Protein kinase C (PKC) is a key enzyme involved in regulation of cell growth and differentiated function and in carcinogenesis. We have developed a model system in which we can define molecular events associated with PKC stimulus-response coupling which has broad implications in understanding a variety of disease processes, including cancer. We have studied PKC activation in cultured rat pituitary cells (GH4CI) in which phorbol dibutyrate (PDBu) mimics the endogenous PKC activator, diacylglycerol (DAG), and stimulates synthesis and secretion of prolactin. Thyrotropin-releasing hormone (TRH), which stimulates inositol lipid metabolism, increases both DAG and Ca2+ levels. Using immunoblot analysis with our alpha- (Type 3) PKC specific monoclonal antibodies (mAbs), we have determined that TRH and PDBu cause increased membrane association of alpha-PKC. Redistribution by TRH and PDBU can also be seen by immunofluorescence. The TRH- and PDBu-mediated membrane association is chelator and detergent stable indicating that PKC is associated with insoluble cytoskeletal (CSK) components. Immunofluorescence experiments verified that alpha-PKC was retained in detergent- and chelator-extracted preparations from TRH- and PDBu-treated, but not resting cultures.This also suggest that activated PKC is associated with the CSK. In support of this,we found that PKC activation causes major changes in CSK organization. Our working hypothesis is that in resting cells, alpha-PKC is inactive and soluble. Activation with PDBu or TRH leads to association with CSK components. Possibly, Ca2+, enhances this association. The CSK association that occurs with activation places PKC in the appropriate place for phosphorylation of CSK proteins. Phosphorylation leads to changes in CSK organization which are associated with observed biological responses.
Our aims are to identify the CSK proteins to which alpha-PKC binds and identify the CSK-associated substrates. We have determined that in addition to the calcium-sensitive PKC isozymes alpha- and beta-, these cells express the calcium-independent isozyme, epsilon-PKC. The purpose of PKC heterogeneity may be to modify isozyme cofactor requirements for activation, substrate specificity, or subcellular localization. Therefore, we have also designed experiments to distinguish between alpha- and non-alpha-PKC mediated phosphorylation events in order to fully define the role of PKC in regulation of prolactin synthesis and secretion.
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