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Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain
1Department of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853.
Abstract:
The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.
Insights
Focal adhesion kinase (FAK) interacts with Src homology 2 (SH2) domain proteins. FAK autophosphorylation is crucial for this binding, mediating signals from the cell surface to the interior.
Area of Science:
- Cellular Biology
- Molecular Signaling
Background:
- Focal adhesion kinase (FAK) is a key signaling molecule involved in cell adhesion and growth factor pathways.
- FAK relays signals from cell surface receptors like integrins to intracellular pathways.
- Proteins with Src homology 2 (SH2) domains are critical intracellular signaling molecules.
Purpose of the Study:
- To investigate the mechanisms by which FAK transmits signals from the cell membrane.
- To identify interactions between FAK and SH2 domain-containing proteins.
Main Methods:
- Immune-complex precipitation using anti-Src antibody in v-Src-transformed NIH3T3 cells.
- Yeast two-hybrid system to detect in vivo interactions.
- In vitro binding assays using recombinant FAK and bacterial fusion proteins with Src SH2/SH3 domains.
- Experiments with kinase-defective FAK mutants and protein-tyrosine phosphatase treatment.
Main Results:
- FAK was found to interact with v-Src in vivo.
- Direct binding of FAK to the Src SH2 domain, but not the SH3 domain, was demonstrated in vitro.
- FAK autophosphorylation sites are essential for SH2 domain interaction.
- Dephosphorylation reduced binding, while in vitro autophosphorylation enhanced it.
Conclusions:
- FAK autophosphorylation is critical for its interaction with SH2 domain-containing proteins.
- FAK mediates cell surface signal transduction through kinase activation and autophosphorylation.
- These interactions facilitate the binding of FAK to other key intracellular signaling molecules.