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

The related adhesion focal tyrosine kinase (RAFTK) is tyrosine-phosphorylated following β1 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.

The related adhesion focal tyrosine kinase (RAFTK) is tyrosine-phosphorylated following ␤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 p130 Cas 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 p130 Cas and HEF1, phosphorylation of p130 Cas , but not of HEF1, was dependent on an intact autophosphorylation site (Tyr 402 ) on RAFTK. To determine if RAFTK phosphorylated p130 Cas and HEF1 directly or through an intermediate, we assayed the ability of RAFTK and of a Tyr 402 mutant to phosphorylate purified HEF1 and p130 Cas domains. RAFTK was able to phosphorylate the substrate domains of both p130 Cas and HEF1, but only the C-terminal domain of p130 Cas . Furthermore, Tyr 402 , which mediates the binding of RAFTK to c-Src kinase, was required for the phosphorylation of the C-terminal domain of p130 Cas . 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 p130 Cas .
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)(2)(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 CAK␤ (6,7), is tyrosine-phosphorylated following ␤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 p125 FAK (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 p130 Cas 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)(13)(14). p130 Cas 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). p130 Cas 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 (RPLP-SPP) 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 p130 Cas 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 p130 Cas (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 p130 Cas 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 p130 Cas (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 p130 Cas and HEF1. Although RAFTK by itself seems sufficient for HEF1 phosphorylation, a cooperation between RAFTK and Src kinase is required for a complete p130 Cas phosphorylation. These studies suggest that p130 Cas , but not HEF1, activity may be regulated in response to the action of Src and Src family tyrosine kinases.
Cell Transfection-Cos-7 cells were transiently transfected using DEAE-dextran, using p130 Cas cDNA subcloned in pSSR␣ and HEF1 cDNA subcloned in pcDNA3 through restriction digests of overlapping clones (13). p130 Cas cDNAs were cloned into a modified version of the pcDL-SR␣296 expression plasmid termed pSP65-SR␣.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 ⌬SD, in which the sequence from amino acids 213 to 514 was deleted; and Cas ⌬SB, in which the sequence from amino acids 638 to 889 (17) (Fig. 1B). The transfected p130 Cas 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 Na 2 VO 4 ) 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 l 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 ␤-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-P TM 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 MnCl 2 , 5 mM MgCl 2 , 100 M Na 2 VO 4 ) 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.

