Mechanisms of CAS Substrate Domain Tyrosine Phosphorylation by FAK and Src

Abstract

Tyrosine phosphorylation of CAS (Crk-associated substrate, p130Cas ) has been implicated as a key signaling step in integrin control of normal cellular behaviors, including motility, proliferation, and survival. Aberrant CAS tyrosine phosphorylation may contribute to cell transformation by certain oncoproteins, including v-Crk and v-Src, and to tumor growth and metastasis. The CAS substrate domain (SD) contains 15 Tyr-X-X-Pro motifs, which are thought to represent the major tyrosine phosphorylation sites and to function by recruiting downstream signaling effectors, including c-Crk and Nck. CAS makes multiple interactions, direct and indirect, with the tyrosine kinases Src and focal adhesion kinase (FAK), and as a result of this complexity, several plausible models have been proposed for the mechanism of CAS-SD phosphorylation. The objective of this study was to provide experimental tests of these models in order to determine the most likely mechanism(s) of CAS-SD tyrosine phosphorylation by FAK and Src. In vitro kinase assays indicated that FAK has a very poor capacity to phosphorylate CAS-SD, relative to Src. However, FAK expression along with Src was found to be important for achieving high levels of CAS tyrosine phosphorylation in COS-7 cells, as well as recovery of CAS-associated Src activity toward the SD. Structure-functional studies for both FAK and CAS further indicated that FAK plays a major role in regulating CAS-SD phosphorylation by acting as a docking or scaffolding protein to recruit Src to phosphorylate CAS, while a secondary FAK-independent mechanism involves Src directly bound to the CAS Src-binding domain (SBD). Our results do not support models in which FAK either phosphorylates CAS-SD directly or phosphorylates CAS-SBD to promote Src binding to this site. CAS (Crk-associated substate, p130Cas ) was first recognized as a tyrosine-phosphorylated protein in cells transformed by v-Crk or v-Src (27, 38, 52) and later characterized as a docking protein containing multiple protein-protein interaction domains, including a Src-homology 3 (SH3) domain at the N terminus, a Src-binding domain (SBD) near the C terminus, and a large interior substrate domain (SD) (40,47,52). The CAS SH3 domain may function as a molecular switch regulating CAS tyrosine phosphorylation since it interacts with tyrosine kinases focal adhesion kinase (FAK) (22,47,48) and the FAK-related kinase PYK2 (also known as CAK␤, RAFTK, and CADTK) (3, 42) and also with tyrosine phosphatases PTP-1B (35) and PTP-PEST (18). The SBD represents a second site of CAS interaction with tyrosine kinases and consists of a proline-rich motif, RPLPSPP (amino acid residues 639 to 645 in mouse CAS), that can interact with the SH3 domains of Src-family kinases (SFKs) and a nearby tyrosine phosphorylation site (Tyr-668 and/or Tyr-670) that can promote an interaction with the Src-homology 2 (SH2) domain of SFKs (37, 40). CAS-SD, the major region of tyrosine phosphorylation, is characterized by 15 tyrosines present in Tyr-X-X-Pro (YXXP) motifs. When phospho...

Involvement of focal adhesion kinase in cellular invasion of head and neck squamous cell carcinomas via regulation of MMP-2 expression

“…FRNK expression did not affect the expression of paxillin, a FAK-associated focal adhesion component. However, the phosphorylation of paxillin at Tyr 118 decreased in FRNK cells as compared with pWZL cells, consistent with a model of FAK as an upstream modulator of focal adhesion components phosphorylation (Ruest et al, 2001). Stable pool populations of SCC38 cells were also obtained and shown to behave as described for SCC40 cells (data not shown).…”

The substrate domain of BCAR1 is essential for anti-estrogen-resistant proliferation of human breast cancer cells

“…The SH3 domains of BCAR1 and HEF1 exhibit extensive sequence homology (75% identity) and have been found to bind to many different proteins, including FAK, which is involved in the phosporylation of BCAR1 [32]. N-terminal deletion of BCAR1, which eliminates the SH3 domains, did show partially impaired growth of the transfected cells.…”

“…1). A proline rich sequence (RPLPSPP) and a tyrosine phosphorylation site (YDYV) in BCAR1 have been demonstrated to represent a binding site for the SRC kinase and to contribute to cellular functions [32,45]. Individual mutations were introduced into BCAR1 to prevent the binding of SH3 domain-containing proteins to the proline-rich sequence motif and to prohibit phosphorylation of the specific tyrosine residues.…”

“…SRC was shown to bind to the Src-binding site and to phosphorylate several YXXP motifs of BCAR1 [30][31][32][33]. These experiments have shown that BCAR1 phosphorylation may be important for some processes (cell migration), but not essential for anchorage-independent growth of SRC transformed fibroblasts [7,23,24,[34][35][36].…”

Src Points the Way to Biomarkers and Chemotherapeutic Targets

“…47,48 At the C-terminus of Cas, Tyr668 (in the sequence pYDYV) fits the consensus sequence for high-affinity Src SH2 domain binding. Both Tyr668 and the C-terminal polyproline sequence of Cas are known to be binding sites for Src, [49][50][51][52] and mutations in this region result in impaired phosphorylation of Cas in vivo. Other kinases and signaling molecules interact with the C-terminal domain of Cas and related proteins.…”