The Related Adhesion Focal Tyrosine Kinase Differentially Phosphorylates p130Cas and the Cas-like Protein, p105HEF1*

(Received for publication, April 15, 1997, and in revised form, May 15, 1997)

Anne Astier Dagger §, Serge N. Manié Dagger §, Hava Avraham Dagger , Hisamaru Hirai par , Susan F. Law **, Yuzhu Zhang **, Erica A. Golemis **, Yigong Fu , Brian J. Druker Dagger Dagger , Nilou Haghayeghi §, Arnold S. Freedman Dagger §¶¶ and Shalom Avraham Dagger

From the Dagger  Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115,  Division of Experimental Medicine and Hematology/Oncology Research, Beth Israel-Deaconess Medical Center, Boston, Massachusetts 02115, par  Third Department of Internal Medicine, University of Tokyo, Hongo, Tokyo 113, Japan, ** Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, Dagger Dagger  Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland Oregon 97201, and § Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The related adhesion focal tyrosine kinase (RAFTK) is tyrosine-phosphorylated following beta 1 integrin or B cell antigen receptor stimulation in human B cells. Two substrates that are tyrosine-phosphorylated following integrin ligation in B cells are p130Cas and the Cas family member human enhancer of filamentation 1 (HEF1), both of which can associate with RAFTK. In this report we observed that RAFTK was involved in the phosphorylation of these two proteins. While a catalytically active RAFTK was required for both p130Cas and HEF1, phosphorylation of p130Cas, but not of HEF1, was dependent on an intact autophosphorylation site (Tyr402) on RAFTK. To determine if RAFTK phosphorylated p130Cas and HEF1 directly or through an intermediate, we assayed the ability of RAFTK and of a Tyr402 mutant to phosphorylate purified HEF1 and p130Cas domains. RAFTK was able to phosphorylate the substrate domains of both p130Cas and HEF1, but only the C-terminal domain of p130Cas. Furthermore, Tyr402, which mediates the binding of RAFTK to c-Src kinase, was required for the phosphorylation of the C-terminal domain of p130Cas. These data suggest that RAFTK itself is sufficient for HEF1 phosphorylation, whereas a cooperation between RAFTK and Src kinases is required for the complete phosphorylation of p130Cas.


INTRODUCTION

The integrin family of adhesion receptors are involved in transducing signals into cells that result in diverse biologic events, such as the modulation of cell viability, proliferation, and differentiation (1-3). One of the intracellular signaling events initiated by integrins is the activation of a cascade of tyrosine phosphorylation (4). We have previously reported that the related adhesion focal tyrosine kinase, RAFTK,1 (5) also known as PYK2 and CAKbeta (6, 7), is tyrosine-phosphorylated following beta 1 integrin or B cell antigen receptor-mediated (BCR) stimulation in both transformed and normal human B cells (8). This kinase is preferentially expressed in hematopoietic cells and neurons and is distinct from p125FAK (focal adhesion kinase, FAK). Similar to FAK, RAFTK lacks a transmembrane region, does not contain any SH2 or SH3 domains, but does have a proline-rich region in its C terminus (9).

RAFTK interacts with several proteins involved in integrin signaling including paxillin and Src kinases (9-11). RAFTK, like FAK, also interacts constitutively with p130Cas and the Cas-like molecule human enhancer of filamentation 1 (HEF1), two proteins which are tyrosine-phosphorylated after integrin stimulation in lymphoid cells (8, 12-14). p130Cas belongs to a new family of structurally related proteins which are thought to act as "docking molecules," which also includes HEF1/Cas-L and Efs/Sin (15, 16). p130Cas is an SH3 domain-containing molecule with 15 potential Crk-SH2-binding motifs (substrate domain, SD domain), an SH3 binding motif located near the N-terminal region as well as a proline-rich sequence (RPLPSPP) and a YDYV motif, which have been shown to bind to Src SH3 and SH2 domains, respectively, in its C-terminal region (17). HEF1 is 64% homologous to p130Cas and contains also an SH3 domain and multiple SH2 binding motifs, but lacks the proline-rich sequences located near the N-terminal SH3 domain and in the C-terminal region of p130Cas (13). Binding of Crk family members to tyrosine phosphorylated Cas and HEF1 illustrates the assembly of signaling complexes, since the SH3 domain of Crk proteins can bind in turn to a number of proteins including two guanine nucleotide exchange factors, Sos and C3G, which regulate Ras and Rap1 activation, respectively (18-21). The Cas and HEF1 signaling complexes are potentially involved in the propagation of downstream signals.

