Cas-L (pp105), a Crk-associated substrate (p130Cas )-related protein, was first identified as a 105-kDa protein that is tyrosine-phosphorylated following ␤1 integrin cross-linking in T cells. Cas-L contains possible multiple binding sites for the Src homology (SH) 2 domains of various signaling molecules, and appears to be involved in signal transduction through phosphorylated tyrosine-mediated protein-protein interaction. Since Cas-L is preferentially expressed in lymphocytes, it is conceivable that Cas-L plays an important role in lymphocytespecific signals. Here, we show the involvement of Cas-L in the T cell receptor (TCR)/CD3 signaling pathway. Cas-L is transiently phosphorylated following CD3 cross-linking, and tyrosine-phosphorylated Cas-L binds to Crk and C3G. Furthermore, a Cas-L mutant that lacks the SH3 domain, the binding site for focal adhesion kinase (FAK), is also tyrosine-phosphorylated upon CD3 cross-linking, but not upon ␤1 integrin crosslinking, suggesting that FAK is not involved in CD3-dependent Cas-L phosphorylation. Taken together, the present study indicates a novel signaling pathway mediated by tyrosine-phosphorylated Cas-L upon the TCR/CD3 stimulation. T cell receptor (TCR)1 -antigen binding induces gene expression, cytokine production, and cell proliferation in T lymphocytes (1). These TCR-dependent signals are mediated by tyrosine phosphorylation of various proteins including CD3␦, CD3⑀, CD3␥, Shc, Vav,. These signaling molecules appear to be involved in the phosphorylated tyrosinemediated protein-protein interaction and the recruitment of the other signaling molecules containing Src homology (SH) 2 domains (3). The recruitment of these signaling molecules is essential to induce various signals to the downstream events such as the activation of mitogen-activated protein kinases, Ca 2ϩ influx, and transcriptional activation of various genes (1).Thus, protein tyrosine phosphorylation plays a key role during the initial phase of TCR-mediated T cell activation. However, the essential phosphorylated molecule and the precise function of each phosphorylated molecule for these signaling pathwayshave not yet been clarified. Cas-L (pp105) was first identified as a 105-kDa protein that is tyrosine-phosphorylated upon ␤1 integrin cross-linking (4). Cas-L belongs to Cas-family along with p130Cas and Efs/Sin (5-7). The three proteins share the structural characteristics of an N-terminal SH3 domain, followed by multiple (8 to 15) YXXP motifs, which are putative binding sites for the Crk SH2 domain, and a conserved YDYVHL sequence that possibly binds to the Src SH2 domain. (5-7). Cas-L was shown to associate with the C-terminal domain of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase that is localized to focal adhesions (5,8). Recently, we reported that the conserved YDYVHL sequence of Cas-L was phosphorylated by FAK following ␤1 integrin cross-linking and that an Src family tyrosine kinase is recruited to the phosphorylated YDYVHL sequence and causes further tyrosine phosphorylation of ...

Integrin-ligand binding induces the tyrosine phosphorylation of various proteins including focal adhesion kinase (pp125 FAK ) 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 (1-4). 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 (7-10). 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.One of the prime candidates for this tyrosine kinase is focal adhesion kinase, pp125 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 (25-27). Subsequently, a 105-110-kDa p130Cas -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 direc...

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