Induced focal adhesion kinase (FAK) expression in FAK-null cells enhances cell spreading and migration requiring both auto- and activation loop phosphorylation sites and inhibits adhesion-dependent tyrosine phosphorylation of Pyk2.

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 FAK-null 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 FAK-related kinase Pyk2/CAKbeta/RAFTK/CadTK.

Mechanisms of CAS substrate domain tyrosine phosphorylation by FAK and Src.

Tyrosine phosphorylation of CAS (Crk-associated substrate, p130(Cas)) 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.

Cloning, expression, purification, and characterization of the murine lysosomal acid alpha-mannosidase.

Catabolism of alpha-linked mannose residues on eukaryotic glycoproteins is accomplished by a broad specificity lysosomal alpha-mannosidase (EC 3.2.1.24). Based on regions of protein sequence conservation between the lysosomal alpha-mannosidase from Dictyostelium discoideum and the murine Golgi glycoprotein processing alpha 1,3/1,6-mannosidase, alpha-mannosidase II, we have cloned a cDNA encoding the murine lysosomal alpha-mannosidase. The longest of the clones was 3.1 kb in length and encoded a polypeptide of 992 amino acids containing a putative NH2-terminal signal sequence and 11 potential N-glycosylation sites. The deduced amino acid sequence was 76.5% identical to the human lysosomal alpha-mannosidase and 38.1% identical to the lysosomal alpha-mannosidase from D. discoideum. Expression of the cDNA in Pichia pastoris resulted in the secretion of an alpha-mannosidase activity into the culture medium. This recombinant expression product was purified and was shown to have enzymatic characteristics highly similar to the enzyme purified from mammalian sources and to the human lysosomal alpha-mannosidase cDNA expressed in Pichia. These characteristics include a similar pH optimum, Km, Vmax, inhibition by swainsonine, and activity toward natural substrates. Northern blots identified a major 3.5 kb RNA transcript in all murine tissues tested. A minor transcript of 5.4 kb was also detected in some murine tissues similar to the alternatively spliced transcripts that have been previously identified in human tissues.

Phosphospecific antibodies reveal focal adhesion kinase activation loop phosphorylation in nascent and mature focal adhesions and requirement for the autophosphorylation site.

Focal adhesion kinase (FAK) is a key signaling molecule regulating cellular responses to integrin-mediated adhesion. Integrin engagement promotes FAK phosphorylation at multiple sites to achieve full FAK activation. Phosphorylation of FAK Tyr-397 creates a binding site for Src-family kinases, and phosphorylation of FAK Tyr-576/Tyr-577 in the kinase domain activation loop enhances catalytic activity. Using novel phosphospecific antibody reagents, we show that FAK activation loop phosphorylation is significantly elevated in cells expressing activated Src and is an early event following cell adhesion to fibronectin. In both cases, this regulation is largely dependent on Tyr-397. We also show that the FAK activation loop tyrosines are required for maximal Tyr-397 phosphorylation. Finally, immunostaining analyses revealed that tyrosine-phosphorylated forms of FAK are present in both newly forming and mature focal adhesions. Our findings support a model for reciprocal activation of FAK and Src-family kinases and suggest that FAK/Src signaling may occur during both focal adhesion assembly and turnover.

Focal adhesion kinase promotes phospholipase C-gamma1 activity.

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)-gamma1 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-gamma1 in COS-7 cells increases PLC-gamma1 enzymatic activity and tyrosine phosphorylation, also dependent on FAK Tyr-397. However, FAK appears incapable of directly phosphorylating PLC-gamma1. These observations suggest a role for FAK in recruiting PLC-gamma1 to the plasma membrane at sites of cell-matrix adhesion and there promoting its enzymatic activity, possibly by releasing the repression caused by intramolecular interactions of the PLC-gamma1 Src homology domains and/or by positioning it for phosphorylation by associated Src-family kinases. These findings expand the known signaling functions of FAK and provide mechanistic insight into integrin-stimulation of PLC-gamma1.