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1 in its interaction with the insulin receptor
Address correspondence to Michel Bernier, Diabetes Section, National Institute on Aging, Gerontology Research Center, 5600 Nathan Shock Drive, Box 23, Baltimore, MD 21224-6825. Tel.: (410) 558-8199. Fax: (410) 558-8381. email: Bernierm{at}vax.grc.nia.nih.gov
A thiol-reactive membrane-associated protein (TRAP) binds covalently to the cytoplasmic domain of the human insulin receptor (IR) ß-subunit when cells are treated with the homobifunctional cross-linker reagent 1,6-bismaleimidohexane. Here, TRAP was found to be phospholipase C
1 (PLC1) by mass spectrometry analysis. PLC1 associated with the IR both in cultured cell lines and in a primary culture of rat hepatocytes. Insulin increased PLC1 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 PLC1 Src homology 2 domains, and instead required the pleckstrin homology (PH)–EF-hand domain. Expression of the PH–EF construct blocked endogenous PLC1 binding to the IR and inhibited insulin-dependent phosphorylation of mitogen-activated protein kinase (MAPK), but not AKT. Silencing PLC1 expression using small interfering RNA markedly reduced insulin-dependent MAPK regulation in HepG2 cells. Conversely, reconstitution of PLC1 in PLC1-/- fibroblasts improved MAPK activation by insulin. Our results show that PLC1 is a thiol-reactive protein whose association with the IR could contribute to the activation of MAPK signaling by insulin.
Key Words: receptors; PLC; signal transduction; cultured cells; mass spectrometry
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