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) ...

The diverse biological actions of insulin and insulin-like growth factor I (IGF-I) are initiated by binding of the polypeptides to their respective cell surface tyrosine kinase receptors. These activated receptors phosphorylate a series of endogenous substrates on tyrosine, amongst which the insulin receptor substrate (IRS) proteins are the best characterized. Their phosphotyrosine-containing motifs become binding sites for Src homology 2 (SH2) domains on proteins such as SH2 domain-containing protein-tyrosine-phosphatase (SHP)-2/Syp, growth factor receptor bound-2 protien, (Grb-2), and phosphatidyl inositol 3 kinase (PI3 kinase), which participate in activation of specific signaling cascades. However, the IRS molecules are not only platforms for signaling molecules, they also orchestrate the generation of signal specificity, integration of signals induced by several extracellular stimuli, and signal termination and modulation. An extensive review is beyond the scope of the present article, which will be centered on our own contribution and reflect our biases.

Phosphatidylinositol 3-kinase is a key step in the metabolic actions of insulin. Two amino acid substitutions have been identified in the gene for the regulatory subunit of human p85␣, Met-326Ile, and Asn-330Asp, and the former has been associated with alterations in glucose͞insulin homeostasis. When the four human p85␣ proteins were expressed in yeast, a 27% decrease occurred in the level of protein expression of p85␣ Ile/Asp (P ‫؍‬ 0.03) and a 43% decrease in p85␣ Ile/Asn (P ‫؍‬ 0.08) as compared with p85␣ Met/Asp . Both p85␣ Ile/Asp and p85␣ Ile/Asn also exhibited increased binding to phospho-insulin receptor substrate-1 by 41% and 83%, respectively (P < 0.001), as compared with p85␣ Met/Asp . The expression of p85␣ Ile was also slightly decreased and the binding to insulin receptor substrate-1 slightly increased in brown preadipocytes derived from p85␣ knockout mice. Both p85␣ Met and p85␣ Ile had similar effects on AKT activity and were able to reconstitute differentiation of the preadipocytes, although the triglyceride concentration in fully differentiated adipocytes and insulin-stimulated 2-deoxyglucose uptake were slightly lower than in adipocytes expressing p85␣ Met . Thus, the Met-326Ile variant of p85␣ is functional for intracellular signaling and adipocyte differentiation but has small alterations in protein expression and activity that could play a role in modifying insulin action.A major pathway for the metabolic effects of insulin and other growth factors is the phosphatidylinositol 3-kinase (PI3-kinase). This enzyme plays important roles in the stimulation of glucose transport, p70 S6 kinase, glycogen synthesis, and lipolysis (1). The PI3-kinases are heterodimeric enzymes composed of a regulatory subunit (p85) and a catalytic subunit (p110␣ or p110␤). Recent work has demonstrated that the regulatory subunit of PI3-kinase exists in several isoforms derived from the p85␣ gene, including two short forms termed p50␣ and p55␣͞ AS53 ( Fig. 1), as well as subunits encoded by the p85␤ and p55 PIK ͞p55␥ genes (1-3). The SH2 domains of the PI3-kinase regulatory subunits bind to phosphotyrosine residues in specific sequence motifs possessing the sequence YMXM or YXXM, in all four insulin receptor substrates (IRS-1, -2, -3, and -4) (4, 5).The gene encoding p85␣ is an obvious candidate gene for the development of diabetes, because of its key role in insulin signaling and studies showing a decrease in IRS-1-associated PI3-kinase in tissues of the type 2 diabetic (6-8). A nucleotide substitution has been detected which predicts a change of methionine to isoleucine at codon 326 (9). This change occurs 6 amino acids from the N-terminal SH2 domain and is present in p85␣, p55␣͞AS53, and p50␣ (3) (Fig. 1). A study in Danish Caucasian subjects has shown that, although the Met-326Ile variant is frequent (allelic frequency Ϸ15-16%) and occurs with similar frequencies in diabetic and healthy subjects, homozygous carriers for this variant (Ϸ2% in this population) are characterized by reduction in whole-body glucose effec...