Both the Catalytic and Regulatory Domains of Protein Kinase C Chimeras Modulate the Proliferative Properties of NIH 3T3 Cells

Péter Ács,Qiming J Wang,Krisztina Bögi,Adriana M Marquez,Patricia S Lorenzo,Tamás Bı́Ró,Zoltán Szállási,J Frederic Mushinski,Peter M Blumberg

doi:10.1074/jbc.272.45.28793

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, PKCdelta arrests cell growth whereas PKCepsilon 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 PKCalpha, -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 PKCepsilon 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 PKCepsilon regulatory domain also gave rise to colonies in soft agar; the role of the catalytic domain of PKCepsilon was evident in the PMA-treated cells that overexpressed the PKC chimera containing the delta regulatory and the epsilon catalytic domains (PKCdelta/epsilon). The important contribution of the PKCepsilon catalytic domain to the growth of PKCdelta/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 PKCepsilon, we have shown that most chimeras with PKCalpha 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.

Highlights

Protein kinase C (PKC) isozymes exhibit important differences in terms of their regulation and biological functions
To confirm that the growth alterations observed in these cells were due to the presence of overexpressed PKC isozymes, we examined the effect of an inhibitor of PKC catalytic activity, GF 109203X
The protein kinase C molecule consists of two functional domains; an N-terminal regulatory domain that binds phosphatidylserine, diacylglycerol and, in the case of the classic isozymes, calcium; and a C-terminal catalytic domain that contains the catalytic center of the enzyme [1, 8]

Summary

Introduction

Protein kinase C (PKC) isozymes exhibit important differences in terms of their regulation and biological functions. Aside from the growth-promoting effect of PKC⑀, we have shown that most chimeras with PKC␣ and -␦ regulatory domains inhibit cell growth These results underscore the complex contributions of the regulatory and catalytic domains to the overall behavior of PKC. It is crucial to emphasize that the effect of PKC isozymes is greatly influenced by the host cell; PKC␦ enhances tumorigenicity of breast cancer cells and may promote tumor progression in that system [16] This cell type-specific function holds for the PKC␣ and -␤II isotypes, which play distinct roles in the transduction of proliferative and differentiating signals in K-562 erythroleukemia cells; PKC␣ inhibits whereas PKC␤II promotes cell growth [17]. We found that both the regulatory and the catalytic domains of PKC contributed to the isotype-specific effects on cell growth

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Conclusion