The impact of overexpression of mouse protein kinase C beta-II cDNA in C3H 10T1/2 mouse fibroblasts
Abstract
Protein Kinase C (PKC) is a phospholipid- and calcium-dependent protein kinase which phosphorylates proteins on serine or threonine residues. PKC was originally found to be a high affinity intracellular receptor for phorbol esters, a family of tumor promoters, and later found involved in cell-surface signal transduction and control of cellular growth and differentiation. The cDNA of mouse PKC $\beta$-II, containing the whole coding sequence, was cloned from a mouse brain cDNA library. A site-directed mutagenesis was induced at the ATP-binding site to change the conserved residue Lys to Ile. Both the wild-type and the mutant PKC $\beta$-II cDNA were expressed in C3H 10T1/2 mouse fibroblasts. Cells overexpressing wide-type PKC $\beta$-II exhibited enhanced growth in medium with low serum, but no morphologically altered foci formed neither the anchorage-independent growth was detected. The mutant PKC molecule was identified kinase-deficient. Cell lines overexpressing this mutant PKC appeared to have a decreased rate of growth. Thus, although PKC $\beta$-II overexpression does not have the same impact on neoplastic phenotype as that of oncogenes, these results suggest PKC $\beta$-II is involved in the regulation of cell proliferation, and the mutant PKC molecules antagonized the action of endogenous PKC. Autophosphorylation of PKC was hypothesized to be a trigger of PKC down-regulation. TPA treatment of PKC $\beta$-II expressing cells induced down-regulation of the kinase-defective PKC $\beta$-II in the same manner as wild-type PKC $\beta$-II and endogenous PKC in parental C3H 10T1/2 cells. This result indicates that intrapeptide autophosphorylation of PKC is not required for down-regulation. In mitogen-stimulated cells rasGAP becomes phosphorylated on tyrosine and associates with proteins in the particulate cell fraction. C3H 10T1/2 cell lines overexpressing wild-type PKC $\beta$-II, but not the kinase-defective mutant PKC $\beta$-II, showed enhanced GAP translocation to the particulate fraction after mitogenic stimulation with TPA. This indicates that PKC mediates this component of the mitogenic action of TPA.
Degree
Ph.D.
Advisors
Ashendel, Purdue University.
Subject Area
Molecular biology|Pharmacology|Biochemistry
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