Role of the pleckstrin homology domain of PLCγ1 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) β-subunit when cells are treated with the homobifunctional cross-linker reagent 1,6-bismaleimidohexane. Here, TRAP was found to be phospholipase C γ1 (PLCγ1) by mass spectrometry analysis. PLCγ1 associated with the IR both in cultured cell lines and in a primary culture of rat hepatocytes. Insulin increased PLCγ1 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 PLCγ1 Src homology 2 domains, and instead required the pleckstrin homology (PH)–EF-hand domain. Expression of the PH–EF construct blocked endogenous PLCγ1 binding to the IR and inhibited insulin-dependent phosphorylation of mitogen-activated protein kinase (MAPK), but not AKT. Silencing PLCγ1 expression using small interfering RNA markedly reduced insulin-dependent MAPK regulation in HepG2 cells. Conversely, reconstitution of PLCγ1 in PLCγ1−/− fibroblasts improved MAPK activation by insulin. Our results show that PLCγ1 is a thiol-reactive protein whose association with the IR could contribute to the activation of MAPK signaling by insulin

Insight into the association between phospholipase C-γ1 and the insulin receptor

La identificació de senyals intermediaris que interaccionen directament amb el receptor d'insulina (IR) és actualment objecte d'una intensa recerca, ja que sembla estar relacionada amb l'acció de la insulina. Recentment, el nostre grup de recerca va establir l'associació induïble de la fosfolipasa C (PLC)-γ1 amb l'IR i la seva implicació en la transducció de senyal mitjançada per la insulina a través de l'activació de la ruta de la cinasa regulada per Ras. En aquest estudi hem generat diversos mutants de la PCLγ1 per identificar i definir la funció dels residus tiol PCLγ1 en el control de la interacció IR-PCLγ1 i determinar l'impacte que el redox cell. ular té en el procés. Per aconseguir aquests objectius, hem utilitzat dos mètodes que demostren la presència de sulfhidrils en la PCLγ1 sensibles a l'oxidació en cèll. ules CHO que expressen IR. Diversos experiments de mutagènesi dirigida varen demostrar la modificació selectiva de les cisteïnes 8 i 12 de PCLγ1 després de l'estimulació amb peròxid d'hidrogen i la inhibició d'oxidases de membrana productores de ROS. Aquestes dades suggereixen que la regió aminoterminal de la PCLγ1 conté cisteïnes nucleofíliques particularment sensibles a la regulació redox i que són necessàries per a la interacció eficient amb l'IR. La importància d'aquestes troballes s'ha confirmat també en estudis sobre el paper regulador de la redox cell. ular en la transducció de senyal de la insulina.An intense investigation is underway to identify the signaling intermediates that interact directly with the insulin receptor (IR) as they appear to be associated with the transmission of insulin action.We recently reported the inducible association of phospholipase C (PLC)γ1 with the IR and its involvement in the transduction of insulin-mediated signals through activation of the Ras/extracellular-regulated kinase pathway. Akin to protein phosphorylation, redox regulation represents an important metabolic modulator of cellular functions. In this study, various mutant forms of PCLγ1 were generated to identify putative key redox-sensitive cysteines that provide sites for a number of post-translational modifications. Toward this aim, two methods were used that demonstrate the presence of oxidation-sensitive cysteine residues in PCLγ1. Using site-directed mutagenesis experiments we found that two cysteines in PCLγ1 (Cys-8 and Cys-12) were modified with hydrogen peroxide and protected against a host of reactive oxygen species through pharmacological inhibition of cellular membrane oxidases. The data suggest that the amino-terminal region of PCLγ1 contains reactive cysteine-SH groups that are exquisitely sensitive to redox regulation and which are associated with efficient interaction with the IR. The importance of these findings is already asserting themselves in studies reporting the regulatory role of cellular redox in insulin signal transduction