have previously shown to be phosphorylated by p210(9) . The interaction between p210, CRKL, and paxillin could contribute to the pathogenesis of CML.


MATERIALS AND METHODS

Cell Lines and Cell Culture

The 32Dcl3 cell line was obtained from Dr. Joel Greenberger (University of Pittsburgh) and 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(18) . Several clones were used for the experiments, and a typical clone, 32D.p210.26, is shown in the data section. K562 cells were obtained from the ATCC and were cultured in RPMI 1640 with 10% fetal calf serum. Human neutrophils were isolated from volunteer donors or subjects with CML after obtaining appropriate informed consent on approved protocols(19) .

Immunoblotting and Immunoprecipitations

32Dcl3 cells were starved and stimulated with IL-3 (10 ng/ml, murine, 10 min) as described previously(18) . Similarly, isolated neutrophils were unstimulated or stimulated with GM-CSF (10 ng/ml, human, 10 min)(19) . Lysates were then prepared(4) , and immunoprecipitations or immunoblots were performed with anti-CRKL rabbit polyclonal sera (Santa Cruz or CH16 (20) antibody, 1:1000), anti-paxillin mouse monoclonal antibody (Zymed Laboratories, Inc., South San Francisco, CA, 1:5000), anti-CRK mouse monoclonal antibody (Transduction Laboratories, KY, 1:500), or anti-ABL mouse monoclonal antibody (clone AB-3, Oncogene Sciences, Cambridge, MA, 1:500) as described previously(9) . Protein samples were separated under reducing conditions by SDS-polyacrylamide gel electrophoresis (PAGE, 6%-12% gradient gel) and electrophoretically transferred and immunoblotted as described(9) .

GST Fusion Protein Production and Precipitations

GST fusion proteins containing full-length CRKL and GST-CRKL-SH2-SH3(N) have been described previously(20) . GST-CRKL-SH2 was generated by inserting a 360-base pair TaqI-RsaI fragment of a CRKL cDNA into pGEX-2T. GST-CRKL-SH3(N)-SH3(C) was obtained by cloning a blunt-ended 630-base pair BsmI fragment into pGEX-3X. Precipitations were performed with 1-10 µg of fusion protein on glutathione beads using the lysate from 5-10 times 10^6 cells as described(9) .

Tyrosine to Phenylalanine Mutants of Paxillin and Binding to GST-CRKL-SH2 Domain

Individual tyrosine to phenylalanine mutations of human paxillin at amino acid positions 31, 118, and 181 were performed via a polymerase chain reaction-based strategy(21) . The final constructs were entirely sequenced. The full-length wild type human paxillin (amino acids 1-527) and tyrosine mutant paxillin were expressed in the vector pGEX-2TK (Pharmacia Biotech Inc.), and paxillin fusion proteins were produced. For the CRKL-SH2 fusion protein binding experiment, 1 µg of wild type or mutant GST-paxillin fusion protein on glutathione beads was phosphorylated for 30 min at 25 °C in vitro with 2 units of v-Abl kinase (Oncogene Science) in 100 µl of kinase reaction buffer (containing 1 mM MgCl(2), 1 mM MnCl(2), 0.1 mM ATP in 20 mM HEPES solution, pH 7.2). After the kinase reaction, the v-Abl and ATP were removed by washing the beads with lysis buffer at least five times. Tyrosine phosphorylation of the paxillin fusion protein was evaluated by an anti-phosphotyrosine immunoblot. The tyrosine-phosphorylated paxillin-GST protein was cleaved with thrombin(22) . The thrombin cleavage product of the paxillin fusion protein was checked on an SDS-PAGE gel with Coomassie staining for amount of protein cleaved and on an immunoblot with anti-paxillin antibody to confirm the amount of cleaved product to be used for binding assay. An equivalent amount of thrombin-cleaved paxillin protein (approximately 0.3 µg) was used in precipitations with GST-CRKL-SH2 (1 µg) protein. Finally, the precipitated samples were subjected to SDS-PAGE, electrotransferred to Immobilon-polyvinylidene difluoride membranes, and immunoblotted with anti-paxillin antibody.


