The association of insulin-elicited phosphotyrosine proteins with src homology 2 domains.
The interactions of the phosphotyrosine (Tyr(P))-containing proteins in basal and insulin-stimulated 3T3-L1 adipocytes with src homology 2 (SH2) domains from phosphatidylinositol 3-kinase (PI3K), ras GTPase-activating protein (GAP), and phospholipase C gamma have been examined. The Tyr(P) forms of the insulin receptor and its 160-kDa substrate protein (pp160) associated with fusion proteins containing either or both the SH2 domains of PI3K, but not with fusion proteins containing the two SH2 domains of GAP or phospholipase C gamma. These results demonstrate a specificity for the association of the Tyr(P) form of the insulin receptor and pp160 with SH2 domains that parallels the reported effects of insulin on PI3K, GAP, and phospholipase C gamma in vivo. Immunoprecipitates of pp160 from the cytosol of insulin-treated, but not basal, 3T3-L1 adipocytes contained PI3K activity. Moreover, the Tyr(P) form of pp160 with associated PI3K activity migrated at 10 S on a sucrose velocity gradient, whereas the Tyr(P) form without associated activity migrated at 6 S. These findings indicate that the Tyr(P) form of pp160 associates directly with PI3K in vivo.
Lavan
B E
BE
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844.
Kuhné
M R
MR
Garner
C W
CW
Anderson
D
D
Reedijk
M
M
Pawson
T
T
Lienhard
G E
GE
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D020690:GTPase-Activating Proteins; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases; D010770:Phosphotransferases / Q000378:metabolism; D019000:Phosphotyrosine; D011233:Precipitin Tests; D011506:Proteins / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D007328:Insulin; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin; D015688:Oncogene Protein pp60(v-src); D010738:Type C Phospholipases