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...

By reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry, MGSA-alpha, -beta, -gamma, and CXCR2 mRNA expression and proteins are detected in 7 out of 10 human melanoma lesions. The biological consequence of constitutive expression of the MGSA/GRO chemokine in immortalized melanocytes was tested in SCID and nude mouse models. Continuous expression of MGSA/GRO-alpha, -beta, or -gamma in immortalized melan-a mouse melanocytes results in nearly 100% tumor formation for each of the clones tested, whereas clones expressing only the neomycin resistance vector form tumors <10% of the time. Moreover, antibodies to the MGSA/GRO proteins slow or inhibit the formation of tumors in the SCID mouse model and block the angiogenic response to conditioned medium from the tumor-producing clones. Transcription of the MGSA/GRO chemokines is regulated by an enhancesome-like complex comprised of the nuclear factor-kappaB (NF-kappaB), HMG(I)Y, IUR, and Sp1 elements. In Hs294T melanoma cells the half life of the IKB protein is shortened in comparison to normal retinal epithelial cells, facilitating the endogenous nuclear localization of NF-kappaB. We propose that this endogenous nuclear NF-kappaB, working in concert with the 115-kDa IUR-binding factor, promotes constitutive expression of MGSA/GRO genes.

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.

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The Duffy antigen receptor for chemokines (DARC) is a receptor for both CXC and CC chemokines. We have cloned a mouse gene with a predicted amino acid sequence homology of approximately 63% to human DARC and localized this gene to mouse chromosome 1 between the Xmv41 and D1Mit166 loci. We further demonstrated that, like the human gene, the mouse gene exhibits a single intron of 462 bp which interrupts the open reading frame between the codons for the seventh and eighth amino acid residues. Northern blot analyses revealed that putative mDARC mRNA is highly expressed in adult mouse spleen and skeletal muscle and in whole embryos between embryonic days 8.5 to 12. Northern blot analysis of hemangiosarcomas which develop spontaneously in the spleen of the Eker rat reveal expression of mRNAs which hybridize with both DARC and CXCR2 probes, suggesting a potential role of these receptors in the angiogenesis associated with tumor formation.