Integrin-mediated phosphorylation of p130Cas has been shown to be primarily mediated by c-Src in fibroblasts (22). The autophosphorylation site of FAK, tyrosine 397, serves as a Src binding site (23). Following FAK autophosphorylation, Src family kinases are recruited and then phosphorylate p130Cas (24). Similarly, in PC12 cells, the autophosphorylation site of RAFTK, tyrosine 402, binds to Src kinase upon bradykinin activation (11). In this study, we present evidence that RAFTK is involved in the tyrosine phosphorylation of p130Cas and HEF1. Although RAFTK by itself seems sufficient for HEF1 phosphorylation, a cooperation between RAFTK and Src kinase is required for a complete p130Cas phosphorylation. These studies suggest that p130Cas, but not HEF1, activity may be regulated in response to the action of Src and Src family tyrosine kinases.


EXPERIMENTAL PROCEDURES

Cell Lines and Materials

Cos-7 cells were maintained in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum. Antibodies used in this study were directed against phosphotyrosine (4G10 mAb); p130Cas (Cas), c-Src (rabbit polyclonal IgG, Santa Cruz Biotechnology, Santa Cruz, CA); hemagglutinin (HA mAb; Boehringer Mannheim); Flag mAb (M5 antibodies, Eastman Kodak Co.). HEF1 polyclonal antibodies (anti-HEF1) have been previously described (13). The anti-C/H mAb recognizes both p130Cas and HEF1 (p130Cas mAb, Transduction laboratories, Lexington, KY). Affinity-purified rabbit anti-mouse Igs (RAM) were obtained from Jackson Laboratories (West Grove, PA). The following GST fusion proteins were used: substrate domain of p130Cas (SD Cas), amino acids 213-514; C-terminal domain of p130Cas containing the Src SH2 and SH3 binding motifs (CT Cas/SB), amino acids 691-786 (17); substrate domain of HEF1 (SD HEF1), amino acids 82-398, cloned in pGEX1lambdaT vector at EcoRI/XhoI sites; C-terminal domain of HEF1 (CT HEF1), amino acids 626-832 (13) (Fig. 1A).


Fig. 1. Schematic representation of the various constructions used. A, representation of the GST fusion proteins containing the various domains of p130Cas and HEF1 used: substrate domain of p130Cas (SD Cas), amino acids 213-514; C-terminal domain of p130Cas containing the Src SH2 and SH3 binding motifs (CT Cas/SB), amino acids 691-786; substrate domain of HEF1 (SD HEF1), amino acids 82-398; C-terminal domain of HEF1 (CT HEF1), amino acids 626-832. B, p130Cas constructs used for eukaryotic expression vector: Delta SD in which the substrate domain was deleted; Delta SB in which the C-terminal domain containing the Src SH3 and Src SH2 binding motif was deleted.
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Cell Transfection

Cos-7 cells were transiently transfected using DEAE-dextran, using p130Cas cDNA subcloned in pSSRalpha and HEF1 cDNA subcloned in pcDNA3 through restriction digests of overlapping clones (13). p130Cas cDNAs were cloned into a modified version of the pcDL-SRalpha 296 expression plasmid termed pSP65-SRalpha .2-HAtag-Hygro containing a hygromycin B-phosphotransferase gene and an HA epitope tag sequence in frame with the Cas cDNAs (25), using restriction sites XbaI and EcoRI. RAFTK constructs were subcloned into pcDNA3/Flag vector as already described (26). The mutants of Cas included: Cas Delta SD, in which the sequence from amino acids 213 to 514 was deleted; and Cas Delta SB, in which the sequence from amino acids 638 to 889 (17) (Fig. 1B). The transfected p130Cas proteins were fused to the HA peptide, whereas RAFTK was fused to a Flag peptide located downstream of the proteins. Cells were lysed (in 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetamide, 10 mM NaF, 0.4 mM Na2VO4) 48 h after transfection, and immunoprecipitations were then performed.

