The association of phosphoinositide-specific phospholipase C (PLC)-gamma1 with the insulin receptor (IR) is considered to be responsible for inducing a variety of biological responses upon insulin stimulation. Our recent work showed that mouse embryonic fibroblasts (MEF) from PLCg1 knockout animals have low insulin-mediated ERK phosphorylation, which significantly increases upon reintroduction of HA-tagged PLCg1. Interestingly, MEF PLCg1-/- cells remain very sensitive to insulin with regard to AKT phosphorylation. Conversely, siRNA-mediated reduction in PLCg1 expression in HepG2 cells was accompanied by selective attenuation of insulin-dependent ERK phosphorylation, with only minimal effect toward phosphorylation of AKT. Despite these advances, little is known however on the molecular mechanisms leading to insulin-mediated PLCg1 activation, its cellular redistribution, and role in insulin signaling. PLCg1 is an enzyme that plays a pivotal role in transmembrane signaling, notably the regulation of extracellular signal-regulated kinase (ERK) 1/2. PLCg1 contains several domains through which it can interact with actin, signaling proteins and lipid products. Through this network of interactions, PLCg1 is activated and redistributed within the cell where it exerts its essential role in mammalian growth and differentiation. We found here that insulin promoted formation of a ternary complex encompassing the IR, PLCg1 and caveolin-1 in CHO-IR and 3T3-L1 cells. These proteins were largely present in caveolae/lipid rafts. GST-pulldown experiment shows insulin-dependent association of tyrosine-phosphorylated caveolin-1 to the SH2 domains of PLCg1. Stable knockdown of caveolin-1 by RNA interference in 3T3-L1 cells (Cav-/-) sharply increased constitutive tyrosine phosphorylation of PLCg1 and its association with the IR despite the absence of IR activation. This result is consistent with our previous finding showing the requirement of the N-terminal PH-EF domain in functional interaction between PLCg1 with the IR. Of interest, constitutive and insulin-stimulated phosphorylation of ERK was significantly higher in Cav-/- cells and was dependent on the enzyme p60Src, which phosphorylates PLCg1. In many cases, it has been shown that interaction of caveolin-1 with signaling molecules, including Src family kinases, exerts an inhibitory tone on their activities. We are currently investigating the relative contribution of PLCg1 and caveolin-1 on insulin-dependent Elk-1 transcription, using Elk-1 promoter-luciferase construct. To further address the mechanism by which PLCg1 is regulated, insulin-stimulated 3T3-L1 cells were lysed and subjected to immunoprecipitation with mAb anti-PLCg1 followed by protein identification by peptide sequencing using mass spectrometry (LC-MS/MS). Several structural proteins were found associated with PLCg1, which included gelsolin, vimentin and g-actin. The protein mortalin-2 was also found to be present in PLCg1 immunoprecipitates. Mortalin-2 has been assigned multiple functions ranging from intracellular trafficking, control of cell proliferation, differentiation and tumorigenesis. Because of its role in these dynamic and regulatory events, mortalin-2?s interaction with PLCg1 was then evaluated. Several domains, including the two SH2 domains and the SH3 domain have been implicated in interaction of PLCg1 with adapter proteins. Using GST-pull down assays, our data showed that this association occurred through the SH3 domain of PLCg1; the interaction was significantly greater using extracts from Cav-/- cells. We found that the insulin-induced phosphorylation of PLCg1 and activation of the Shc/ERK 1/2 cascade were selectively reduced by knocking down mortalin-2 expression using siRNA methodology in 3T3-L1 cells. In contrast, insulin-stimulated IRS-1 phosphorylation and activation of the PI3K/AKT pathway was largely unaffected by the reduction in mortalin-2 expression. Moreover, when immunofluorescence studies were performed, the constitutive colocalization of caveolin-1 with PLCg1 into intracellular vesicular structures was found to be sharply reduced following cell treatment with mortalin-2 siRNA but not with that of Gab-1. Taken together, these results indicate that mortalin-2 and caveolin-1 play important regulatory roles in insulin signaling possibly through activation and subcellular redistribution of PLCg1. The process of adipogenesis involves a complex program of gene expression. We are planning to investigate further the relationship between PLCg1 and PLCg1-interacting molecules (caveolin-1 and mortalin-2) with adipogenesis of 3T3-L1 fibroblasts. A special emphasis will be put on the use of siRNAs to mediate reduction of mRNA encoding for PLCg1 and other relevant signaling molecules. The pSIREN-siLUC and pSIREN-siPLCg1, and pSIREN-siMortalin-2 retroviral constructs will be generated. Production of retroviral supernatant fluid and infection of 3T3-L1 cells will be carried out followed by puromycin selection. In addition to differentiation assays, changes in the activation of insulin metabolic and mitogenic signaling will be explored. The possibility that PLCg1, caveolin-1 and/or mortalin-2 down-regulation is critical for adipogenesis remains to be determined. Nevertheless, the identification of novel modulators of PLCg1 signaling should provide insights into the mechanisms through which this phospholipase exerts its pleiotropic effects on cell growth and differentiation in general, and in adipogenesis in particular.
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