RESULTS

p39, but Not c-CRK, Is Tyrosine-phosphorylated in p210 Cells

We used immunoblotting to investigate the expression and tyrosine phosphorylation of CRKL and c-CRK in myeloid cell lines. p39 was detected in untransformed murine 32Dcl3 cells, 32Dcl3 cells transformed with BCR/ABL, and the Philadelphia chromosome-positive human cell line K562 (Fig. 1). p39 could readily be immunoprecipitated with an anti-phosphotyrosine monoclonal antibody (4G10) from p210-containing cells, but not from untransformed 32Dcl3 cells. Similarly, both CRK-I and CRK-II (the two forms of c-CRK) were found to be expressed in 32Dcl3 and K562 cells. However there was no detectable tyrosine phosphorylation of c-CRK in normal or BCR/ABL-transformed cell lines (Fig. 2). The reverse experiment, using an anti-CRKL or anti-CRK immunoprecipitation and phosphotyrosine immunoblotting yielded the same results (data not shown).


Figure 1: Expression and tyrosine phosphorylation of CRKL in myeloid cells. Whole cell lysates (W.C.L., 5 times 10^6 cells, first four lanes) or immunoprecipitates of cell lysates (I.P., 20 times 10^6 cells, last eight lanes) with anti-phosphotyrosine (p-Tyr I.P.) or immunoprecipitates with control antibody against -interferon, 3c11c8 (Cntrl I.P.) were processed as described under ``Materials and Methods'' and applied to a gradient SDS-PAGE gel (6-12%) and transferred to Immobilon-P membrane. The membrane was immunoblotted with anti-CRKL antibody. The cells used are unstimulated 32Dcl3 cells (32D(-)), 32Dcl3 cells stimulated with IL-3 (32D(+)), 32D.p210.26 cells (32D.p210), and K562 cells (K562). Molecular masses are shown in kilodaltons.




Figure 2: c-CRK is expressed but not tyrosine-phosphorylated or associated with paxillin in myeloid cells. Whole cell lysates (W.C.L., 5 times 10^6 cells) or immunoprecipitates of cell lysates (20 times 10^6) with anti-phosphotyrosine (p-Tyr I.P.) or immunoprecipitates with anti-paxillin (Paxillin I.P.) were processed as described. Membrane was immunoblotted with anti-CRK antibody. Shown are the bands for CRK-I and CRK-II (c-CRK). The cells used are unstimulated 32Dcl3 cells (32D(-)), 32Dcl3 cells stimulated with IL-3 (32D(+)), 32D.p210.26 cells (32D.p210), and K562 cells (K562). Molecular masses are shown in kilodaltons.



CRKL Binds Paxillin in p210-containing Cell Lines and Primary Neutrophils from Patients with CML

Since CRKL has the predicted structure of an adaptor protein, we looked for CRKL-associated proteins by immunoprecipitating CRKL with a rabbit polyclonal antibody (CH-16) followed by immunoblotting with anti-phosphotyrosine (Fig. 3). Several phosphotyrosine-containing proteins coprecipitated with CRKL in 32Dp210 and K562 cells, but not in untransformed 32Dcl3 cells (Fig. 3). The major tyrosine phosphoproteins observed after SDS-PAGE had estimated molecular masses of 210, 140, 120, 68, 52, and 39 kDa. The 68-kDa protein was consistently observed in anti-CRKL immunoprecipitates in p210-containing cell lines. Since we have recently identified paxillin as a 68-kDa protein which is phosphorylated on tyrosine residues in p210-containing cell lines(9) , we asked if the 68-kDa protein which coprecipitates with anti-CRKL was paxillin. Anti-CRKL immune complexes were separated on SDS-PAGE and then immunoblotted with anti-paxillin antibody. These immune complexes contain paxillin in p210BCR/ABL-containing cells, but not in untransformed myeloid cell lines (Fig. 4). The reverse experiment, paxillin immunoprecipitation followed by anti-CRKL immunoblotting, gave identical results. Interestingly, we did not detect coprecipitation of c-CRK and paxillin in these same cells (Fig. 2), suggesting that this interaction is unique to CRKL in BCR/ABL-transformed cells.


