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.
Kwon
Yong-Kook
YK
Diabetes Section, Laboratory of Clinical Investigation, National Institute on Aging, National Institute of Health, Baltimore, MD 21224-6825, USA.
Jang
Hyeung-Jin
HJ
Kole
Sutapa
S
He
Hua-Jun
HJ
Bernier
Michel
M
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