The Crk-associated substrate (Cas) is a unique docking protein that possesses a repetitive stretch of tyrosine-containing motifs and an Src homology 3 (SH3) domain. Embryonic fibroblasts lacking Cas demonstrated resistance to Src-induced transformation along with impaired actin bundling and cell motility, indicating critical roles of Cas in actin cytoskeleton organization, cell migration, and oncogenesis. To gain further insight into roles of each domain of Cas in these processes, a compensation assay was performed by expressing a series of Cas mutants in Cas-deficient fibroblasts. The results showed that motifs containing YDxP were indispensable for actin cytoskeleton organization and cell migration, suggesting that CrkII-mediated signaling regulates these biological processes. The C-terminal Src-binding domain played essential roles in cell migration and membrane localization of Cas, although it was dispensable in the organization of actin stress fibers. Furthermore, the Src-binding domain was also a prerequisite for Src transformation possibly, because of its crucial role in the phosphorylation of Cas during transformation. Overall, differential uses of the Cas domains in individual biological processes were demonstrated. Cas1 docking protein was initially identified and cloned as a major phosphotyrosine-containing protein in cells transformed by v-src and v-crk oncogenes (1, 2). It has a structure with a number of protein-protein interaction domains, including an N-terminal Src homology 3 (SH3) domain, a substrate domain (SD) that consists of a cluster of YxxP motifs (one YLVP, four YQxPs, nine YDxPs, and one YAVP), and a C-terminal Srcbinding domain containing motifs YDYV (amino acids 762-765) and RPLPSPP (2). The substrate domain offers docking sites for several molecules including adaptor proteins Crk, Nck, and an inositol 5Ј-phosphatase, SHIP2 (SH2-containing inositol 5Ј-phosphatase), through their SH2 domains in a phosphorylation dependent manner (3-5). Motifs RPLPSPP and YDYV in the SB domain serve as direct binding sites for SH3 and SH2

Src family kinases are major regulators of various integrin-mediated biological processes, although their functional roles and substrates in cancer metastasis are unknown. We explored the roles of Src family tyrosine kinases in cell migration and the spread of K-1735 murine melanoma cell lines with low or high metastatic potential. Corresponding to elevated cell motility and spreading ability, Fyn was selectively activated among Src family kinases, and the cell motility was blocked by an inhibitor of Src family kinases. Significant tyrosine phosphorylation of cortactin, stable complex formation between activated Fyn and cortactin, and co-localization of cortactin with Fyn at cell membranes were all observed only in cells with high metastatic potential. Both integrin-mediated Fyn activation and hyperphosphorylation of cortactin were observed 2-5 h after stimulation in highly metastatic cells, and they required de novo protein synthesis. We demonstrate that cortactin is a specific substrate and cooperative effector of Fyn in integrin-mediated signaling processes regulating metastatic potential.Malignant tumors are thought to contain subpopulations of cells with differential metastatic capabilities (1). Processes of tumor metastasis consist of multiple steps linked together, including invasion, detachment, intravasation, circulation, adhesion, extravasation, and growth in distant organs (2). Cell locomotion and spreading are key functions of the cells required in most of these processes. Analysis of differences in expression and modification of signaling molecules associated with cell migration and spreading among tumor sublines with different metastatic potentials in a tumor is expected to provide precise information for understanding the molecular mechanisms underlying the development of cancer metastasis.There are studies demonstrating that Src family tyrosine kinases play essential roles in the signaling of integrin-mediated biological processes such as actin organization and cell migration (3-7). In addition, recent reports show that Src is highly activated in colon cancers, particularly in those metastatic to the liver (8). Involvement of Src kinase during metastatic spread of carcinoma cells in NBT-II rat carcinoma cell lines has also been suggested (5). Fyn has been suggested a factor governing the metastatic potential of tumors including murine methylcholanthrene-induced fibrosarcoma cells (9), although the precise mechanism is unknown.Increasing numbers of studies have shown that Src family kinases function through collaboration with their substrates, such as FAK, cortactin, p130Cas (a Crk-associated substrate), and paxillin in cytoskeleton organization and cell migration. FAK is activated upon cell binding to extracellular matrix proteins and forms transient signaling complexes with Src family kinases (10). Cortactin plays essential roles in cortical actin cytoskeleton organization, primarily affecting cell motility and invasion (11-13). A docking protein, p130Cas , plays essential roles in cell attachme...

Crk-associated substrate (Cas) is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of the actin cytoskeletal organization and cell migration in fibroblasts. The function of Cas in neurons, however, is poorly understood. Here we report that Cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. During cerebellar development, Cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. Cas coimmunoprecipitates with Src family protein tyrosine kinases, Crk, and cell adhesion molecules and colocalizes with these proteins in granule cells. The axon extension of granule cells is inhibited by either RNA interference knockdown of Cas or overexpression of the Cas mutant lacking the YDxP motifs, which are tyrosine phosphorylated and thereby interact with Crk. These findings demonstrate that Cas acts as a key scaffold that links the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation.

The regulation of cytoskeletal components in the dendritic shaft core is critical for dendrite elongation and branching. Here, we report that a brain-specific Ras guanine nucleotide exchange factor (RasGEF) carrying two kinase non-catalytic C-lobe domains (KINDs), very-KIND (v-KIND), regulates microtubule-associated protein 2 (MAP2). v-KIND is expressed in developing mouse brain, predominantly in the cerebellar granule cells. v-KIND not only activates Ras small GTPases via the C-terminal RasGEF domain, but also specifically binds to MAP2 via the second KIND domain (KIND2), leading to threonine phosphorylation of MAP2. v-KIND overexpression suppresses dendritic extension and branching of hippocampal neurons and cerebellar granule cells, whereas knockdown of endogenous v-KIND expression promotes dendrite growth. These findings suggest that v-KIND mediates a signaling pathway that links Ras and MAP2 to control dendrite growth.

VEGF receptor (VEGFR) signaling plays a key role in tumor angiogenesis. Although some VEGFR signaltargeted drugs have been approved for clinical use, their utility is limited by associated toxicities or resistance to such therapy. To overcome these limitations, we developed TAS-115, a novel VEGFR and hepatocyte growth factor receptor (MET)-targeted kinase inhibitor with an improved safety profile. TAS-115 inhibited the kinase activity of both VEGFR2 and MET and their signal-dependent cell growth as strongly as other known VEGFR or MET inhibitors. On the other hand, kinase selectivity of TAS-115 was more specific than that of sunitinib and TAS-115 produced relatively weak inhibition of growth (GI 50 > 10 mmol/L) in VEGFR signal-or MET signalindependent cells. Furthermore, TAS-115 induced less damage in various normal cells than did other VEGFR inhibitors. These data suggest that TAS-115 is extremely selective and specific, at least in vitro. In in vivo studies, TAS-115 completely suppressed the progression of MET-inactivated tumor by blocking angiogenesis without toxicity when given every day for 6 weeks, even at a serum-saturating dose of TAS-115. The marked selectivity of TAS-115 for kinases and targeted cells was associated with improved tolerability and contributed to the ability to sustain treatment without dose reduction or a washout period. Furthermore, TAS-115 induced marked tumor shrinkage and prolonged survival in MET-amplified human cancer-bearing mice. These data suggest that TAS-115 is a unique VEGFR/MET-targeted inhibitor with improved antitumor efficacy and decreased toxicity. Mol Cancer Ther; 12(12); 2685-96. Ó2013 AACR.