Figure 3: CRKL binds multiple tyrosine-phosphorylated proteins in myeloid cells containing BCR/ABL. Whole cell lysates (W.C.L., 5 times 10^6 cells) or immunoprecipitates of cell lysates (CRKL I.P., 20 times 10^6 cells) with anti-CRKL rabbit polyclonal antibody were processed as described. The membrane was immunoblotted with anti-phosphotyrosine (4G10) antibody. An arrow marks the band at 68 kDa. The cells used are unstimulated 32Dcl3 cells (32D(-)), 32Dcl3 cells stimulated with IL-3 (32D(+)), 32D.p210.26 cells (32D.p210), and K562 cells (K562). Molecular masses are shown in kilodaltons.




Figure 4: CRKL binds paxillin in BCR/ABL-containing myeloid cells. A, CRKL coimmunoprecipitates with paxillin in BCR/ABL expressing cells. Whole cell lysates (W.C.L., 5 times 10^6 cells) or immunoprecipitates of cell lysates (15 times 10^6 cells) with control antibody using normal rabbit pre-immune serum (Cntrl. I.P.) or anti-CRKL rabbit polyclonal antibody (CRKL I.P.) were processed as described. The membrane was immunoblotted with anti-paxillin antibody. The cells used are unstimulated 32Dcl3 cells (32D(-)), 32Dcl3 cells stimulated with IL-3 (32D(+)), 32D.p210.26 cells (32D.p210), and K562 cells (K562). Molecular mass is shown in kilodaltons. B, paxillin immunoprecipitates with CRKL in BCR/ABL expressing cells. Immunoprecipitates of cell lysates (15 times 10^6 cells) with anti-paxillin antibody were applied to a gradient gel and transferred. The membrane was probed with anti-CRKL antibody. The cells used are unstimulated 32Dcl3 cells (32D(-)), 32Dcl3 cells stimulated with IL-3 (32D(+)), 32D.p210.26 cells (32D.p210), and K562 cells (K562).



The possible association of CRKL and paxillin was also investigated in freshly isolated, primary neutrophil samples from seven patients with CML and compared with neutrophils from four normal subjects (Fig. 5). Again, anti-CRKL immune complexes contained paxillin in all CML stable phase neutrophil samples tested, but not when neutrophils from normal individuals were examined. Since the neutrophils from the normal subjects were isolated in a quiescent state, we also stimulated normal neutrophils with the potent neutrophil-activating cytokine GM-CSF (10 ng/ml for 10 min) prior to repeating the immunoprecipitation experiments. GM-CSF stimulation did not induce CRKL and paxillin coprecipitation in normal neutrophils (Fig. 5). In contrast, paxillin coprecipitated with CRKL both before and after GM-CSF stimulation of CML neutrophils. After stimulation with GM-CSF, CRKL coimmunoprecipitated with both the slower and faster migrating forms of paxillin (phosphorylated forms of paxillin have slower migration(9) ). These results suggest that the interaction of CRKL and paxillin in myeloid cells is restricted to cells expressing an active BCR/ABL protein.


Figure 5: CRKL binds to paxillin in neutrophils from stable phase CML but not in normal neutrophils. A, CRKL coimmunopreciptates with paxillin in CML neutrophils. Immunoprecipitates of neutrophil lysates (20 times 10^6 cells) with anti-CRKL rabbit polyclonal antibody were applied to a gradient gel and transferred. The membrane was probed with anti-paxillin antibody. The lower panel shows the same membrane stripped and reprobed with anti-CRKL antibody. IgH represents the immunoglobulin heavy chain. Shown are neutrophil lysate immunoprecipitates from two different normal subjects (lanes 1 and 2) and four different CML stable phase subjects (lanes 3-6). B, GM-CSF stimulation of neutrophils and coimmunoprecipitation of CRKL with paxillin. Immunoprecipitates of neutrophil lysates unstimulated (-) or stimulated with GM-CSF (10 ng/ml, 10 min, +) with anti-CRKL rabbit polyclonal antibody were applied to a gradient gel and transferred. The membrane was probed with anti-paxillin antibody. Shown are neutrophil lysate immunoprecipitates from two different normal subjects (lanes 1 and 2) and three different CML stable phase subjects (lanes 3-5).



