The overall goal of this proposal is to elucidate the role of diacylglycerol (DAG) metabolism and of protein kinase C (PKC) activity in oncogenic transformation. DAG is the endogenous activator of PKC, which is also the receptor for the tumor- promoting phorbol esters. This laboratory has proposed that some oncogenes might transform cells by de-regulating DAG metabolism, so as to constitutively activate PKC. In support of this hypothesis, DAG levels were found to be evaluated in fibroblasts transformed by ras, src or fms and PKC was partially activated. Surprisingly, however, these transformed cells are also partially densensitized to the effects of phorbol esters, and revertant lines, resistant to transformation by v-ras, display a more exaggerated desensitization. These results suggest that desensitization may represent an attempt by the cell to overcome the constitutive proliferative signals generated by the oncogenes. To determine the mechanism responsible for constitutive elevation of DAG, metabolic labeling studies will be performed using a 3T3 cell-line transfected with a temperature-sensitive v- src oncogene. Investigation of the control of DAG metabolism at the molecular level will focus on DAG kinase, which has been found to exist in multiple isotypes, and which translocates rapidly to membranes in response to serum or phorbol esters. Monoclonal antibodies are being generated to the different isotypes to facilitate studies of phosphorylation, tissue specificity and processing. Secondly, the kinetics and mechanism of PKC desensitization will be determined. (Is desensitization produced by changes in phosphatase activity, PKC inhibitor production, PKC phosphorylation, alterations in membrane association of PKC or in the PKC substrates themselves?) The role of PKC in the mediation of transformation by v-src will also be investigated. Phenotypic changes to be studied in detail include activation of c- myc expression, initiation of DNA synthesis early changes in protein phosphorylation and synthesis, and epidermal growth factor (EGF) receptor down-modulation. This last response appears to be PKC-independent and involves two distinct, separable mechanisms, one of which is cycloheximide-sensitive. The role of other protein kinases, and of autocrine factors, in promoting down-modulation will be elucidated, using both whole cell and membrane preparations. These experiments will provide insight into the molecular mechanism by which certain oncogenes mediate transformation, and the mechanism by which transformation can be effectively blocked.
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