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Complex formation with focal adhesion kinase: A mechanism to regulate activity and subcellular localization of Src
M D Schaller1, J D Hildebrand, J T Parsons
1Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Abstract:
Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60(c-src) or p59(fyn) results in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60(c-src) or p59(fyn) to induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60(c-src) is hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60(c-src) from a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60(c-src) to focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.
Insights
Focal adhesion kinase (FAK) recruits Src kinases to focal adhesions, enhancing their activity and directing the phosphorylation of substrates like paxillin. This interaction is crucial for regulating cell adhesion and kinase localization.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Focal adhesion kinase (FAK) plays a key role in cell adhesion and signaling.
- Src family kinases are involved in various cellular processes, including cell growth and migration.
- The interaction between FAK and Src kinases is critical for regulating cellular functions.
Purpose of the Study:
- To investigate the role of FAK in regulating Src kinase activity and substrate phosphorylation.
- To elucidate the mechanism by which FAK influences the localization and enzymatic activity of Src kinases.
- To determine how FAK-Src complex formation affects downstream signaling pathways.
Main Methods:
- Co-expression of FAK and Src kinases (pp60(c-src), p59(fyn)) in cellular systems.
- Analysis of tyrosine phosphorylation of cellular substrates, including paxillin.
- Utilizing FAK mutants defective for Src binding, focal adhesion targeting, or catalytic activity.
- Assessment of Src kinase localization and phosphorylation status.
Main Results:
- Simultaneous overexpression of FAK and Src kinases enhanced tyrosine phosphorylation of substrates like paxillin in a cell adhesion-dependent manner.
- FAK mutants defective for Src binding or focal adhesion targeting failed to induce paxillin phosphorylation.
- Catalytically inactive FAK mutants could still direct paxillin phosphorylation.
- FAK association led to hypophosphorylation of pp60(c-src) at its negative regulatory site and its redistribution to focal adhesions.
- FAK mutants defective for Src binding impaired the translocation of pp60(c-src) to focal adhesions.
Conclusions:
- FAK association alters Src kinase localization and enhances their enzymatic activity.
- FAK acts as a scaffold to recruit Src kinases to focal adhesions, directing substrate phosphorylation.
- This FAK-mediated recruitment mechanism is essential for regulating cellular signaling pathways involved in cell adhesion and migration.