CRKL-SH2 Domain Binds to Paxillin

To partially map the domains of CRKL involved in binding to paxillin in BCR/ABL-containing cells, GST-CRKL fusion proteins were used for precipitation experiments. GST-CRKL (full length), GST-CRKL-SH2, and GST-CRKL-SH2-(N)SH3 effectively precipitated paxillin from cells transformed by BCR/ABL, while GST-CRKL-(N)SH3-(C)SH3 precipitated only a very small amount of paxillin (lower panel, Fig. 6). In contrast, the same blot stripped and reprobed with anti-ABL indicated that fusion proteins containing one or more SH3 domains of CRKL can precipitate c-ABL and BCR/ABL, whereas GST-CRKL-SH2 did not. These results suggest that the CRKL-SH2 domain can interact with paxillin while one or both of the SH3 domains interact with BCR/ABL, and the very minor binding of the GST-CRKL-SH3 domains to paxillin could be indirect through c-ABL or BCR/ABL.


Figure 6: Binding of CRKL subdomains to tyrosine-phosphorylated proteins in BCR/ABL expressing 32Dcl3 cells. Precipitations with various GST fusion proteins of CRKL and its subdomains with 32D.p210.26 cell lysates were processed as described. The top panel shows Western blot with anti-phosphotyrosine antibody (4G10). The same membrane was stripped and reprobed with anti-ABL antibody (middle panel) and thereafter re-stripped and probed with anti-paxillin antibody (lower panel). Shown are precipitations with GST protein alone, GST-CRKL (full length), GST-CRKL-SH2, GST-CRKL-SH3(N)-SH3(C), and GST-CRKL-SH2-SH3(N). Molecular mass is shown in kilodaltons.



Tyrosines at Amino Acid Positions 31 and 118, but Not 181, of Paxillin Are Important in Binding to the CRKL-SH2 Domain in Vitro

SH2 domains are known to bind to specific motifs containing a phosphotyrosine residue. However, the optimum binding motif for CRKL has not yet been determined. Since the structures of the c-CRK and CRKL-SH2 domains are quite similar, we examined paxillin for possible c-CRK-SH2 binding motifs, based on the predicted optimum sequence, Y-X-X-P(23) . Three such motifs were identified, at amino acid positions (for the tyrosine residue) 31, 118, and 181. Tyrosine to phenylalanine mutants of paxillin were constructed at each residue, and binding of these paxillin mutants to GST-CRKL-SH2 was examined in vitro after the mutant paxillin proteins were phosphorylated with v-Abl. Mutations of either Tyr-31 or Tyr-118, but not Tyr-181, of paxillin significantly reduced binding of GST-CRKL-SH2 (Fig. 7). The lack of binding of GST-CRKL-SH2 to the mutant paxillin proteins was more than would be expected if the two motifs around Tyr-31 and Tyr-118 contributed equally and independently to binding. It is possible that one of the mutations has an effect that alters the structure of the actual binding site. At the present time, the explanation for this phenomenon is unknown and is being explored with additional mutants.


Figure 7: In vitro binding showing amino acid tyrosines at positions 31 and 118, but not 181, of paxillin are required to bind to the SH2 domain of CRKL. Full-length paxillin (wild type, W.T.) and tyrosine to phenylalanine mutants at amino acid positions 31 (Y31F), 118 (Y118F), or 181 (Y181F) were all expressed in the vector pGEX-2TK. Fusion proteins were isolated as described under ``Materials and Methods'' and thereafter tyrosine-phosphorylated by v-Abl in vitro. The phosphorylated protein product was then thrombin-cleaved and applied to a gradient SDS-PAGE gel (A), transferred, and immunoblotted with anti-paxillin. The same amount of phosphorylated thrombin-cleaved product was then precipitated with 1 µg of SH2-CRKL-GST fusion protein and applied to another SDS-PAGE gradient gel, transferred, and immunoblotted with anti-paxillin (B).




