Tyrosine Phosphorylation of Crk-associated Substrates by Focal Adhesion Kinase

Abstract

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...

Alterations in the focal adhesion kinase/Src signal transduction pathway correlate with increased migratory capacity of prostate carcinoma cells

“…In addition, substrates of the FAK/Src bipartite kinase complex have been identi®ed as regulators of migration (Cary et al, 1998;Klemke et al, 1998;Petit et al, 2000). Speci®cally, FAK/Srcmediated phosphorylation of Cas or paxillin creates binding sites for the adapter protein Crk (Hamasaki et al, 1996;Harte et al, 1996;Schaller and Parsons, 1995;Tachibana et al, 1997), which binds to DOCK180 (Kiyokawa et al, 1998b;Matsuda et al, 1996;Posern et al, 1998), an activator of Rac (Kiyokawa et al, 1998a;Nolan et al, 1998), thereby directing Rac activity to sites of integrin engagement with the ECM to stimulate membrane ru ing and cell migration (Cheresh et al, 1999;Hasegawa et al, 1996;Reddien and Horvitz, 2000;Wu and Horvitz, 1998). Indeed, Cas over expression in FG carcinoma cells and Chinese hamster ovary cells stimulates motility (Cary et al, 1998;Klemke et al, 1998).…”

Attenuation of Adhesion-dependent Signaling and Cell Spreading in Transformed Fibroblasts Lacking Protein Tyrosine Phosphatase-1B

“…Upon integrin stimulation, the adapter protein p130 Cas also becomes tyrosine phosphorylated by Src and/or p125 FAK (8,13,14). Tyrosine-phosphorylated p130…”

Temporal Dissection of β1-Integrin Signaling Indicates a Role for p130Cas-Crk in Filopodia Formation

“…FAK is a tyrosine kinase that is activated early in response to integrin stimulation and is involved in regulating the turnover of cell-matrix adhesions (24,25). This kinase, together with members of the Src family of cytosolic tyrosine kinases, binds and phosphorylates the docking proteins p130Cas and paxillin (26,27). Phosphorylated p130Cas can interact with Crk, which in turn binds Dock180, which is an unconventional Rac-guanine nucleotide exchange factor that activates Rac1 (28 -32).…”