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.
Ruest
P J
PJ
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Roy
S
S
Shi
E
E
Mernaugh
R L
RL
Hanks
S K
SK
D016475:3T3 Cells; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites / Q000276:immunology; D019556:COS Cells; D015815:Cell Adhesion Molecules / Q000276:immunology / Q000378:metabolism*; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016023:Integrins / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000276:immunology*; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism
D000906:Antibodies; D015815:Cell Adhesion Molecules; D016023:Integrins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D019061:src-Family Kinases