Role of the pleckstrin homology domain of PLCgamma1 in its interaction with the insulin receptor.
A thiol-reactive membrane-associated protein (TRAP) binds covalently to the cytoplasmic domain of the human insulin receptor (IR) beta-subunit when cells are treated with the homobifunctional cross-linker reagent 1,6-bismaleimidohexane. Here, TRAP was found to be phospholipase C gamma1 (PLCgamma1) by mass spectrometry analysis. PLCgamma1 associated with the IR both in cultured cell lines and in a primary culture of rat hepatocytes. Insulin increased PLCgamma1 tyrosine phosphorylation at Tyr-783 and its colocalization with the IR in punctated structures enriched in cortical actin at the dorsal plasma membrane. This association was found to be independent of PLCgamma1 Src homology 2 domains, and instead required the pleckstrin homology (PH)-EF-hand domain. Expression of the PH-EF construct blocked endogenous PLCgamma1 binding to the IR and inhibited insulin-dependent phosphorylation of mitogen-activated protein kinase (MAPK), but not AKT. Silencing PLCgamma1 expression using small interfering RNA markedly reduced insulin-dependent MAPK regulation in HepG2 cells. Conversely, reconstitution of PLCgamma1 in PLCgamma1-/- fibroblasts improved MAPK activation by insulin. Our results show that PLCgamma1 is a thiol-reactive protein whose association with the IR could contribute to the activation of MAPK signaling by insulin.
Diabetes Section, Laboratory of Clinical Investigation, National Institute on Aging, National Institute of Health, Baltimore, MD 21224-6825, USA.
Yong-Kook
YK
Kwon
Hyeung-Jin
HJ
Jang
Sutapa
S
Kole
Hua-Jun
HJ
He
Michel
M
Bernier
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism*; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D008970:Molecular Weight; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D011972:Receptor, Insulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010738:Type C Phospholipases / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
D001798:Blood Proteins; D003432:Cross-Linking Reagents; D007328:Insulin; D008301:Maleimides; D010750:Phosphoproteins; D034741:RNA, Small Interfering; C039077:platelet protein P47; C075594:1,6-bismaleimidohexane; D011972:Receptor, Insulin; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma