Interaction of wild type and dominant-negative p55PIK regulatory subunit of phosphatidylinositol 3-kinase with insulin-like growth factor-1 signaling proteins.

In a first series of experiments done in the yeast two-hybrid system, we investigated the nature of protein-protein interaction between the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), p55PIK, and several of its potential signaling partners. The region between the Src homology 2 (SH2) domains of p55PIK bound to the NH2 terminus region of p110alpha, as previously shown for p85alpha. Moreover, we found that the insulin-like growth factor-1 receptor (IGF-IR) bound to p55PIK; the interaction occurred at the receptor tyrosine 1316 and involved both p55PIK SH2 domains.

Identification of Stat 5B as a substrate of the insulin receptor.

We have screened a human placenta library using the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. Doing so, we trapped a cDNA clone which encodes the Stat 5B region comprising amino acids 469 to 786. We show that interaction between Stat 5B and the receptor requires a functional insulin-receptor kinase, Tyr960 of insulin receptor is implicated in the interaction with Stat 5B, whereas asparagine and proline forming the NPEY960-motif are not, and Stat 5B mutated at Thr684, a potential phosphorylation site of mitogen-activated protein kinase, loses its ability to interact with the insulin receptor.

Tyr624 and Tyr628 in insulin receptor substrate-2 mediate its association with the insulin receptor.

In addition to the pleckstrin homology domain and the phosphotyrosine binding domain in insulin receptor substrate (IRS)-1 and IRS-2, a region between amino acids 591 and 786 in IRS-2 (IRS-2-(591-786)) binds to the insulin receptor. Based on peptide competition studies, this region interacts with the phosphorylated regulatory loop of the insulin receptor; we designate this region the kinase regulatory loop binding (KRLB) domain. Two tyrosine residues in the KRLB domain at positions 624 and 628 are crucial for this interaction.

Insulin receptor-induced phosphorylation of cellular and synthetic substrates is regulated by the receptor beta-subunit C-terminus.

The transmembrane beta-subunits of the insulin receptor possess hormone-sensitive tyrosine kinase activity. To study the role of the C-terminus domain, a rabbit antipeptide antibody directed to the 1294-1317 domain was produced. The antipeptide antibody inhibited the receptor-induced phosphorylation of poly (Glu, Tyr) and synthetic peptides corresponding to the receptor autophosphorylation sites.

Phosphorylation of insulin receptor substrate-1 on multiple serine residues, 612, 632, 662, and 731, modulates insulin action.

Okadaic acid has been described previously as being a negative regulator of insulin signaling, as it inhibits insulin stimulation of glucose transport. In addition, this drug induces on insulin receptor substrate-1 (IRS-1) a decrease in tyrosine phosphorylation, concomitantly with an increase in serine/threonine phosphorylation. The present work was aimed at the identification of the serine/threonine residues that, upon phosphorylation, might be involved in modulating insulin signaling.