title: Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.
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.
authors.affiliation: Department of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853.
authors.forename: Z
authors.initials: Z
authors.lastname: Xing
authors.forename: H C
authors.initials: HC
authors.lastname: Chen
authors.forename: J K
authors.initials: JK
authors.lastname: Nowlen
authors.forename: S J
authors.initials: SJ
authors.lastname: Taylor
authors.forename: D
authors.initials: D
authors.lastname: Shalloway
authors.forename: J L
authors.initials: JL
authors.lastname: Guan
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002461:Cell Line, Transformed; D002645:Chickens; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051379:Mice; D009036:Moths; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism; D015003:Yeasts / Q000235:genetics
chemical_list: D015815:Cell Adhesion Molecules; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); C496051:Ptk2 protein, mouse