Phosphorylation of protein kinase C: Implications for signal transduction and tumor promotion

Carlos Antonio Molina, Purdue University

Abstract

Our approach to understanding the function of PKC in tumor promotion and signal transduction is to determine in detail its biochemical regulation. We have demonstrated that PKC is phosphorylated on many sites similar to that of autophosphorylation of purified PKC. The extent and sites of phosphorylation of purified PKC and PKC in cells was dramatically affected by activation of the PKC. Phosphorylation on the regulatory domain occurs independently of phosphatidylserine and other enzyme activators, and phosphorylation of the remaining sites is dependent on the presence of activators. The increased incorporation of phosphate (by autophosphorylation) into certain sites on PKC is sufficient to convert PKC from one to another of two forms that are separable by DEAE-cellulose chromatography. Comparison of tryptic phosphopeptide maps of rat brain PKC autophosphorylated in the absence or presence of calcium and phosphatidylserine revealed the presence of phosphorylation sites unique to activator-dependent autophosphorylation. These distinct phosphorylations were found to be the molecular basis for the chromatographic heterogeneity of PKC detected by elution from DEAE-cellulose first with 25 mM PIPES pH 6.5 buffer followed by 125 mM NaCl in 20 mM Tris pH 7.4 buffer. These distinct states of phosphorylated PKC were observed when PKC from untreated C3H 10T$1\over2$ cells metabolically labeled with $\sp{32}$P-phosphate was resolved on DEAE-cellulose and immunoprecipitated and the chromatographic fractions compared by tryptic phosphopeptide mapping. Treatment of these cells with the PKC activators phorbol ester or sn-1,2-dioctanoylglycerol (DiC$\sb8$) induced a conversion of PKC from the PIPES-eluting form to the NaCl-eluting form. Brief treatment of C3H 10T$1\over2$ cells with DiC$\sb8$ or low doses of phorbol ester sufficient to activate PKC and to alter the chromatographic behavior of PKC did not, with extended treatments, down-regulate PKC, indicating that phosphorylation of PKC is not sufficient to induce down-regulation. These results characterize a novel method for monitoring the extent of activation of PKC in cells. Another approach to monitor PKC activation is to study the effect of the PKC inhibitor staurosporine on PKC phosphorylation. Staurosporine was found to induce phosphorylation and down-regulation of PKC without causing PKC translocation to the membrane; conversely, it inhibits activator-dependent autophosphorylation and down-regulation of PKC. Staurosporine causes a conformational change on PKC which mimics the conformation of activated PKC. We concluded that autophosphorylation of PKC is directly related to activation of the enzyme, but is neither involved in translocation of PKC to the membrane, nor to down-regulation. Detailed mutagenesis and expression experiments are essential to assess fully the significance of PKC autophosphorylation in the regulation of PKC and promotion of cell transformation.

Degree

Ph.D.

Advisors

Ashendel, Purdue University.

Subject Area

Biochemistry

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