Immunoprecipitations and Western Blots

Cell lysates were precleared with protein G-Sepharose beads (Pharmacia Biotech Inc.), then incubated with specific antibody for 1 h at 4 °C followed by the addition of 50 µl of protein G-Sepharose beads for 1 h at 4 °C. After four washes with lysis buffer, proteins were eluted with sample buffer (2% SDS, 10% glycerol, 0.7 M beta -mercaptoethanol, 0.1 M Tris, pH 6.8, 0.02% bromphenol blue) and analyzed by 7% SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to Immobilon-PTM membranes (Millipore, Bedford, MA). Membranes were blocked using 5% bovine serum albumin in TBS-T (20 mM Tris, pH 7.6, 130 mM NaCl, 0.1% Tween-20) for anti-phosphotyrosine blotting and with 5% non-fat dried milk in TBS-T for all other antibodies and incubated for 1 h with specific antibodies. Immunoreactive bands were visualized by using secondary horseradish peroxidase-conjugated antibodies (Promega, Madison, WI) and chemiluminescence (Renaissance, DuPont NEN). Densitometric analysis was performed with an LKB Ultrascan XL densitometer.

In Vitro Kinase Assay

Immunoprecipitated proteins or GST fusion proteins were washed twice with lysis buffer, twice with kinase buffer (10 mM Hepes, pH 7.3, 50 mM NaCl, 5 mM MnCl2, 5 mM MgCl2, 100 µM Na2VO4) and then incubated for 30 min at room temperature in kinase buffer containing 0.1 mM ATP (Sigma). Proteins were then eluted by boiling with 0.5% SDS in 50 mM Tris, pH 7.0, 5 mM EDTA, 10 mM dithiothreitol, and subjected to reimmunoprecipitation or boiled directly in sample buffer.


RESULTS

RAFTK Phosphorylates Both p130Cas and HEF1 in Vitro and in Vivo

Since RAFTK can associate with p130Cas as well as HEF1, we investigated whether RAFTK is involved in their tyrosine phosphorylation. Cos-7 cells were transiently transfected with plasmids encoding HEF1, HA-tagged p130Cas, or Flag-tagged RAFTK, and after 2 days cells were harvested and lysed. p130Cas and HEF1 were immunoprecipitated using anti-HA and anti-HEF1 antibodies, respectively, and each immunoprecipitate was divided in two parts. Transfected RAFTK was immunoprecipitated using anti-Flag antibodies and divided into four aliquots. Two aliquots of the immunoprecipitated Flag-RAFTK were subjected to an in vitro kinase assay (- or + ATP) (Fig. 2A) and analyzed by Western blot with anti-phosphotyrosine antibody to assess the autophosphorylation of the kinase (upper panel). As seen in Fig. 2A, an increase in the tyrosine phosphorylation of RAFTK was seen in the presence of ATP. Immunoprecipitated p130Cas or HEF1 was subjected to an in vitro kinase assay in the absence (0) or presence of the remaining parts of immunoprecipitated Flag-RAFTK (+R) (Fig. 2B). As phosphorylated p130Cas and HEF1 comigrate with RAFTK, to enable their detection, proteins were eluted with SDS following in vitro kinase assays. p130Cas or HEF1 were then reimmunoprecipitated using antibody specific for each protein and analyzed by Western blotting using anti-phosphotyrosine antibodies (Fig. 2B, upper panel). As seen in Fig. 2B (upper panel), the incubation of immunoprecipitated p130Cas or HEF1 with RAFTK led to prominent tyrosine phosphorylation of p130Cas and HEF1. The membranes were stripped and reprobed with anti-Flag antibody (Fig. 2A, lower panel) or with anti-C/H antibody, which recognizes both p130Cas and HEF1 (Fig. 2B, lower panel), to confirm that equivalent amounts of protein were loaded in each lane.


