Tyrosine Phosphorylation of Crk-associated Substrates by Focal Adhesion Kinase

Integrin-ligand binding induces the tyrosine phosphorylation of various proteins including focal adhesion kinase (pp125FAK) and Crk-associated substrate (Cas). FAK is activated and autophosphorylated by the ligation of integrins, although the substrate of FAK has not been revealed. We show here that p130Cas and Cas-L are FAK substrates. FAK directly phosphorylates Cas proteins primarily at the YDYVHL sequence that is conserved among all Cas proteins. Furthermore, the phosphorylated YDYVHL sequence is a binding site for Src family protein-tyrosine kinases, and the recruited Src family kinase phosphorylates the other tyrosine residues within Cas. The Cas-L YDYVHL sequence is phosphorylated upon integrin-ligand binding, and this integrin-mediated tyrosine phosphorylation is inhibited by the cotransfection of the FAK COOH-terminal domain that does not contain a kinase domain. These findings strongly suggest that FAK initiates integrin-mediated tyrosine phosphorylation of Cas proteins; then, Src family tyrosine kinases, which are recruited to phosphorylated Cas and FAK, further phosphorylate Cas proteins.

Integrin-ligand binding induces various biological and biochemical signals in addition to cell adhesion (1)(2)(3)(4). We and others showed the costimulatory effect of the ligation of ␤1 integrin in CD3-dependent T cell proliferation, indicating that integrin-ligand binding can promote cell proliferation (5,6). To reveal the mechanism of the integrin-mediated biological signals, numbers of laboratories have studied the role of protein tyrosine phosphorylation in various cell types (7)(8)(9)(10). However, how proteins are tyrosine phosphorylated upon integrin stimulation is currently unknown. Since integrin themselves are not a tyrosine kinase, a tyrosine kinase(s) that is functionally linked to integrins is essential for the integrin signaling pathway.
Crk-associated substrates (Cas) form a protein family of at least three distinct proteins. p130 Cas is a 120 -130-kDa protein that was first identified as a highly tyrosine-phosphorylated protein in both v-Src-and v-Crk-transformed fibroblasts (25)(26)(27). Subsequently, a 105-110-kDa p130 Cas -related protein (Cas-L or HEF1) was identified as a 105-kDa protein that is tyrosine phosphorylated upon the ligation of ␤1 integrins in T lymphocytes (28,29). Furthermore, an 83-kDa Cas-related protein, Efs (or Sin), was reported as the binding proteins for the Src homology 3 (SH3) domains of Fyn and Src (30,31). All Cas proteins contain one SH3 domain in the NH 2 -terminal region, and they all contain multiple putative binding sites for Src homology 2 (SH2) domains, including 8 -15 binding sites for the Crk SH2 domain. All three Cas proteins also contain a YDYVHL sequence, which is a potential binding site for the Src SH2 domain (31,32). We and others reported that both p130 Cas and Cas-L were tyrosine phosphorylated upon the ligation of integrins and that tyrosine-phosphorylated Cas proteins bind to the SH2 domain-containing proteins Crk, Nck, and SHP-2 (28,33,34). Furthermore, Cas proteins bind directly to FAK (28,29,35,36). These findings suggest the role of Cas proteins in integrin-mediated signaling, especially in the recruitment of Crk and the other signaling molecules containing SH2 domains.
In this study, we show that FAK phosphorylates the YDYVHL motif within Cas proteins and initiates tyrosine phosphorylation of Cas by pleural tyrosine kinases. We also demonstrate the putative involvement of FAK in integrin-mediated tyrosine phosphorylation of Cas proteins.

MATERIALS AND METHODS
Cell Culture and Transfection-Cos-1 cells and 293T cells were obtained from ATCC and Dr. David Baltimore (MIT, Cambridge, MA), respectively. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 20 M Gentamicin (Life Technologies, Inc.). Plasmid DNAs were transfected into Cos-1 cells by the DEAE-Dextran method previously described (28) and transfected into 293T cells by the calcium-phosphate coprecipitation method (37). Usually, 3 g of Cas plasmid DNAs and 4 g of FAK plasmid DNAs were transfected into cells cultured in a 10-cm dish. For fibronectin (FN) stimulation, 293T cells were detached from the substrata by pipetting, washed in Dulbecco's modified Eagle's medium, cultured for 1 h in a dish coated with human FN (Life Technologies, Inc.), and blocked with 1% bovine serum albumin (Sigma) (17).
