Potent Activation of Phosphatidylinositol 3'-Kinase by Simple Phosphotyrosine Peptides Derived from Insulin Receptor Substrate 1 Containing Two YMXM Motifs for binding SH2 Domains

Abstract

The phosphotyrosine form of the major substrate for the insulin receptor tyrosine kinase, insulin receptor substrate 1 (IRS-1), associates with and activates the enzyme phosphatidylinositol 3'-kinase (PtdIns 3'-kinase). IRS-1 contains nine potential tyrosine phosphorylation sites within YMXM or YXXM sequences known to bind to the two SH2 domains on the 85-kDa regulatory subunit of PtdIns 3'-kinase. We used sequences within IRS-1 as a model for synthesizing phosphotyrosine and nonhydrolyzable phosphonotyrosine peptides containing two YMXM motifs and tested them for their ability to bind to the SH2 domains of PtdIns 3'-kinase and stimulate its activity. We demonstrated for the first time that IRS-1-derived peptides containing two tyrosine phosphorylated YMXM motifs are capable of stimulating PtdIns 3'-kinase activity in the cytosol of 3T3-L1 adipocytes at nanomolar concentrations, similar to that required by purified phosphoryl-IRS-1 [Lamphere, M., Carpenter, C. L., Sheng, Z., Kallen, R. G., & Lienhard, G. E. (1994) Am. J. Physiol. 266 (Endocrinol. Metab. 29), E486-E489] and the extent of activation by these peptides was similar to that seen by maximal stimulation of cells with insulin. In contrast, those phosphotyrosine peptides containing only a single YMXM motif were able to stimulate PtdIns 3'-kinase activity only at concentrations over 10 microM. We conclude from these results that the high-affinity activation of PtdIns 3'-kinase requires the simultaneous binding of two phosphorylated YMXM motifs on IRS-1 to the two SH2 domains of PtdIns 3'-kinase.

Involvement of Secretory and Endosomal Compartments in Presentation of an Exogenous Self-Glycolipid to Type II NKT Cells

“…These data further suggests that phosphorylation of the tyrosine motif plays an important role in trafficking of CD1d. Indeed, the phospho-tyrosine motif, pYXXM present in insulin receptor substrate-1, plateletderived growth factor receptor, and ErbB2, 3 receptors binds with the SH2 domain of the p85 subunit of PI 3-kinase (52)(53)(54)(55)(56)(57)(58). Additional studies in AP-3Ϫ/Ϫmice are needed to further clarify specific interaction of PI 3-kinase on trafficking of CD1d protein.…”

Mitogenic Signals and Transforming Potential of Nyk, a Newly Identified Neural Cell Adhesion Molecule-Related Receptor Tyrosine Kinase

“…By contrast, Nyk does not induce phosphorylation of GAP and GAP-associated p62 and p190 proteins but is a potent activator of PI3K. The ability of Nyk to activate PI3K may be related to the presence of a PI3K SH2-binding consensus sequence (YMXM) (34,37) encompassing amino acids 830 to 833 of Nyk. The activation of PI3K by Nyk is further supported by the heightened activity of p70 S6 kinase, a serine/threonine kinase believed to be downstream from PI3K (15).…”

Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site

“…The latter site corresponds to tyrosine 779 of the Ufo/Axl receptor. Using tyrosine 779 as a low a nity docking site for the second SH2 domain of p85 proteins could be of functional signi®cance, since various research groups have shown that simultaneous binding of two SH2 domains is required for full activation of PI3-kinase activity (Rordorf-Nikolic et al, 1995;Herbst et al, 1994;Carpenter et al, 1993). Determination of PI3-kinase activity associated with the respective receptor mutants may resolve this issue.…”