Fig. 2. RAFTK phosphorylates p130Cas and HEF1 in vitro and in vivo. Cos-7 cells were transiently transfected with an expression vector encoding HA-tagged p130Cas or HEF1, or with a vector encoding Flag-tagged RAFTK and lysed after 2 days. Half of immunoprecipitated Flag-RAFTK was subjected to an in vitro kinase assay with (+) or without (-) ATP to assess its autophosphorylation, analyzed by Western blotting with anti-Tyr(P) (P-tyr) antibodies and anti-Flag antibodies (A). Transfected p130Cas or HEF1 were immunoprecipitated and mixed with buffer alone (0) or with the remaining half of the immunoprecipitated RAFTK (+R), and an in vitro kinase assay was performed. p130Cas or HEF1 were then reimmunoprecipitated and analyzed by Western blot with anti-Tyr(P) (P-tyr) antibodies. The membranes were then stripped and reblotted with an antibody that recognizes both Cas and HEF (anti-C/H) (B). C, co-transfections were performed in Cos-7 cells with p130Cas (C) or HEF1 (H) along with the vector encoding Flag alone (F) or Flag-RAFTK (R). Transfected p130Cas or HEF1 were immunoprecipitated and analyzed by Western blot with anti-Tyr(P) (P-tyr) antibodies. The membrane was then stripped and reblotted with anti-C/H antibody.
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To determine whether RAFTK was able to phosphorylate p130Cas and HEF1 in vivo, we performed co-transfections in Cos-7 cells of p130Cas (C) or HEF1 (H) with the vector encoding Flag-RAFTK (C/R or H/R) or with a vector Flag control (C/F or H/F). After 2 days, transfected p130Cas or HEF1 were immunoprecipitated using anti-HA or anti-HEF1 antibodies, respectively, and analyzed by Western blot using anti-phosphotyrosine antibodies. As seen in Fig. 2C (upper panels), co-transfection of RAFTK along with p130Cas or HEF1 led to the in vivo tyrosine phosphorylation of the two proteins. The membrane was stripped and reprobed with anti-C/H antibody to confirm that equivalent amounts of p130Cas and HEF1 proteins were loaded in each lane (Fig. 2C, lower panels). These results indicate that RAFTK participates in Cas and HEF1 phosphorylation in vitro and in vivo.

Involvement of the Autophosphorylation Site (Tyr402) and Catalytic Activity of RAFTK for p130Cas and HEF1 Phosphorylation

It has been previously reported that the autophosphorylation site of FAK (Tyr397) interacts with the Src SH2 domain, thus recruiting Src kinase to phosphorylate p130Cas (24). This tyrosine is conserved in RAFTK (Tyr402) and has also been shown to be a Src SH2 binding motif (11). We compared the ability of wild type (WT) RAFTK, a Y402F RAFTK mutant, and a K457R RAFTK mutant that abolishes the RAFTK catalytic activity, to induce the phosphorylation of p130Cas and HEF1. Co-transfection experiments were performed in Cos-7 cells using p130Cas or HEF1 and each individual RAFTK construct (WT = R, Y402F = 02, K457R = 57) or the vector-Flag alone (F). Transfected RAFTK (Fig. 3A), p130Cas or HEF1 (Fig. 3B) were immunoprecipitated, using anti-Flag, anti-HA, or anti-HEF1 antibodies respectively, and analyzed by Western blot with anti-phosphotyrosine antibody.


Fig. 3. Involvement of Tyr402 of RAFTK in p130Cas phosphorylation. Cos-7 cells were co-transfected with the vector encoding Flag alone (F), wild-type RAFTK (R), a Y402F mutated RAFTK (02), a K457R mutated RAFTK (57), and p130Cas (C) or HEF1 (H). Transfected RAFTK (A) and p130Cas or HEF1 (B) were immunoprecipitated and analyzed by Western blot with anti-Tyr(P) (P-tyr) antibodies. The membrane was then stripped and reblotted with anti-C/H antibodies or anti-Flag antibodies as indicated.
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The analysis by Western blot with anti-phosphotyrosine of the different Flag-RAFTK mutants showed that a basal RAFTK tyrosine phosphorylation was observed with the WT RAFTK (C/R and H/R), whereas no basal phosphorylation was observed with the two mutants of RAFTK expressed in Cos-7 cells (Fig. 3A). Reblotting of the membrane demonstrated that equivalent amounts of RAFTK proteins were expressed and immunoprecipitated (Fig. 3A, lower panel, anti-Flag blot).

