Structural and functional studies of protein kinase C.

It has now become clear that protein kinase C (PKC) plays a fundamental role in cellular growth control in higher eukaryotes. In addition, since PKC has also been shown to be the high-affinity intracellular receptor for several classes of tumor promoters, the study of PKC has also become a central focus of current work in cancer research. We have isolated cDNA clones encoding several forms of this enzyme, and we have used these clones to begin to study in detail the role of PKC in growth control and tumor promotion. The complete primary structure of one of these forms, designated PKC beta 1, exhibits structural and functional characteristics which are shared among all of the currently identified forms of PKC. These include an amino terminal cysteine-rich domain which mediates Ca2+ and phospholipid binding, tumor promoter binding, and membrane association, and a carboxy terminal catalytic domain which possesses serine/threonine protein kinase activity. To further characterize the function of PKC, we have generated a series of rat fibroblast cell lines which stably overexpress a full-length cDNA encoding the beta 1 form of this enzyme. These cell lines contain a 20- to 53-fold increase in PKC activity, and also have an increase in high affinity phorbol ester receptors, relative to control cells. They also exhibit dramatically enhanced morphologic changes in response to treatment with the tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA). These cell lines grow to a higher saturation density in monolayer culture and, when maintained at post-confluence, develop small, dense foci. In contrast to the control cells, which display complete anchorage dependence, the cell lines that overproduce PKC form small colonies in soft agar in the absence of TPA, and larger colonies in the presence of TPA. Thus, the mere overproduction of a single form of PKC is sufficient to confer anchorage independent growth and other growth abnormalities in rat fibroblasts. Taken together, these results provide direct evidence that PKC plays a critical role in normal cellular growth control and that it mediates several, and perhaps all of the cellular effects of the phorbol ester tumor promoters.