Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAKnull mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130 Cas , were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAKrelated kinase Pyk2/CAK␤/RAFTK/CadTK. FAK (focal adhesion kinase) is a widely expressed nonreceptor protein tyrosine kinase found in focal adhesions of cultured cells (23,61). FAK becomes activated by tyrosine phosphorylation in response to integrin clustering achieved by cell adhesion or antibody cross-linking (5,19,23,34,40). FAK Tyr-397 is an autophosphorylation site and a high-affinity binding site for Src homology 2 (SH2) domains of Src family kinases, including c- Src and Fyn (48,62,80). This interaction could contribute both to the recruitment of Src family kinases to sites of cell adhesion and to their catalytic activation through C-terminal tail displacement. Other adhesion-regulated sites of FAK phosphorylation are tyrosines 407, 576, 577, 861, and 925 (7, 8, 65). These tyrosines do not appear to be autophosphorylation sites but are efficiently phosphorylated by c-Src in vitro and elevated in Src-transformed cells (7,8,66). Tyr-397 can also be phosphorylated by c-Src (7); hence, it is not strictly an autophosphorylation site. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and mutation of these residues reduces FAK catalytic activity (7, 42). The potential for reciprocal activation of FAK and Src family kinases suggests a mechanism for signal amplification following an initial integrin-induced FAK autophosphorylation event. Other sites of FAK tyrosine phosphorylation are likely to participate in downstream signaling through recruitment of additional SH2-containing proteins. Indeed, phosphoryl...

During sexual reproduction in most animals, oocytes arrest in meiotic prophase and resume meiosis (meiotic maturation) in response to sperm or somatic cell signals. Despite progress in delineating mitogen-activated protein kinase (MAPK) and CDK/cyclin activation pathways involved in meiotic maturation, it is less clear how these pathways are regulated at the cell surface. The Caenorhabditis elegans major sperm protein (MSP) signals oocytes, which are arrested in meiotic prophase, to resume meiosis and ovulate. We used DNA microarray data and an in situ binding assay to identify the VAB-1 Eph receptor protein-tyrosine kinase as an MSP receptor. We show that VAB-1 and a somatic gonadal sheath cell-dependent pathway, defined by the CEH-18 POU-class homeoprotein, negatively regulate meiotic maturation and MAPK activation. MSP antagonizes these inhibitory signaling circuits, in part by binding VAB-1 on oocytes and sheath cells. Our results define a sperm-sensing control mechanism that inhibits oocyte maturation, MAPK activation, and ovulation when sperm are unavailable for fertilization. MSP-domain proteins are found in diverse animal taxa, where they may regulate contact-dependent Eph receptor signaling pathways. Sexual reproduction requires meiosis to generate haploid (1n) gamete nuclei, which unite after fertilization to form the diploid (2n) totipotent embryo. Despite this universal requirement, meiosis is regulated differently in sperm and oocytes. Whereas sperm proceed through the meiotic divisions uninterrupted, oocytes almost invariably arrest during one, and sometimes two stages following premeiotic DNA replication and meiotic recombination, depending on the species. Therefore, the completion of meiosis in oocytes must be coordinated with development and fertilization to ensure successful reproduction. To achieve this coordination, sperm and somatic cell signals regulate oocyte meiotic progression by activating downstream cyclin-dependent kinase regulatory pathways, which mediate cell cycle transitions in eukaryotes (for review, see Ferrell 1999; Masui 2001).The oocytes of most animals, including the early-diverging sponges and cnidarians (Masui 1985), arrest during meiotic prophase, suggesting that this regulatory mechanism represents a fundamental metazoan reproductive strategy. Human oocytes can remain arrested in prophase for several decades, and aberrant regulation of the first meiotic division is a major cause of infertility, miscarriage, and chromosomal nondisjunction (for review, see Jacobs 1992; Hunt and LeMaire-Adkins 1998). In most animals examined, meiosis resumes in response to nonautonomous signals through a process termed meiotic maturation, which prepares the oocyte for fertilization and embryogenesis. The hallmarks of meiotic maturation include nuclear envelope breakdown, cortical cytoskeletal rearrangement, and meiotic spindle assembly. Studies of Xenopus have identified two key intracellular enzymes, maturation-promoting factor (MPF), a complex consisting of the regulatory protein cyclin B...

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...

The nonreceptor tyrosine kinase FAK (''focal adhesion kinase'') is a key mediator of integrin signaling events controlling cellular responses to the extracellular matrix, including spreading, migration, proliferation, and survival. Integrin-ligand interactions stimulate FAK tyrosine phosphorylation and activation of FAK signaling functions. Here evidence is presented that the FAK autophosphorylation site Tyr-397 mediates a direct interaction with the C-terminal Src homology 2 domain of phospholipase C (PLC)-␥1 and that this is required for both adhesion-dependent association of the two molecules and increased inositol phosphate production in mouse embryo fibroblasts. Overexpression of FAK and PLC-␥1 in COS-7 cells increases PLC-␥1 enzymatic activity and tyrosine phosphorylation, also dependent on FAK Tyr-397. However, FAK appears incapable of directly phosphorylating PLC-␥1. These observations suggest a role for FAK in recruiting PLC-␥1 to the plasma membrane at sites of cellmatrix adhesion and there promoting its enzymatic activity, possibly by releasing the repression caused by intramolecular interactions of the PLC-␥1 Src homology domains and͞or by positioning it for phosphorylation by associated Src-family kinases. These findings expand the known signaling functions of FAK and provide mechanistic insight into integrinstimulation of PLC-␥1.

Paxillin is a prominent focal adhesion docking protein that regulates cell adhesion and migration. Although numerous paxillin-binding proteins have been identified and paxillin is required for normal embryogenesis, the precise mechanism by which paxillin functions in vivo has not yet been determined. We identified an ortholog of mammalian paxillin in Drosophila (Dpax) and have undertaken a genetic analysis of paxillin function during development. Overexpression of Dpax disrupted leg and wing development, suggesting a role for paxillin in imaginal disc morphogenesis. These defects may reflect a function for paxillin in regulation of Rho family GTPase signaling as paxillin interacts genetically with Rac and Rho in the developing eye. Moreover, a gain-of-function suppressor screen identified a genetic interaction between Dpax and cdi in wing development. cdi belongs to the cofilin kinase family, which includes the downstream Rho target, LIM kinase (LIMK). Significantly, strong genetic interactions were detected between Dpax and Dlimk, as well as downstream effectors of Dlimk. Supporting these genetic data, biochemical studies indicate that paxillin regulates Rac and Rho activity, positively regulating Rac and negatively regulating Rho. Taken together, these data indicate the importance of paxillin modulation of Rho family GTPases during development and identify the LIMK pathway as a critical target of paxillin-mediated Rho regulation.