DISCUSSION

BCR/ABL is a unique tyrosine kinase oncogene which transforms hematopoietic cells in vivo and in vitro, but does not effectively transform many nonhematopoietic cell lines. The chimeric oncoprotein p210 produced from the Philadelphia chromosome is known to bind the actin cytoskeleton through a conserved C-terminal domain in ABL, and this interaction is believed to be important for transformation(1, 24, 25) . It has been suggested that once p210 translocates to the cytoskeleton, it recruits signaling proteins which result in aberrant regulation of adhesion, proliferation, and viability(26) .

One of the potentially most interesting proteins that interact with p210 is CRKL. Previous studies from three different laboratories have identified CRKL as a major tyrosine-phosphorylated protein in primary cells or cell lines derived from patients with stable phase CML(2, 3, 11) . The CRKL gene was initially identified independently in the process of generating a long range physical map of the region between the BCR gene and the centromere of chromosome 22q11(12) . An exon of an unknown gene was found to encode a possible SH2 domain, and the complete cDNA sequence revealed a novel crk-like gene, termed CRKL. The CRKL gene product has about 60% amino acid identity to c-Crk. CRKL, like c-CRK, has an SH2 domain at the N terminus of the protein, and two tandem SH3 domains at the C terminus, with no other known functional domains(13) . The structural similarity between the SH2 and SH3 domains of CRKL and c-CRK suggests that they could interact with an overlapping set of proteins(27) . However, in vivo, c-CRK and CRKL may or may not have related functions, and it is largely unknown even if they are in the same subcellular compartment(s).

The finding that CRKL is a prominent substrate for the BCR/ABL tyrosine kinase and coprecipitates with p210, suggested that it could function to link BCR/ABL to other cellular signaling proteins. At present, however, very few binding partners are known for either c-CRK or CRKL. Interestingly, Feller et al.(17) have recently showed that both v-Crk and c-Crk could bind in vitro to c-Abl. This interaction was shown to occur at least in part through the SH3 domains of v-Crk or c-Crk, and possible proline-rich Crk-SH3 binding sites have been identified in c-Abl. In this study, we looked for CRKL-associated proteins, in addition to p210 and c-ABL, in cells transformed by BCR/ABL and found that CRKL coprecipitated with several additional cellular proteins. In the current study, one of these proteins has been identified as the focal adhesion protein paxillin. In vitro binding studies indicated that CRKL bound to paxillin predominantly through the CRKL-SH2 domain, but bound to p210 through the CRKL-SH3 domain, the later finding confirming earlier studies(20) . Taken together, these results suggest that CRKL may indeed function as an adaptor protein, linking p210 to paxillin in CML cells, but not in untransformed cells. In support of this hypothesis, we have shown that a small amount of paxillin coprecipitates with p210 and c-ABL in lysates from CML cell lines, but not from untransformed cell lines. These immune complexes also contain CRKL.

The interaction of p210 with paxillin is not unanticipated. Previous studies have shown that p210 binds to actin in transformed fibroblasts(1) , and we have recently shown that p210 in hematopoietic cells is localized to punctate cytoskeletal structures that contain paxillin and vinculin, two proteins characteristic of focal adhesions(28) . Paxillin was originally identified by a monoclonal antibody produced by Glenney and Zokas (29) during a blind screen for potential substrates of the v-Src tyrosine kinase. A cDNA encoding human paxillin has recently been cloned by our group and predicts the presence of several protein-protein interaction domains in this protein(9) . The C-terminal half of paxillin is composed of four tandem LIM domains. A number of transcription factors such as rhombotin-I and -II have LIM domains(30) , and at least two other cytoskeletal proteins also have LIM domains, zyxin, and cysteine-rich protein. These latter two proteins have been shown to bind to each other through their LIM domains, suggesting that LIM domains are capable of mediating protein interactions(31) . Paxillin also contains a proline-rich domain which is a potential binding site for SH3-containing proteins. The binding sites in paxillin for talin, vinculin, and p125 have been partially localized. Paxillin has also been shown to bind to the SH2 domain of v-Crk in vitro, and recent studies indicate that phosphopeptides with pY-X-X-P (where pY indicates the phosphorylated tyrosine residue) motifs can bind to CRK-SH2 domains(23, 32, 33) . Human paxillin has three of these motifs at tyrosine positions 31, 118, and 181. Schaller and Parsons (33) have recently shown that tyrosines at positions 31 and 118 of paxillin may be important in binding the Crk-SH2 domain.

