Association of phosphorylated insulin-like growth factor-I receptor with the SH2 domains of phosphatidylinositol 3-kinase p85.
Insulin-like growth factor-I (IGF-I) stimulates the production of 3-inositides and markedly increases the phosphatidylinositol 3-kinase activity that is immunoprecipitated by anti-phosphotyrosine antibodies, a portion of which is also associated with the IGF-I receptor. In this study, recombinant p85, the regulatory subunit of phosphatidylinositol 3-kinase, and fusion proteins containing various subdomains were used to investigate the association of p85 with the IGF-I receptor and to demonstrate that p85 is a direct in vitro substrate of the IGF-I receptor kinase. Solubilized IGF-I receptor was immobilized on antireceptor antibody-agarose beads. Following in vitro receptor phosphorylation and incubation with cell lysate, immobilized receptor became associated with phosphatidylinositol 3-kinase activity and with protein bands with molecular masses of 85 and 110 kDa, which correspond to the known molecular masses of the subunits of phosphatidylinositol 3-kinase. These associations were inhibited by the addition of recombinant intact p85 or SH2-containing fusion proteins, but not by fusion proteins containing its SH3 domain or breakpoint cluster homology region. A fusion protein containing the SH2 domains of Ras GTPase-activating protein also inhibited the association of phosphatidylinositol 3-kinase activity with immobilized IGF-I receptor, although less effectively than p85, whereas a similar construct containing the SH2 domain of pp60src was without effect. When immobilized phosphorylated IGF-I receptor was incubated with intact p85 or the SH2-containing fusion proteins, it became associated with and phosphorylated these proteins. These results demonstrate that at least in vitro, a tight association occurs between phosphorylated IGF-I receptor and phosphatidylinositol 3-kinase, that the region of phosphatidylinositol 3-kinase that contains its SH2 domains is directly involved in this association, and that this region is a direct substrate for IGF-I receptor tyrosine kinase. Furthermore, these results suggest that Ras GTPase-activating protein can also interact with the IGF-I receptor and that different SH2 domain-containing proteins interact with the IGF-I receptor with widely differing affinities.
Yamamoto
K
K
Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
Altschuler
D
D
Wood
E
E
Horlick
K
K
Jacobs
S
S
Lapetina
E G
EG
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D051379:Mice; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013002:Somatomedins / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins; D013002:Somatomedins; D013438:Sulfhydryl Compounds; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases