Potent activation of phosphatidylinositol 3'-kinase by simple phosphotyrosine peptides derived from insulin receptor substrate 1 containing two YMXM motifs for binding SH2 domains.
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.
Herbst
J J
JJ
Pfizer Central Research, Groton, Connecticut 06340.
Andrews
G
G
Contillo
L
L
Lamphere
L
L
Gardner
J
J
Lienhard
G E
GE
Gibbs
E M
EM
D000595:Amino Acid Sequence; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D055504:Insulin Receptor Substrate Proteins; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism* / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D019000:Phosphotyrosine; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D055504:Insulin Receptor Substrate Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)