Insulin-like growth factor-1-mediated association of p85 phosphatidylinositol 3-kinase with pp 185: requirement of SH2 domains for in vivo interaction.
Insulin-like growth factor-I (IGF-1) stimulates the production of 3-phosphoinositides and increases the phosphatidylinositol 3-kinase activity that is immunoprecipitated by antiphosphotyrosine antibodies, a small portion of which are also associated with the IGF-1 receptor. In vitro reconstitution experiments showed that p85 associates with high affinity to the IGF-1 receptor and this interaction is mediated through the p85 SH2 groups. Moreover, in vitro, p85 is a substrate for the IGF-1 receptor tyrosine kinase activity. In this study, we analyzed the in vivo association of p85 with tyrosyl- phosphorylated proteins and its tyrosyl phosphorylation state, in response to IGF-1. After stimulation with IGF-1, the major tyrosylphosphorylated protein that was associated with p85 was a 185-kilodalton protein, identified as IRS-1. Only a small fraction of p85 was associated with the IGF-1 receptor. In contrast, the PDGF receptor was the major protein associated with p85 upon stimulation. Neither ligand stimulated the tyrosyl phosphorylation of p85 in vivo. In order to determine whether the SH2 domains of p85 were involved in its association with p185 in vivo after IGF-1 stimulation, different SH2-constructs of p85 were expressed in COS-1 cells. After stimulation with IGF-1, the expressed SH2 proteins were immunoprecipitated with specific antibodies, and associated p185 was detected on Western blots. These results show that both the p85 N-SH2 and N+C-SH2 associate with IRS-1 after IGF-1 stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Division of Cell Biology, Burroughs Wellcome Company, Research Triangle Park, North Carolina 27709.
D
D
Altschuler
K
K
Yamamoto
E G
EG
Lapetina
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D005347:Fibroblasts; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects*; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000187:drug effects / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013438:Sulfhydryl Compounds
D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds