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

Abstract

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, PKCδ arrests cell growth whereas PKCε 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 PKCα, -δ, and -ε. 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 PKCε 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 PKCε regulatory domain also gave rise to colonies in soft agar; the role of the catalytic domain of PKCε was evident in the PMA-treated cells that overexpressed the PKC chimera containing the δ regulatory and the ε catalytic domains (PKCδ/ε). The important contribution of the PKCε catalytic domain to the growth of PKCδ/ε-expressing cells was also evident in terms of a significantly increased saturation density in the presence of PMA, their formation of loci upon PMA treatment, and the induction of anchorage-independent growth. Aside from the growth-promoting effect of PKCε, we have shown that most chimeras with PKCα and -δ regulatory domains inhibit cell growth. These results underscore the complex contributions of the regulatory and catalytic domains to the overall behavior of PKC.