As shown in Fig. 3B, when compared with WT RAFTK, the RAFTK Y402F mutation markedly decreased p130Cas phosphorylation, and minimally affected HEF1 phosphorylation. Densitometric analysis indicated that the Tyr402 mutation led to an 80% decrease in p130Cas phosphorylation but only a 20% decrease in HEF1 phosphorylation. Although the anti-C/H antibody does not recognize hyperphosphorylated HEF1 in the H/R and H/02 lanes as well as the unphosphorylated protein in H/F or H/57 lanes, equivalent amounts of protein were observed in H/R and H/02 lanes. Therefore, mutation of the putative binding site of Src kinase markedly decreased the phosphorylation of p130Cas but affected only minimally the phosphorylation of HEF1. No phosphorylation of p130Cas or HEF1 was observed when either were co-transfected with the K457R mutant of RAFTK (Fig. 3B, C/57 and H/57). This indicates that the kinase activity of RAFTK was required for p130Cas and HEF1 phosphorylation. Furthermore, the kinase activity alone was sufficient for HEF1 phosphorylation but not necessarily for p130Cas.

Association of RAFTK and c-Src Mediated by Tyr402

Since we observed a decreased phosphorylation of p130Cas in the presence of the Y402F RAFTK mutant, a binding site for Src kinases, we investigated whether tyrosine 402 of RAFTK recruited c-Src in Cos-7 cells. Cos-7 cells were transfected with the Flag-vector (F) or with the vector encoding each of the Flag-RAFTK proteins (WT = R, Y402F = 02, K457R = 57) and lysed after 2 days. The cell lysates were divided into two aliquots, and c-Src or Flag-RAFTK were immunoprecipitated and analyzed by Western blotting with anti-phosphotyrosine (Fig. 4A). WT RAFTK (R) was co-immunoprecipitated with c-Src, but not the Y402F (02) or the K457R (57) RAFTK mutants as shown by reblotting of the membrane with anti-Flag antibody (Fig. 4B, upper panel). Furthermore, in a reciprocal experiment, c-Src could also be detected in WT RAFTK immunoprecipitate, but not in the Y402F immunoprecipitate (Fig. 4B, bottom panel, blot c-Src). Therefore, when expressed in Cos-7 cells, WT RAFTK co-immunoprecipitated with c-Src, and this binding required tyrosine 402. 


Fig. 4. The association of c-Src and RAFTK is mediated by Tyr402. Cos-7 cells were transfected with the Flag-vector (F), WT RAFTK (R), the Y402F mutant (02), or the K457R mutant (57). c-Src and Flag-RAFTK were immunoprecipitated and analyzed by Western blot with anti-Tyr(P) (P-tyr) antibodies (A). The membrane was then stripped and reblotted with anti-Flag and anti-c-Src antibodies (B).
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RAFTK Phosphorylates the Substrate Domain of p130Cas and HEF1 and the C-terminal Portion of p130Cas but Not of HEF1

In an attempt to understand the differences in p130Cas and HEF1 phosphorylation, we investigated which domains of p130Cas or HEF1 were phosphorylated by RAFTK. Both proteins have a substrate domain which contains multiple YDXP Crk SH2 consensus binding motifs. The C-terminal domain of both p130Cas and HEF1 contains a YDYV Src SH2 consensus binding motif. GST fusion proteins containing the p130Cas or HEF1 substrate domain (SD) (Fig. 5) or the C-terminal domain (CT) (Fig. 6) were used as substrates for RAFTK. Transfected WT RAFTK or mutated RAFTK were immunoprecipitated from Cos-7 cell lysates using anti-Flag antibody and mixed with the different GST fusion proteins as indicated (Figs. 5 and 6). A kinase assay was then performed on each sample for 30 min with (+) or without (-) ATP, and phosphorylation of the GST fusion proteins was assessed by anti-phosphotyrosine immunoblotting. As seen in Fig. 5, an increase in phosphorylation of WT RAFTK (R) (indicated with an arrow) was observed after the kinase assay, as has already been observed in Fig. 2. The Y402F mutant (02) was also phosphorylated in vitro, indicating that the tyrosine 402 is not the only site of in vitro autophosphorylation. However, no phosphorylation of the K457R mutant was observed. The SD domains of both p130Cas and HEF1 were tyrosine-phosphorylated when incubated with WT RAFTK. Similarly, the phosphorylation of the SD domains of p130Cas and HEF1 was unaffected by mutation of tyrosine 402. In contrast, no phosphorylation of GST SD of p130Cas or HEF1 was detected in the presence of the K457R RAFTK mutant with inactivated kinase activity. These data suggest that RAFTK directly phosphorylates the SD domain of p130Cas and HEF1.