We investigated the binding of CRKL-SH2 to paxillin in vitro using wild type and tyrosine mutants of paxillin. Each of the tyrosines with the Y-X-X-P motif (at tyrosines position 31, 118, and 181) in paxillin was mutated to phenylalanine for in vitro binding assays. Decreased binding of CRKL-SH2 to paxillin was observed with mutant tyrosines at positions 31 and 118, but not 181. It is not clear why the data suggest that both tyrosine 31 and 118 need to be intact for binding. These two tyrosines could both be important in binding either a single CRKL protein or two different CRKL proteins. Overall, CRKL could link BCR/ABL and paxillin and thereby contribute to the adhesion defects of CML progenitor cells(34, 35) .

The mechanism of transformation of avian cells by v-crk may have some parallels in the transformation of hematopoietic cells by BCR/ABL. v-Crk activates a cellular tyrosine kinase, now believed to be c-Abl(17, 36) . v-Crk has lost the C-terminal SH3 domain through deletion and with it, a potentially important phosphorylation site at Tyr-221 of c-CRK. It has been suggested that Tyr-221 is phosphorylated in resting, nontransformed cells and forms a binding site for the c-CRK-SH2 domain(13) . This intramolecular interaction would be predicted to make the SH2 domain unavailable for binding to other cellular phosphoproteins and could also potentially interfere with the availability of one or both of the c-CRK-SH3 domains. In contrast, the SH2 domain of v-Crk may constitutively be available for binding, while the SH3 domain interacts with one or more proline-rich domains in c-ABL, linking c-ABL to other cellular proteins. In BCR/ABL-transformed cells, CRKL coprecipitates with p210, and as shown here, binds p210 through one or both CRKL-SH3 domains. Since anti-CRKL immune complexes contain a number of proteins in addition to p210 and CRKL, the SH2 domain of CRKL appears to be available for binding. In this study, we have identified one of these binding proteins as paxillin. Interestingly, we did not detect tyrosine phosphorylation of either CRKL or CRK in resting, untransformed, myeloid cell lines, and it is not yet known if the CRKL-SH2 domain will bind through an intramolecular interaction with the tyrosine residue comparable with Tyr-221 of c-CRK (Tyr-207 of CRKL). Also of interest was the finding that CRK-I and -II were not detectably tyrosine-phosphorylated in cells transformed by BCR/ABL. Interestingly, paxillin is also known to be prominently phosphorylated on tyrosine residues in cells transformed by v-crk(23) , and it is quite possible, based on the current studies, that paxillin is linked to c-Abl by v-Crk, functioning as an adaptor protein in v-crk-transformed cells.

Although most of the studies reported here were performed in cell lines transformed by BCR/ABL, the coprecipitation of CRKL and paxillin was confirmed by studying primary neutrophils from patients with CML stable phase. Paxillin and CRKL were found to coimmunoprecipitate in lysates from CML neutrophils but not from normal neutrophils. This is noteworthy since there are only a few proteins in primary CML cells which contain more phosphotyrosine than in normal cells, and CRKL is one of the two most prominent of such proteins(3) . Further studies will be necessary to determine if these interactions lead to aberrant adhesive properties or other biological effects.