Plasmids-Hemagglutinin (HA) epitope-tagged FAK was generated by the addition of a sequence, TACCCATACGATGTTCCAGATTAC-GCT, to the 5Ј-end of the coding region of the human FAK cDNA (24). c-Myc-tagged p130 Cas and Cas-L were generated by the addition of a sequence, GAACAGAAACTCATCTCTGAAGAGGATCTG, to the 5Ј-end of rat p130 Cas cDNA and human Cas-L cDNA (27,28). Substitution mutants were generated by polymerase chain reaction as described (24). Tagged FAK and Cas-L were inserted into pMT3, and tagged p130 Cas was inserted into pcDL-SR␣. Thrombin-cleavage sites (LVPRGSA) were generated by the replacement of p130 Cas residues 519 -540 or by insertion between Cas-L residues 406/407. pMES-human Fyn was obtained from Dr. Tadashi Yamamoto (Institute of Medical Science, Tokyo University, Tokyo). pMT3-Crk I was generated from chicken Crk I cDNA obtained from Dr. Bruce J. Mayer (Children's Hospital, Boston).
Immunoprecipitation and Immunoblotting-Cells were lysed in 1% Nonidet P-40 lysis buffer as described (24). The c-Myc-tagged proteins were immunoprecipitated with anti-c-Myc-tag mAb (9E10, Oncogene Science Inc., Manhasset, NY) and protein-A conjugated beads (Pharmacia LKB, Uppsala, Sweden) as described (28). The glutathione Stransferase (GST) fusion protein of the Fyn SH2 domain was generated from a plasmid provided from Dr. Gots Baumann (Sandoz Pharma Ltd., Basel, Switzerland) and was used for precipitation. Immunoprecipitants and lysates were fractionated by SDS-polyacrylamide gel electrophoresis and electrotransfered onto nitrocellulose membranes. Immunoblotting was performed with a primary mAb, horseradish peroxidaseconjugated anti-mouse IgG/anti-rabbit IgG (Amersham Corp.), and chemiluminescence reagents (Renaissance, DuPont NEN) or performed with 125 Iodine-labeled anti-phosphotyrosine mAb (4G10, Upstate Biotechnology Inc.) as described (24). Rabbit anti-Cas-L polyclonal antibody was developed using GST-Cas-L fusion protein containing Cas-L residues 419 -524. 2 Thrombin cleavage was performed with immunoprecipitated protein by incubation with thrombin (Boehringer Mannheim) in phosphate-buffered saline at room temperature overnight. Plasmid DNAs for the GST-FAK kinase domain and GST-Cas-L were generated by the insertion of the cDNA fragments into pGEX (Pharmacia LKB). In vitro kinase reaction was performed by the incubation of GST-FAK kinase domain and GST-Cas-L in the in vitro kinase reaction buffer (30 mM Hepes, pH 7.0, 4 mM MnCl 2 , 8 mM MgCl 2 , and 25 M ATP) at room temperature for 10 min.

Tyrosine Phosphorylation of Cas and Cas-L by FAK-
p130 Cas and Cas-L/pp105 are tyrosine phosphorylated upon the ligation of ␤1 integrins. Because Cas proteins bind to FAK, it was conceivable that Cas proteins are tyrosine phosphorylated by activated FAK following ␤1 integrin-ligand binding. To determine whether this was indeed the case, we coexpressed HA-tagged FAK and c-Myc-tagged p130 Cas or Cas-L in Cos-1 cells and analyzed tyrosine phosphorylation of Cas proteins by immunoprecipitation and immunoblotting. As shown in Fig.  1A, both p130 Cas and Cas-L were tyrosine phosphorylated by the coexpression of wild-type FAK, whereas neither p130 Cas nor Cas-L was tyrosine phosphorylated without FAK coexpression. A kinase negative mutant of FAK (FAK-KN) did not induce any tyrosine phosphorylation of Cas proteins, indicating that this Cas phosphorylation was dependent on the kinase activity of FAK. On the other hand, tyrosine phosphorylation of Cas proteins was observed when coexpressed with the FAK-Y397F mutant, although this phosphorylation was slightly reduced compared with phosphorylation by wild-type FAK. FAK-Y397 is an autophosphorylation site that becomes a binding site for the SH2 domains of Src family tyrosine kinases, and the Y397F mutation inhibits the recruitment of Src family kinases to FAK. This result suggests that FAK directly phosphorylates Cas proteins, although FAK-associated tyrosine kinases also may be involved in FAK-dependent Cas phosphorylation. Furthermore, coexpression of FAK-P715/718A, which does not bind to Cas, resulted in Cas phosphorylation, indicating that the stable binding of FAK to Cas is not necessary for FAK-dependent tyrosine phosphorylation of Cas proteins when FAK is overexpressed. However, when the expression level of FAK is low, the binding of FAK to Cas causes a significant difference in tyrosine phosphorylation of Cas (Fig. 1B, third and seventh lanes from left), suggesting the role of direct binding in the FAK-dependent tyrosine phosphorylation of Cas proteins.
Identification of the FAK-dependent Cas Phosphorylation Sites-We wanted to determine which Cas sites are tyrosine phosphorylated by the FAK coexpression by using Cas mutants. The c-Myc-tagged p130 Cas and Cas-L deletion mutants (schematically shown in Fig. 1C) were expressed in the presence or absence of HA-tagged FAK in Cos-1 cells and were analyzed as described before. As shown in Fig. 2A, two p130 Cas mutants, ⌬SD and ⌬SB, were marginally phosphorylated by FAK, whereas wild-type p130 Cas was well phosphorylated. FAK-dependent phosphorylation of two Cas-L mutants, Cas-L⌬SD and Cas-L624, were both markedly reduced compared with that of wild-type Cas-L. p130 Cas ⌬SD lacks p130 Cas residues 119 -420 that contain multiple YXXP motifs, while p130 Cas ⌬SB lacks residues 449 -701 that contain tyrosine residues including the conserved YDYVHL sequence. Cas-L⌬SD lacks Cas-L residues 63-401 containing YXXP motifs, while Cas-L624 lacks Cas-L residues 625 to COOH-terminal containing the YDYVHL sequence. Taken together, these results suggest that at least two Cas domains, YXXP motifs and a domain containing the YDYVHL sequence, are tyrosine phosphorylated when FAK is coexpressed.
To define the phosphorylation site more precisely, we performed peptide mapping using a Cas-L mutant with a thrombin cleavage site between the YXXP motifs and the YDYVHL sequence. This mutant (Cas-L-Thr) was coexpressed with HAtagged FAK, immunoprecipitated, cleaved by thrombin, and analyzed by immunoblotting. As shown in Fig. 2B, c-Myctagged Cas-L-Thr was cleaved by thrombin into a 52-kDa COOH-terminal peptide, which was detected by anti-Cas-L polyclonal antibody, and 60 -65-kDa NH 2 -terminal peptides, which were detected by anti-c-Myc mAb. In contrast, wild-type Cas-L was not specifically cleaved by thrombin. The 52-kDa peptide was highly tyrosine phosphorylated by FAK, while 60 -65-kDa peptides were also tyrosine phosphorylated. We also generated a similar mutant in p130 Cas (Cas-Thr) and performed peptide mapping. As shown in Fig. 2C, 42/44-kDa COOH-terminal peptides were generated from Cas-Thr by thrombin cleavage and were detected by anti-Cas mAb (Transduction Laboratories, Lexington, KY). These 42/44-kDa peptides were tyrosine phosphorylated by wild-type FAK or by FAK-Y397F, while an 80-kDa NH 2 -terminal peptide was also tyrosine phosphorylated and was detected by anti-c-Myc mAb (data not shown). These mapping analyses further indicate that at least two sites in Cas protein are tyrosine phosphorylated by FAK coexpression. Furthermore, the ratio of tyrosinephosphorylated 80-kDa to phosphorylated 42/44-kDa peptides was reduced when coexpressed with FAK-Y397F compared with that when coexpressed with wild-type FAK. This result suggests the direct involvement of FAK in the phosphorylation of the 42/44-kDa peptide and the involvement of a FAK-Y397associated kinase in the phosphorylation of the 80-kDa peptide.
A p130 Cas mutant with a substitution of the YDYVHL to FDFVHL (CasF) was generated and used to determine the phosphorylation sites. As shown in Fig. 2D, the FAK-dependent tyrosine phosphorylation of CasF (fifth lane from left) was markedly reduced compared with phosphorylation of wild-type Cas (second lane from left). Furthermore, coexpression of the FAK-Y397F mutant resulted in a marginal phosphorylation in CasF (sixth lane from left). These results indicate that a tyrosine residue(s) within the p130 Cas YDYVHL sequence is the major site of tyrosine phosphorylation by FAK and that a tyrosine kinase associated with FAK-Y397 phosphorylates another Cas tyrosine residue. A similar result was observed using a Cas-L mutant with the substitution of YDYVHL to FDFVHL (Cas-LF), although Cas-LF was more tyrosine phosphorylated than CasF by FAK-Y397F (data not shown).
In Vitro Phosphorylation of Cas-L by FAK-To further confirm the direct phosphorylation of Cas by FAK, we investigated the in vitro phosphorylation of Cas-L using bacteria-generated c-Myc-tagged p130 Cas -Thr was coexpressed with HA-tagged FAK or FAK-Y397 and was analyzed as described above. A 80-kDa tyrosine-phosphorylated peptide was not detected by anti-Cas mAb, although degraded peptides were migrated close to this peptide. D, c-Myc-tagged p130 Cas and its phenylalanine mutant CasF were coexpressed with HA-tagged FAK, and tyrosine phosphorylation of Cas proteins was detected as described.
GST-FAK kinase domain fusion protein as a kinase and bacteria-generated GST-Cas-L proteins as substrates. As shown in Fig. 3, Cas-L YXXP motifs (SD) and Cas-L residues 419 -661 were tyrosine phosphorylated by the in vitro kinase reaction using GST-FAK, whereas Cas-L residues 419 -624 were not phosphorylated. The only tyrosine residues localized in Cas-L 625-661 are tyrosines within the YDYVHL sequence. These results indicate that the catalytic domain of FAK can phosphorylate tyrosine residues within the YXXP motifs and the YDYVHL sequence of Cas-L.
Recruitment of Src Family Protein-tyrosine Kinases to the Phosphorylated YDYVHL Sequence-CasF was marginally tyrosine phosphorylated by FAK-Y397, whereas p130 Cas was well phosphorylated, suggesting the involvement of the Cas YDYVHL sequence in tyrosine phosphorylation of the Cas YXXP motifs. To elucidate the function of the tyrosine-phosphorylated YDYVHL sequence, we performed peptide mapping using Cas-L-Thr and its FDFVHL mutant, Cas-LF-Thr. As expected, a thrombin-cleaved 52-kDa peptide containing the YDYVHL sequence was tyrosine phosphorylated in Cas-L-Thr by FAK-Y397F but not in Cas-LF-Thr (Fig. 4A). Moreover, the 60 -65-kDa NH 2 -terminal peptides of Cas-L-Thr were more phosphorylated than the 60 -65-kDa peptides of Cas-LF-Thr, indicating the involvement of the phosphorylated YDYVHL sequence in the tyrosine phosphorylation of these 60 -65-kDa peptides. This finding strongly suggests that the Cas-L YXXP motifs are phosphorylated by a tyrosine kinase that is recruited to the phosphorylated YDYVHL sequence.