Fig. 5. RAFTK phosphorylates the substrate domain of both p130Cas and HEF1. GST fusion proteins containing the substrate domain (SD) of p130Cas (SD/Cas) or HEF1 (SD/HEF1) were subjected to an in vitro kinase assay with (+) or without (-) ATP in the presence of the wild-type RAFTK (R), the Y402F mutant (02), or the K457R mutant (57). Proteins were then analyzed by Western blot with anti-Tyr(P) (P-tyr) antibodies. The membrane was then stripped and reblotted with anti-Flag or anti-GST antibodies as indicated.
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Fig. 6. RAFTK is involved in the phosphorylation of the C terminus portion of p130Cas but not of HEF1. GST fusion proteins containing the C terminus part of p130Cas (CT Cas/SB) or HEF1 (CT/HEF1) were subjected to an in vitro kinase assay with (+) or without (-) ATP in presence of the wild-type RAFTK (R), the Y402F mutant (02), or the K457R mutant (57). Proteins were then analyzed by Western blot with anti-Tyr(P) (P-tyr) antibodies. The membrane was then stripped and reblotted with anti-Flag or anti-GST antibodies as indicated.
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We then examined the phosphorylation of the CT domain of p130Cas and HEF1 by RAFTK. In contrast to the SD domains of p130Cas and HEF1, only the CT domain of p130Cas (CT Cas/SB) was phosphorylated by RAFTK and no phosphorylation of the CT domain of HEF1 (CT HEF1) was observed (indicated by arrows) (Fig. 6). GST CT HEF1 did not comigrate with the Ig heavy chain, since we were able to detect the Ig chain above the GST fusion protein when the membrane was reprobed with anti-GST antibodies, as indicated in Fig. 6. Interestingly, the Y402F mutation of RAFTK decreased the phosphorylation of p130Cas GST CT Cas/SB. This may account for the decrease in p130Cas-specific phosphorylation seen with the Tyr402 mutant observed in Fig. 2. This suggests that phosphorylation of the CT domain of p130Cas is dependent upon the presence of the tyrosine 402, and therefore likely involves a Src kinase. It also implies that despite an identical Src SH2 consensus sequence present in the C-terminal region of HEF1, the Src kinase is unable to phosphorylate this domain of HEF1.

The in Vivo Phosphorylation of the CT Region of p130Cas Requires the Presence of Tyr402

The previous results indicated that in vitro, Tyr402 in RAFTK was required to phosphorylate the CT domain of p130Cas. We investigated whether the in vivo phosphorylation of the CT region of p130Cas in vivo also required Tyr402. cDNAs encoding HA-tagged wild-type Cas or deletion mutants of Cas were transiently expressed in Cos-7 cells with the Flag-vector (F) or the vector encoding the WT RAFTK (R) or the Y402F (02) and the K457R (57) mutants. Two deleted-Cas mutants were used (Fig. 1B): Cas Delta SD, in which the sequence from amino acids 213 to 514 containing the substrate domain was deleted; and Cas Delta SB, in which the sequence from amino acids 638 to 889, containing the Src SH2 and SH3 binding motifs in the CT, was deleted (17). Transfected Cas proteins were immunoprecipitated with anti-HA antibody and analyzed by anti-phosphotyrosine immunoblotting (Fig. 7A). As shown previously, a decrease in WT Cas phosphorylation was observed with the Y402F RAFTK mutant, and no phosphorylation was observed with the RAFTK K457R mutant. Co-transfection of WT RAFTK and Cas Delta SD mutant led to the phosphorylation of the Cas mutant. The co-expression of the Cas Delta SD with the Y402F RAFTK led to a dramatic decrease of Cas Delta SD tyrosine phosphorylation. Therefore, Tyr402 is necessary for the phosphorylation of the CT domain of Cas in vivo, strongly supporting the involvement of a Src kinase.


