In recent years, members of the protein kinase family have been discovered at an accelerated pace. Most were first described, not through the traditional biochemical approach of protein purification and enzyme assay, but as putative protein kinase amino acid sequences deduced from the nucleotide sequences of molecularly cloned genes or complementary DNAs. Phylogenetic mapping of the conserved protein kinase catalytic domains can serve as a useful first step in the functional characterization of these newly identified family members.

The eukaryotic protein kinases make up a large superfamily of homologous proteins. They are related by virtue of their kinase domains (also known as catalytic domains), which consist of approximately 250-300 amino acid residues. The kinase domains that define this group of enzymes contain 12 conserved subdomains that fold into a common catalytic core structure, as revealed by the 3-dimensional structures of several protein-serine kinases. There are two main subdivisions within the superfamily: the protein-serine/threonine kinases and the protein-tyrosine kinases. A classification scheme can be founded on a kinase domain phylogeny, which reveals families of enzymes that have related substrate specificities and modes of regulation.

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase implicated in integrin-mediated signal transduction pathways and in the process of oncogenic transformation by v-Src. Elevation of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAK phosphorylation in signal transduction events, we used a tryptic phosphopeptide mapping approach to identify tyrosine sites of phosphorylation responsive to both cell adhesion and Src transformation. We have identified four tyrosines, 397, 407, 576, and 577, which are phosphorylated in mouse BALB/3T3 fibroblasts in an adhesion-dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosines 407, 576, and 577, which are previously unrecognized sites, is significantly elevated in the presence of c-Src in vitro and v-Src in vivo. Tyrosines 576 and 577 lie within catalytic subdomain VIII-a region recognized as a target for phosphorylation-mediated regulation of protein kinase activity. We found that maximal kinase activity of FAK immune complexes requires phosphorylation of both tyrosines 576 and 577. Our results indicate that phosphorylation of FAK by Src (or other Src family kinases) is an important step in the formation of an active signaling complex.Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase (PTK) which localizes to focal adhesion structures found in well-spread cultured cells (15,35). Focal adhesions (reviewed in references 3 and 27) are discrete regions associated with the ventral cell surface where integrin receptors interact with adhesive extracellular matrix proteins of the underlying substratum. Inside the cell, the small cytoplasmic tails of clustered ligand-occupied integrins participate in assembly of a protein complex which functions to anchor actin filament bundles, thereby providing the physical support necessary for cell spreading and locomotion. Integrinextracellular matrix interactions at focal adhesion sites not only modulate cell adhesion and shape changes but also influence gene expression (reviewed in reference 20) and contribute to the phenomenon of anchorage dependency for cell growth (reviewed in reference 38). With its discovery, FAK emerged as a likely key element in signal transduction pathways underlying integrin-mediated changes in cell behavior. A potential role for tyrosine phosphorylation in integrin-mediated signaling was earlier suggested from studies showing that phosphotyrosine-containing proteins are greatly enriched in focal adhesions (28) and that proteins with apparent molecular masses of ϳ115 to 130 kDa become tyrosine phosphorylated following clustering of ␤1 integrins brought about by antibody crosslinking (25) or plating cells onto fibronectin-coated dishes (13).

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