The Cas YDYVHL sequence was reported as a binding site for Src family kinases (31,32). To determine whether tyrosine phosphorylation of the YDYVHL sequence by FAK results in the recruitment of Src family kinases, we coexpressed Fyn, an Src family tyrosine kinase, with Cas-L or Cas-LF in the presence or absence of FAK-P715/718A in Cos cells and analyzed the association of Fyn with Cas-L proteins. Unlike p130 Cas , Cas-L does not contain a proline-rich sequence that is a binding site for the Fyn SH3 domain (32). FAK-P715/718A was used to avoid the coprecipitation of Fyn by its association with FAK. As shown in Fig. 4B, p59 Fyn was coprecipitated with Cas-L when coexpressed with FAK (fourth lane from left), whereas Fyn was not coprecipitated without FAK coexpression. Fyn was not coprecipitated with Cas-LF even with FAK coexpression. These results strongly suggest that Fyn was recruited to the tyrosinephosphorylated YDYVHL sequence of Cas-L. To determine that Fyn binds to the YDYVHL sequence via its SH2 domain, we used the GST-Fyn SH2 domain to precipitate Cas proteins. As shown in Fig. 4C, p130 Cas was precipitated by the GST-Fyn SH2 domain when coexpressed with FAK, whereas p130 Cas was not precipitated without FAK coexpression. In contrast, CasF was marginally precipitated by the GST-Fyn SH2 domain. A similar result was obtained using Cas-L and Cas-LF (data not shown). These results strongly suggest that Src family tyrosine kinases are recruited to Cas-L in a FAK-mediated tyrosine phosphorylation-dependent manner.
Integrin-mediated Tyrosine Phosphorylation of Cas Proteins-Cas proteins are tyrosine phosphorylated by FAK in vitro and in Cos cells, although it still remains unclear if Cas proteins are tyrosine phosphorylated by FAK following integrin stimulation. To elucidate the involvement of FAK in integrinmediated Cas phosphorylation, c-Myc-tagged Cas proteins were expressed in human kidney-derived 293T cells. Cells were detached from the substrata and attached to plates coated with FN or poly-L-lysine (PLL, Sigma) and were then lysed. Cas proteins were analyzed by immunoprecipitation and immunoblotting. As shown in Fig. 5A, both p130 Cas and Cas-L showed increased tyrosine phosphorylation following the incubation of cells on FN-coated plates compared with the case incubated on PLL-coated plates. Cas-L was more strongly phosphorylated by FN stimulation than p130 Cas .
Next, we attempted to determine the phosphorylation site in Cas-L following integrin stimulation. Because the major phosphorylation site by FAK was determined to be within the YDYVHL sequence in Cas proteins, integrin-mediated tyrosine phosphorylation of Cas-LF was compared with that of wildtype Cas-L. As shown in Fig. 5B, Cas-LF was poorly tyrosine phosphorylated following integrin stimulation compared with wild-type Cas-L, indicating that the Cas-L YDYVHL is tyrosine phosphorylated upon the ligation of integrins. To further analyze the integrin-mediated phosphorylation sites in Cas-L, we performed peptide mapping of Cas-L-Thr and Cas-LF-Thr. The COOH-terminal 52-kDa peptide was tyrosine phosphorylated following FN stimulation, whereas the 52-kDa peptide from Cas-LF-Thr was not phosphorylated following FN stimulation, indicating that a tyrosine residue(s) within the Cas-L YDYVHL is the integrin-mediated phosphorylation site (Fig. 5C). The NH 2 -terminal 60 -65-kDa peptides of Cas-L-Thr were marginally tyrosine phosphorylated. A similar result was observed by the coexpression of FAK in Cos-1 cells (Fig. 5D). However, when coexpressed with Src-Y527F, Cas-LF-Thr was highly tyrosine phosphorylated, and the 60 -65-kDa NH 2 -terminal peptides from both Cas-L-Thr and Cas-LF-Thr were hyperphosphorylated and shifted to 65-70 kDa. When Crk-I was coexpressed, the 60 -65-kDa peptides were more strongly tyrosine phosphorylated than the 52-kDa COOH-terminal peptide. These data strongly suggest that FAK phosphorylates Cas-L upon integrin stimulation.