Fig. 7. The in vivo phosphorylation of the SB region of p130Cas depends on the presence of Tyr402 of RAFTK. Cos-7 cells were co-transfected with the Flag-vector (F), wild-type RAFTK (R), the Y402F mutant (02), or the K457R mutant (57) and with cDNAs encoding HA-tagged WT-Cas, Cas Delta SD, or Cas Delta SB. Transfected Cas proteins were immunoprecipitated with anti-HA antibody and analyzed by anti-Tyr(P) (P-tyr) immunoblotting (A). The membrane was then stripped and reblotted with anti-Cas antibodies (B).
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The co-transfection of the Cas Delta SB mutant with WT RAFTK led to the detection of a smear containing two discrete phosphorylated bands, probably corresponding to different levels of phosphorylation. The phosphorylation of the lower band corresponding to the Cas Delta SB mutant after reblotting with anti-Cas antibodies was not affected by the Y402F RAFTK mutation. The phosphorylation of the upper band was decreased in the presence of Tyr402 RAFTK mutant. Therefore we cannot exclude the possibility that some of the phosphorylation of Cas Delta SB was dependent upon the presence of a Src kinase. However, these results suggest that the SD domain phosphorylation was largely unaffected by the Tyr402 mutation. Comparable levels of expression of WT Cas and Cas mutants (Fig. 7B) were obtained regardless of the co-transfection conditions.


DISCUSSION

p130Cas is a major tyrosine phosphorylated substrate following integrin ligation in several cell types (24, 27-29). Following integrin stimulation of normal B cells and B cell lines corresponding to various stages of ontogeny, the Cas-like protein HEF1 is the major tyrosine-phosphorylated substrate (14). However, in terminally differentiated B cell lines, p130Cas is also a prominent substrate after integrin stimulation. Therefore, these proteins are differentially phosphorylated in B cells, and probably mediate distinct functions. We previously showed that RAFTK, a tyrosine kinase with significant homology with the FAK was tyrosine-phosphorylated after integrin or BCR stimulation in human B cells. In addition, RAFTK can associate with either HEF1 or p130Cas (8, 14). In this report we observed that RAFTK is differentially involved in the phosphorylation of HEF1 and p130Cas. Our results suggest that, at least in COS-7 cells under the conditions used, RAFTK by itself appeared sufficient for HEF1 phosphorylation, whereas cooperation between RAFTK and Src kinases was required for the complete phosphorylation of p130Cas.

In fibroblasts p130Cas phosphorylation has been reported to be mediated by Src kinases and that the autophosphorylation site of FAK (Tyr397) may act to recruit and/or activate a Src kinase (24). p130Cas phosphorylation was reduced in fibroblasts lacking Src kinases but remained unaffected in fibroblasts lacking FAK (22, 24). It has been suggested that Src kinases act as a "bridge" between p130Cas and FAK, since the expression of a mutated Src containing only the SH2 and SH3 domains but no catalytic domain, was able to restore the phosphorylation of p130Cas in Src- fibroblasts (30). Similarly, our results suggest that one of the autophosphorylation sites of RAFTK, the tyrosine 402, recruits a Src kinase, c-Src in Cos-7 cells, that will phosphorylate the C-terminal part of p130Cas. Alternatively, the binding of Src kinase to Tyr402 in RAFTK could also lead to an enhanced kinase activity of RAFTK, as described for FAK (31). However, it has also been reported that the Src-mediated enhanced FAK/Cas association was observed in the absence of the autophosphorylation site of FAK and of the SH2 domain of Src kinase (32). We found that RAFTK was still able to phosphorylate a Cas mutant lacking the C-terminal Src binding region. This result suggests that RAFTK could still directly phosphorylate Cas without binding via a Src kinase to the C-terminal Src binding region. A possible explanation for this is that RAFTK can bind to the SH3 domain of p130Cas (data not shown) through its SH3 binding motif.