RAFTK Phosphorylates Both p130 Cas and HEF1 in Vitro and
in Vivo-Since RAFTK can associate with p130 Cas 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 p130 Cas , or Flag-tagged RAFTK, and after 2 days cells were harvested and lysed. p130 Cas 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 (Ϫ or ϩ 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 p130 Cas or HEF1 was subjected to an in vitro kinase assay in the absence (0) or presence of the remaining parts of immunoprecipitated Flag-RAFTK (ϩR) (Fig. 2B). As phosphorylated p130 Cas and HEF1 comigrate with RAFTK, to enable their detection, proteins were eluted with SDS following in vitro kinase assays. p130 Cas 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 p130 Cas or HEF1 with RAFTK led to prominent tyrosine phosphorylation of p130 Cas 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 p130 Cas and HEF1 (Fig. 2B, lower panel), to confirm that equivalent amounts of protein were loaded in each lane.
To determine whether RAFTK was able to phosphorylate p130 Cas and HEF1 in vivo, we performed co-transfections in Cos-7 cells of p130 Cas (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 p130 Cas 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), cotransfection of RAFTK along with p130 Cas 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 p130 Cas 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 (Tyr 402 ) and
Catalytic Activity of RAFTK for p130 Cas and HEF1 Phosphorylation-It has been previously reported that the autophosphorylation site of FAK (Tyr 397 ) interacts with the Src SH2 domain, thus recruiting Src kinase to phosphorylate p130 Cas (24). This tyrosine is conserved in RAFTK (Tyr 402 ) 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 p130 Cas and HEF1. Co-transfection experiments were performed in Cos-7 cells using p130 Cas or HEF1 and each individual RAFTK construct (WT ϭ R, Y402F ϭ 02, K457R ϭ 57) or the vector-Flag alone (F). Transfected RAFTK (Fig. 3A), p130 Cas 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.
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 p130 Cas phosphorylation, and minimally affected HEF1 phosphorylation. Densitometric analysis indicated that the Tyr 402 mutation led to an 80% decrease in p130 Cas phosphorylation but only a 20% de- crease 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 p130 Cas but affected only minimally the phosphorylation of HEF1. No phosphorylation of p130 Cas 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 p130 Cas and HEF1 phosphorylation. Furthermore, the kinase activity alone was sufficient for HEF1 phosphorylation but not necessarily for p130 Cas .
Association of RAFTK and c-Src Mediated by Tyr 402 -Since we observed a decreased phosphorylation of p130 Cas 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 ϭ R, Y402F ϭ 02, K457R ϭ 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.
RAFTK Phosphorylates the Substrate Domain of p130 Cas and HEF1 and the C-terminal Portion of p130 Cas but Not of HEF1-In an attempt to understand the differences in p130 Cas and HEF1 phosphorylation, we investigated which domains of p130 Cas 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 p130 Cas and HEF1 contains a YDYV Src SH2 consensus binding motif. GST fusion proteins containing the p130 Cas 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 (ϩ) or without (Ϫ) 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 p130 Cas and HEF1 were tyrosine-phosphorylated when incubated with WT RAFTK. Similarly, the phosphorylation of the SD domains of p130 Cas and HEF1 was unaffected by mutation of tyrosine 402. In contrast, no phosphorylation of GST SD of p130 Cas 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 p130 Cas and HEF1.
We then examined the phosphorylation of the CT domain of p130 Cas and HEF1 by RAFTK. In contrast to the SD domains of p130 Cas and HEF1, only the CT domain of p130 Cas (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 p130 Cas GST CT Cas/SB. This may account for the decrease in p130 Cas -specific phosphorylation seen with the Tyr 402 mutant observed in Fig. 2. This suggests that phosphorylation of the CT domain of p130 Cas 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 p130 Cas Requires the Presence of Tyr 402 -The previous results indicated that in vitro, Tyr 402 in RAFTK was required to phosphorylate the CT domain of p130 Cas . We investigated whether the in vivo phosphorylation of the CT region of p130 Cas in vivo also required Tyr 402 . 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 ⌬SD, in which the sequence from amino acids 213 to 514 containing the substrate domain was deleted; and Cas ⌬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 ⌬SD mutant led to the phosphorylation of the Cas mutant. The co-expression of the Cas ⌬SD with the Y402F RAFTK led to a dramatic decrease of Cas ⌬SD tyrosine phosphorylation. Therefore, Tyr 402 is necessary for the phosphorylation of the CT domain of Cas in vivo, strongly supporting the involvement of a Src kinase.
The co-transfection of the Cas ⌬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 corre-sponding to the Cas ⌬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 Tyr 402 RAFTK mutant. Therefore we cannot exclude the possibility that some of the phosphorylation of Cas ⌬SB was dependent upon the presence of a Src kinase. However, these results suggest that the SD domain phosphorylation was largely unaffected by the Tyr 402 mutation. Comparable levels of expression of WT Cas and Cas mutants (Fig. 7B) were obtained regardless of the co-transfection conditions. DISCUSSION p130 Cas is a major tyrosine phosphorylated substrate following integrin ligation in several cell types (24,(27)(28)(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, p130 Cas 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 p130 Cas (8,14). In this report we observed that RAFTK is differentially involved in the phosphorylation of HEF1 and p130 Cas . 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 p130 Cas .
In fibroblasts p130 Cas phosphorylation has been reported to be mediated by Src kinases and that the autophosphorylation site of FAK (Tyr 397 ) may act to recruit and/or activate a Src kinase (24). p130 Cas 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 p130 Cas 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 p130 Cas in Src Ϫ 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 p130 Cas . Alternatively, the binding of Src kinase to Tyr 402 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 p130 Cas (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 p130 Cas 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 p130 Cas . We found that RAFTK could not phosphorylate the YDYV motif present in the CT region of HEF1, whereas it phosphorylated the same motif in p130 Cas . Although similar in the C-terminal region, HEF1 does not contain the Src SH3 binding motif present in the p130 Cas 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 p130 Cas and HEF1 to focal contacts. Optimal p130 Cas and HEF1 phosphorylation requires an intact cytoskeleton, since inhibitors of cytoskeletal assembly also inhibit integrin-mediated p130 Cas 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, Tyr 402 is the major Src binding site present in RAFTK.
Our results indicate a distinct role of RAFTK in the differential phosphorylation of HEF1and p130 Cas . Both HEF1 and p130 Cas 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 ␤1 integrin ligation. Moreover, HEF1 is also phosphorylated following BCR ligation, whereas p130 Cas is minimally phosphorylated. RAFTK is also phosphorylated following both ␤1 integrin and BCR stimulation. The finding that RAFTK has a direct role in p130 Cas 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.