The available data suggest a model in which p210 in the cytoskeleton binds to the CRKL-SH3 domain. The SH2 domain of CRKL is free to interact with other proteins such as paxillin. The tyrosines at positions 31 and 118 of paxillin appear to be important in binding to the CRKL-SH2 domain. This trimeric complex may be able to transduce signals which are normally regulated by either integrin or growth factor receptor activation. One testable hypothesis is that p210 mimics or interferes with the signaling normally induced by integrin activation. There are multiple potential biological consequences of such interactions, including altering adhesive properties mediated by integrins, and it has been shown previously that CML cells have characteristically decreased adhesion to fibronectin(34, 35) . Also, interfering with integrin signaling could have effects on cell viability. For example, in some hematopoietic cells, it has been shown that adherence to fibronectin leads to increased apoptosis(37) . Interference with the appropriate signaling pathway could therefore lead to resistance to apoptosis, another property of CML cells. We suggest that the interaction of p210 with paxillin, through CRKL, could contribute to these signaling abnormalities.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants CA60821 (to R. S.), CA36167 (to J. D. G.), CA47456 (to J. G.), a grant from the Deutsche Forschungsgemeinschaft (to M. S.), and a Career Development Award (to R. d. J.). 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.

§
To whom correspondence should be addressed. Tel.: 617-632-3360; Fax: 617-632-4388.

(^1)
The abbreviations used are: CML, chronic myelogenous leukemia; GM-CSF, granulocyte-macrophage colony-stimulating factor; GST, glutathione S-transferase; IL, interleukin; SH2, Src homology region-2; SH3, Src homology region-3; Y-X-X-P, tyrosine-X-X-proline motifs; PAGE, polyacrylamide gel electrophoresis.


