Insulin Receptor Substrate-1 as a Signaling Molecule for Focal Adhesion Kinase pp125FAK and pp60

Abstract

Insulin receptor substrate-1 (IRS-1) is a major substrate of insulin and insulin-like growth factor-I receptors, which upon phosphorylation on tyrosine docks several signaling molecules. Recently, IRS-1 was found to interact with ␣ v ␤ 3 integrins upon insulin stimulation. Integrins are transmembrane proteins that play an important role in adhesion between cells and between cells and extracellular matrix. One of the major proteins implicated in integrin signaling is pp125 FAK , a cytosolic tyrosine kinase, which upon integrin engagement becomes tyrosine-phosphorylated and subsequently binds to c-Src. Here, we established a mammalian two-hybrid system to show that pp125 FAK binds to IRS-1. This association depends largely on the C terminus of pp125 FAK but not on pp125 FAK tyrosine kinase activity. Furthermore, we observed co-immunoprecipitation of pp125 FAK with IRS-1 in 293 cells, suggesting a possible biological function of this association. When IRS-1 was expressed in 293 cells together with pp125 FAK or Src, we found extensive IRS-1 tyrosine phosphorylation. In pp125 FAKexpressing cells, this was concomitant with increased association of IRS-1 with Src homology 2-containing proteins such as growth factor receptor-bound protein 2, phosphatidylinositol (PI) 3-kinase p85␣ subunit, and Src homology 2-containing protein-tyrosine phosphatase-2. In addition, pp125 FAK -induced association of IRS-1 with PI 3-kinase resulted in increased PI 3-kinase activity. In contrast, no change in mitogen-activated protein kinase activity was observed, indicating that pp125 FAK -induced association between IRS-1 and growth factor receptor-bound protein 2 does not affect the mitogen-activated protein kinase pathway. Moreover, we found that engagement of integrins induced IRS-1 tyrosine phosphorylation. Considering our results together, we suggest that integrins and insulin/insulinlike growth factor-I receptor signaling pathways converge at an early point in the signaling cascade, which is the IRS-1 protein.Integrins are transmembrane proteins expressed in most tissues. They are involved in key biological functions including cell migration and adhesion, embryogenic development, prevention of programmed cell death, wound repair, and angiogenesis (1-7). Protein phosphorylation on tyrosine is an immediate event after integrin engagement following cell interaction with the extracellular matrix. One of the major phosphorylated proteins is the cytosolic tyrosine kinase (focal adhesion kinase) pp125 FAK , 1 which upon integrin engagement becomes phosphorylated on tyrosine and activated (8 -10). pp125 FAK contains a central kinase domain flanked by large N-and C-terminal regions, but it lacks canonical interaction motifs such as pleckstrin homology (PH), phosphotyrosine binding (PTB), and SH2 and SH3 (Src homology) domains. The pp125 FAK sequence, which allows targeting of the kinase to the focal adhesions, is located in the C terminus (11). The potential physiological substrates of pp125 FAK are the cytoskeletal proteins paxillin (12-14) ...

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