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 i soforms 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 dom ains of PKC isozymes to isozyme-specific responses, we prepared chimer as between the regulatory and the catalytic domains of PKC alpha, -del ta, 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 c ells 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 gro wth in the absence or presence of phorbol 12-myristate 13-acetate (PMA ), and, in the presence of PMA, all chimeras with the PKC epsilon regu latory 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 cont ribution of the PKC epsilon catalytic domain to the growth of PKC delt a/epsilon-expressing cells was also evident in terms of a significantl y increased saturation density in the presence of PMA, their formation of foci upon PMA treatment, and the induction of anchorage-independen t growth. Aside from the growth-promoting effect of PKC epsilon, we ha ve shown that most chimeras with PKC alpha and -delta regulatory domai ns inhibit cell growth. These results underscore the complex contribut ions of the regulatory and catalytic domains to the overall behavior o f PKC.