REFERENCES

  1. McWhirter, J., and Wang, J. (1991) Mol. Cell. Biol. 11, 1553-1565 [Medline] [Order article via Infotrieve]
  2. ten Hoeve, J., Arlinghaus, R. B., Guo, J. Q., Heisterkamp, N., and Groffen, J. (1994) Blood 84, 1731-1736 [Abstract/Free Full Text]
  3. Oda, T., Heaney, C., Hagopian, J. R., Okuda, K., Griffin, J. D., and Druker, B. J. (1994) J. Biol. Chem. 269, 22925-22928 [Abstract/Free Full Text]
  4. Druker, B., Okuda, K., Matulonis, U., Salgia, R., Roberts, T., and Griffin, J. (1992) Blood 79, 2215-2220 [Abstract]
  5. Andoniou, C. E., Thien, C. B. F., and Langdon, W. Y. (1994) EMBO J. 13, 4515-4523 [Abstract]
  6. Matsuguchi, T., Salgia, R., Hallek, M., Eder, M., Druker, B., Ernst, T., and Griffin, J. (1994) J. Biol. Chem. 269, 5016-5021 [Abstract/Free Full Text]
  7. Ernst, T. J., Slattery, K. E., and Griffin, J. D. (1994) J. Biol. Chem. 269, 5764-5769 [Abstract/Free Full Text]
  8. Matsuguchi, T., Inhorn, R. C., Carlesso, N., Xu, G., Druker, B., and Griffin, J. D. (1995) EMBO J. 14, 257-265 [Abstract]
  9. Salgia, R., Li, J. L., Lo, S. H., Brunkhorst, B., Kansas, G. S., Sobhany, E. S., Sun, Y., Pisick, E., Hallek, M., Ernst, T., Tantravahi, R., Chen, L. B., and Griffin, J. D. (1995) J. Biol. Chem. 270, 5039-5047 [Abstract/Free Full Text]
  10. Tauchi, T., Feng, G. S., Shen, R., Song, H. Y., Donner, D., Pawson, T., and Broxmeyer, H. E. (1994) J. Biol. Chem. 269, 15381-15387 [Abstract/Free Full Text]
  11. Nichols, G. L., Raines, M. A., Vera, J. C., Lacomis, L., Tempst, P., and Golde, D. W. (1994) Blood 84, 2912-2918 [Abstract/Free Full Text]
  12. ten Hoeve, J., Morris, C., Heisterkamp, N., and Groffen, J. (1993) Oncogene 8, 2469-2474 [Medline] [Order article via Infotrieve]
  13. Feller, S. M., Ren, R. B., Hanafusa, H., and Baltimore, D. (1994) Trends Biochem. Sci. 19, 453-458 [CrossRef][Medline] [Order article via Infotrieve]
  14. Mayer, B. J., Hamaguchi, M., and Hanafusa, H. (1988) Nature 32, 272-275
  15. Sawasdikosol, S., Ravichandran, K. S., Lee, K. K., Chang, J.-H., and Burakoff, S. J. (1995) J. Biol. Chem. 270, 2893-2896 [Abstract/Free Full Text]
  16. Mayer, B. J., and Hanafusa, H. (1990) J. Virol. 64, 3581-3589 [Medline] [Order article via Infotrieve]
  17. Feller, S. M., Knudsen, B., and Hanafusa, H. (1994) EMBO J. 13, 2341-2351 [Abstract]
  18. Matulonis, U., Salgia, R., Okuda, K., Druker, B., and Griffin, J. (1993) Exp. Hematol. 21, 1460-1466 [Medline] [Order article via Infotrieve]
  19. Griffin, J. D., Spertini, O., Ernst, T. J., Belvin, M. P., Levine, H. B., Kanakura, Y., and Tedder, T. F. (1990) J. Immunol. 145, 576-584 [Abstract/Free Full Text]
  20. ten Hoeve, J., Kaartinen, V., Fioretos, T., Haataja, L., Voncken, J. W., Heisterkamp, N., and Groffen, J. (1994) Cancer Res. 54, 2563-2567 [Abstract]
  21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  22. Schlaepfer, D. D., Hanks, S. K., Hunter, T., and van der Geer, P. (1994) Nature 372, 786-791 [Medline] [Order article via Infotrieve]
  23. Birge, R. B., Fajardo, J. E., Reichman, C., Shoelson, S. E., Songyang, Z., Cantley, L. C., and Hanafusa, H. (1993) Mol. Cell. Biol. 13, 4648-4656 [Abstract]
  24. Van Etten, R., Jackson, P., Baltimore, D., Sanders, M., Matsudaira, P., and Janmey, P. (1994) J. Cell Biol. 124, 325-340 [Abstract]
  25. McWhirter, J., and Wang, J. (1993) EMBO J. 12, 1533-1546 [Abstract]
  26. Renshaw, M. W., McWhirter, J. R., and Wang, J. (1995) Mol. Cell. Biol. 15, 1286-1293 [Abstract]
  27. Feller, S. M., Knudsen, B., and Hanafusa, H. (1995) Oncogene 10, 1465-1473 [Medline] [Order article via Infotrieve]
  28. Salgia, R., Brunkhorst, B., Pisick, E., Li, J.-L., Lo, S. H., Chen, L. B., and Griffin, J. D. (1995) Oncogene 11, 1149-1155 [Medline] [Order article via Infotrieve]
  29. Glenney, J., and Zokas, L. (1989) J. Cell Biol. 108, 2401-2408 [Abstract]
  30. Sanchez-Garcia, I., and Rabbitts, T. H. (1993) Semin. Cancer Biol. 4, 349-358 [Medline] [Order article via Infotrieve]
  31. Schmeichel, K., and Beckerle, M. (1994) Cell 79, 211-219 [Medline] [Order article via Infotrieve]
  32. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778 [Medline] [Order article via Infotrieve]
  33. Schaller, M., and Parsons, J. (1995) Mol. Cell. Biol. 15, 2635-2645 [Abstract]
  34. Gordon, M. Y., Dowding, C. R., Riley, G. P., Goldman, J. M., and Greaves, M. F. (1987) Nature 328, 342-344 [CrossRef][Medline] [Order article via Infotrieve]
  35. Verfaillie, C. M., McCarthy, J. B., and McGlave, P. B. (1992) J. Clin. Invest. 90, 1232-1241 [Medline] [Order article via Infotrieve]
  36. Ren, R., Ye, Z. S., and Baltimore, D. (1994) Genes & Dev. 8, 783-795
  37. Sugahara, H., Kanakura, Y., Furitsu, T., Ishihara, K., Oritani, K., Ikeda, H., Kitayama, H., Ishikawa, J., Hashimoto, K., Kanayama, Y., and Matsuzawa, Y. (1994) J. Exp. Med. 179, 1757-1766 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.