To further elucidate the involvement of FAK in the integrinmediated Cas phosphorylation, a FAK deletion mutant that contains the FAK COOH-terminal domain (FAK-CT) was coexpressed with Cas-L, and the effect on integrin-mediated Cas-L phosphorylation was investigated. FAK-CT binds to Cas-L (28) and is localized to focal adhesions (24), although FAK-CT does not contain a kinase domain or Tyr-397 (Fig. 1C). As shown in Fig. 5E, the coexpression of FAK-CT completely inhibited in-tegrin-mediated tyrosine phosphorylation of Cas-L. This result strongly suggests the involvement of FAK in the integrinmediated tyrosine phosphorylation of Cas proteins. DISCUSSION Focal adhesion kinase has been thought to play a key role in integrin-mediated protein tyrosine phosphorylation, although no direct evidence for phosphorylation by FAK has been presented. Two FAK-binding proteins, paxillin and Cas, are the candidates for FAK substrate. We have observed that Cas-L is tyrosine phosphorylated following integrin stimulation in H9 cells (10,28), whereas Cas-L is not tyrosine phosphorylated upon integrin stimulation in Hut78 cells, which are the parental cells for H9. 3 FAK is well expressed in H9 cells, whereas FAK is marginally expressed in Hut78 cells. From this finding, we hypothesized that FAK is essential for the integrin-mediated tyrosine phosphorylation of Cas-L in H9 cells and that FAK may directly phosphorylate Cas-L. However, since Src family tyrosine kinases, Fyn and Src, can stably bind to autophosphorylated FAK (18,21), it is necessary to distinguish FAK kinase activity from FAK-associated Src family kinase activity.
In  (38), although it is necessary to rule out the possible FAK-associated kinase, such as Fyn, in this kind of experiment. We determined that the major phosphorylation site by FAK is within the Cas YDYVHL sequence, and we further demonstrated that an Src family kinase, Fyn, binds to the tyrosine-phosphorylated YDYVHL sequence. FAK has a very stringent substrate specificity, and the only established substrate of FAK is its own Tyr-397. We have demonstrated a second FAK substrate, Cas YDYVHL, which is also a binding site for the Fyn SH2 domain. These findings indicate the strong relevance between FAK substrate specificity and the binding specificity for the SH2 domains of Src family tyrosine kinases. Furthermore, we have shown tyrosine phosphorylation of Cas YXXP motifs by a tyrosine kinase recruited to phosphorylated Cas-YDYVHL and/or FAK-Y397. FAK also can phosphorylate the Cas YXXP motifs at least in vitro, although direct phosphorylation by FAK is weak compared with indirect phosphorylation by a kinase recruited to phosphorylated Cas and FAK in Cos cells.
We have also demonstrated that the Cas-YDYVHL sequence is phosphorylated upon FN stimulation in 293T cells, in which FAK is activated and phosphorylated upon FN stimulation (data not shown). Moreover, this integrin-mediated phosphorylation of Cas-L is inhibited by the overexpression of FAK-CT, further suggesting the involvement of FAK in the integrinmediated Cas phosphorylation. It was also reported that overexpression of the FAK COOH-terminal domain decreases tyrosine phosphorylation of paxillin and tensin and decreases the anti-Tyr(P) staining of focal adhesions (39,40). Taken together, our model for integrin-mediated tyrosine phosphorylation of Cas is as follows. First, integrin-mediated cell adhesion activates FAK. Activated FAK phosphorylates the Cas-YDYVHL and FAK-Y397. Then, an Src family kinase(s) is recruited to the phosphorylation sites, and it phosphorylates Cas YXXP motifs. Tyrosine-phosphorylated YXXP motifs are binding sites for the Crk SH2 domain. These results indicate that a cooperative Cas phosphorylation by FAK and Src family kinases is essential for integrin-mediated recruitment of Crk to Cas. Recent reports demonstrated that overexpression of Crk induces activation of c-Jun NH 2 -terminal kinase (JNK)/stress-activated protein kinase (SAPK) via C3G, a guanine nucleotide exchange factor (41,42). It was reported that Pyk2/Cak␤, a FAK-related tyrosine kinase (43,44), is involved in the activation of JNK/SAPK (45). These findings suggest that FAK may be involved in the activation of JNK/SAPK through tyrosine phosphorylation of Cas proteins and the recruitment of Crk and C3G.
