Both the catalytic and regulatory domains of protein kinase C chimeras modulate the proliferative properties of NIH 3T3 cells

Protein kinase C (PKC) isozymes exhibit important differences in terms of their regulation and biological functions, Not only may some PKC isoforms be active and others not for a given response, but the actions of different isoforms may even be antagonistic, In NIH 3T3 cells, for example, PKC delta arrests cell growth whereas PKC epsilon stimulates it, To probe the contribution of the regulatory and the catalytic domains of PKC isozymes to isozyme-specific responses, we prepared chimeras between the regulatory and the catalytic domains of PKC alpha, -delta, and -epsilon. These chimeras, which preserve the overall structure of the native PKC enzymes, were stably expressed in mouse fibroblasts. A major objective was to characterize the growth properties of the cells that overexpress the various PKC constructs. Our data demonstrate that both the regulatory and the catalytic domains play roles in cell proliferation, The regulatory domain of PKC epsilon enhanced cell growth in the absence or presence of phorbol 12-myristate 13-acetate (PMA), and, in the presence of PMA, all chimeras with the PKC epsilon regulatory domain also gave rise to colonies in soft agar; the role of the catalytic domain of PKC epsilon was evident in the PMA-treated cells that overexpressed the PKC chimera containing the delta regulatory and the epsilon catalytic domains (PKC delta/epsilon). The important contribution of the PKC epsilon catalytic domain to the growth of PKC delta/epsilon-expressing cells was also evident in terms of a significantly increased saturation density in the presence of PMA, their formation of foci upon PMA treatment, and the induction of anchorage-independent growth. Aside from the growth-promoting effect of PKC epsilon, we have shown that most chimeras with PKC alpha and -delta regulatory domains inhibit cell growth. These results underscore the complex contributions of the regulatory and catalytic domains to the overall behavior of PKC.