The role of protein kinase C inras-oncogene signaling

Crystal Mindy Weyman, Purdue University

Abstract

Both the phorbol ester receptor, protein kinase C (PKC), and the protein product of the ras oncogene, p21$\sp{ras}$, have been implicated as key transducers of extracellular signals. A molecular link for the tandem activity of these two protein products has been postulated by data correlating p21$\sp{ras}$ activity with increased levels of sn-1,2-diacylglycerol (DAG), the endogenous activator of PKC. C3H 10T$1\over2$ mouse embryo fibroblasts represent an excellent system for studying the molecular mechanism responsible for the interaction of phorbol esters and the ras-oncogene in multistep transformation. Comparison of C3H 10T$1\over2$ cells and C3H 10T$1\over2$ cells expressing a transfected human Ha-ras oncogene revealed a partial loss of PKC activity and protein in the ras-transfected cells. This loss was reminiscent of the more complete loss observed subsequent to the persistent activation of PKC by phorbol ester. However, the ras-transfected cells contained PKC which was no more active than the PKC from untreated control cells. The PKC from the ras-transfected cells displayed chromatographic behavior identical to the PKC from control cells when resolved on hydroxylapatite indicating that neither cell line possessed isozymes $\beta$ or $\gamma$. Decreasing PKC activity in the ras-transfected cells by treatment with phorbol ester or the inhibitor staurosporine reduced their efficiency of growth in soft agarose without affecting the level of p21$\sp{ras}$ expression suggesting a role for PKC in the maintenance of the p21$\sp{ras}$-induced transformed phenotype in C3H 10T$1\over2$ cells. C3H 10T$1\over2$-derived 23A2 myoblasts were used to study the role of PKC in muscle differentiation and in the ras-induced inhibition of muscle differentiation. Comparison of 23A2 myoblasts and ras-transfected 23A2 myoblasts revealed an increased level of PKC in the ras-transfected cells. A reduced level of PKC was observed after normal differentiation of 23A2 myoblasts, but down-regulation of PKC to levels at or below the level in differentiated 23A2 cells did not abrogate the p21$\sp{ras}$-induced inhibition of differentiation nor did it affect the normal myogenesis of control 23A2 cells. However, down-regulation of PKC prior to the expression of oncogenic ras blocked establishment of the differentiation-defective phenotype suggesting a role for PKC in the initiation of the ras-induced myogenic block.

Degree

Ph.D.

Advisors

Ashendel, Purdue University.

Subject Area

Biochemistry

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