In contrast to our result, it was reported that FAK may not be involved in integrin-mediated tyrosine phosphorylation of p130 Cas based on the analysis of FAK Ϫ/Ϫ cells (46,47). However, we observed that a 120-kDa tyrosine-phosphorylated protein, which binds to paxillin and Cas, is expressed in FAK Ϫ/Ϫ cells (data not shown). Tyrosine phosphorylation of this protein was induced upon integrin stimulation. We identified that this FIG. 5. Integrin-mediated tyrosine phosphorylation of Cas-L. A, c-Myc-tagged p130 Cas and Cas-L were transfected into 293T cells. Then, cells were incubated in the plates coated with FN or PLL. Cas proteins were immunoprecipitated with anti-c-Myc mAb (9E10) and analyzed by immunoblotting. B, c-Myc-tagged Cas-L and Cas-LF were expressed in 293T cells, and tyrosine phosphorylation of Cas-L proteins following FN stimulation was analyzed by the method described. C, mapping of the integrin-mediated phosphorylation site in Cas-L. 293T cells expressing c-Myc-tagged Cas-L-Thr or Cas-LF-Thr were incubated in the plates coated with PLL or FN and then lysed. Cas-L-Thr was immunoprecipitated with anti-c-Myc mAb, cleaved by thrombin, and analyzed by immunoblotting with anti-Cas-L antibody and anti-Tyr(P). D, mapping of the phosphorylation sites in Cas-L. c-Myc-tagged Cas-L-Thr/Cas-LF-Thr were cotransfected with 2 g of pMT3-Crk I, 4 g of pMT3-HA-tagged FAK, or 1 g of pMT3-Src-Y527F into Cos-1 cells. Tyrosine phosphorylation sites were analyzed using anti-Tyr(P). E, c-Myc-tagged Cas-L was transfected into 293T cells with or without c-Myc-tagged FAK-CT. The effect of FAK-CT coexpression was analyzed by the integrin-mediated tyrosine phosphorylation of Cas-L. protein is different from FAK. However, it is highly conceivable that this 120-kDa protein is a FAK-related kinase, such as Pyk2/Cak␤ (43,44), because Pyk2/Cak␤ binds to paxillin and p130 Cas . This 120-kDa protein may substitute for FAK in the integrin-mediated protein tyrosine phosphorylation in FAK Ϫ/Ϫ cells. It was also reported that integrin-mediated p130 Cas phosphorylation is impaired in Src Ϫ/Ϫ cells (38,46,47). Three possible explanations are (i) c-Src recruitment to phosphorylated p130 Cas and FAK may be essential for the detectable phosphorylation of p130 Cas YXXP motifs. Therefore, it may be hard to detect p130 Cas tyrosine phosphorylation upon integrin stimulation in Src Ϫ/Ϫ cells. (ii) c-Src may be involved in FAK activation. Calalb et al. (48) and others (49) showed that v-Src may positively regulate FAK kinase activity, suggesting that c-Src may be essential for FAK activation. (iii) c-Src may regulate ligand-binding activity of integrins, since v-Src phosphorylates the ␤1 integrin cytoplasmic region (50).
Although we have demonstrated that p130 Cas and Cas-L are tyrosine phosphorylated in a similar manner, we observed several differences between these two proteins. The major structural difference between p130 Cas and Cas-L is that p130 Cas contains a proline-rich sequence upstream of the YDYVHL sequence, whereas Cas-L does not contain a proline-rich sequence. This sequence is a binding site for the Src SH3 domain (32) 3 and is suggested to be involved in Src-mediated tyrosine phosphorylation of p130 Cas (32). However, we found that tyrosine phosphorylation of p130 Cas either upon integrin stimulation in 293T cells or by FAK coexpression in Cos cells is much weaker than that of Cas-L. These findings suggest that the Src SH3 domain-mediated interaction is not essential for the recruitment of Src family kinases to Cas proteins or for the Cas phosphorylation by Src family kinases upon integrin stimulation. Putative phosphorylation sites, such as the YDYVHL sequence and the YXXP motifs, are conserved between p130 Cas and Cas-L, suggesting that tyrosine phosphorylation of p130 Cas may be regulated by a slightly different mechanism from that of Cas-L.