Phosphatidylinositol 3'-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase.
Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3'-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase-IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate.
Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
K A
KA
Cengel
R E
RE
Kason
G G
GG
Freund
D000730:Androstadienes / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D014443:Tyrosine / Q000378:metabolism; D000077191:Wortmannin; D018909:src Homology Domains
D000730:Androstadienes; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D011972:Receptor, Insulin; D011993:Recombinant Fusion Proteins; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D014443:Tyrosine; D000077191:Wortmannin; C526292:IRS1 protein, human; C116037:PAS domain kinases; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human