The impact of overexpression of mouse protein kinase C beta-II cDNA in C3H 10T1/2 mouse fibroblasts
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.