RAFTK was observed to be involved in the phosphorylation of the substrate domains of both Cas and HEF1, and this was dependent on the kinase activity of RAFTK. The phosphorylation of the substrate domains of p130Cas and HEF1 by RAFTK can then lead to their potential associations with Crk family members, allowing the propagation of the signals toward Ras activation (30). Although we previously reported that Cas associated with RAFTK is mainly nonphosphorylated on tyrosine residues, following phosphorylation Cas and RAFTK may rapidly dissociate. In contrast, RAFTK appears to have a different role in the phosphorylation of the C-terminal region of HEF1 and p130Cas. We found that RAFTK could not phosphorylate the YDYV motif present in the CT region of HEF1, whereas it phosphorylated the same motif in p130Cas. Although similar in the C-terminal region, HEF1 does not contain the Src SH3 binding motif present in the p130Cas C terminus (SB) region (13). The lack of Src SH3 binding motif in HEF1 might explain the inability of RAFTK, in association with a Src kinase, to phosphorylate the CT region. However, we could not exclude that the conformation of the CT HEF1 GST fusion protein folds in a way that makes the tyrosine inaccessible to the kinases. RAFTK has been shown to localize to focal contact-like structures in adherent megakaryocytic cell line CMK and in RAFTK-transfected COS cells adherent to fibronectin (26). Therefore, one function of RAFTK may be to localize p130Cas and HEF1 to focal contacts. Optimal p130Cas and HEF1 phosphorylation requires an intact cytoskeleton, since inhibitors of cytoskeletal assembly also inhibit integrin-mediated p130Cas and HEF1 tyrosine phosphorylations (14). However, the phosphorylation of the C-terminal region of HEF1 may require another interacting protein(s) despite the presence of RAFTK and Src kinases.

The in vitro kinase assays performed with RAFTK suggested that the tyrosine 402 was not the only site of autophosphorylation of RAFTK, since an increase in phosphorylation was observed with the Y402F mutant. However, no basal phosphorylation of the Y402F mutant was observed in vivo in Cos-7 cells, indicating that the tyrosine 402 is probably the principal site of autophosphorylation in vivo. Furthermore, this tyrosine is the only site of binding for c-Src kinases, since no binding could be observed with the Y402F mutant. Therefore, Tyr402 is the major Src binding site present in RAFTK.

Our results indicate a distinct role of RAFTK in the differential phosphorylation of HEF1and p130Cas. Both HEF1 and p130Cas are expressed in normal and neoplastic B cells and most B cell lines. HEF1 is the major phosphorylated substrate in normal tonsillar B cells and most B cell lines, whereas Cas is the predominant substrate which is phosphorylated in terminally differentiated B cell lines following beta 1 integrin ligation. Moreover, HEF1 is also phosphorylated following BCR ligation, whereas p130Cas is minimally phosphorylated. RAFTK is also phosphorylated following both beta 1 integrin and BCR stimulation. The finding that RAFTK has a direct role in p130Cas and HEF1 phosphorylation might allow a better understanding of these distinct signaling pathways, and further insights into the potential cross-talk that has been observed between integrin and antigen receptor signaling.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grants CA55207 and CA66996, American Cancer Society (ACS) Grant DHP-145 (to A. S. F.), a Fellowship from the Lymphoma Foundation of America (to S. N. M.), National Institutes of Health Grants HL55445 (to S. A.) and HL51456 (to H. A.), National Institutes of Health Training Grants CA09035 (to S. F. L.) and T32 CA09035 (to Y. Z), ACS Grant CB-74749, and National Institutes of Health Grant R29-CA63366 (to E. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶¶   To whom correspondence and reprint requests should be addressed: Division of Hematologic Malignancies, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3441; Fax: 617-632-5167; E-mail: arney_freedman{at}dfci.harvard.edu.
1   The abbreviations used are: RAFTK, related adhesion focal tyrosine kinase; p125FAK, FAK, focal adhesion kinase; p105HEF1, HEF1, human enhancer of filamentation 1; SH, Src homology; BCR, B cell antigen receptor; HA, hemagglutinin; RAM, rabbit anti-mouse Ig; mAb, monoclonal antibody; GST, glutathione S-transferase; SD, substrate domain; CT, C-terminal domain.

ACKNOWLEDGEMENTS

We greatly appreciate Dr. Andreas Beck for helping in preparing p130Cas constructs. We also thank Dr. Bernard Mari for helpful discussions. We thank Janet Walsh for assistance in preparing the manuscript.


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