Inhibition of expression of protein kinase C alpha by antisense cDNA inhibits phorbol ester-mediated arachidonate release

C Godson,K.S Bell,P.A Insel

doi:10.1016/s0021-9258(19)50291-5

C Godson, K.S Bell + Show 1 more

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https://doi.org/10.1016/s0021-9258(19)50291-5

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Abstract

A major unresolved issue in the area of signal transduction relates to the role of particular isoforms of protein kinase C (PKC) in mediating cellular responses subsequent to activation of that enzyme. We have addressed this issue by the use of antisense technology. We have stably transfected Madin-Darby canine kidney cells with antisense PKC alpha, PKC beta, or both PKC alpha and -beta cDNAs. The transfected cDNA was integrated and expressed. We have isolated cells in which expression of PKC alpha is inhibited. In cells transfected with antisense PKC alpha or both PKC alpha and -beta, phorbol ester-stimulated release of arachidonate and its metabolites was inhibited, whereas in cells transfected with antisense PKC beta cDNA alone, phorbol ester-stimulated arachidonate release was not significantly different from control cells. We thus demonstrate the use of a novel technique to inhibit PKC isoform expression. We show that inhibition of expression of PKC alpha causes a loss in phospholipase A2-mediated arachidonate release. Antisense-inhibited expression of PKC isoforms may provide a useful approach to define additional functions of particular PKC isoforms.

Highlights

A major unresolved issue in the area of signal transe-ncoded by individual genes
In cells transfected with antisense PKCa or both PKCa and -6, phorbol esterstimulated release of arachidonate andits metabolites was inhibited, whereas in cells transfected with antisense PKCB cDNA alone, phorbol ester-stimulated arent
The more recently described 6, t, 5; q, and L Protein kinase C (PKC) isoforms are dependent on phospholipid and diacylglycerolfor theiractivation, but lacking the putative calcium binding site expressed in the regulatory region of PKCa, -P and -7,they arecalcium-independachidonatereleasewas notsignificantlydifferent from ent andshow distinct in vitro substrate specificities [6]

Summary

Introduction

A major unresolved issue in the area of signal transe-ncoded by individual genes. The a, p, and y forms of PKC duction relates to the role of particular isoforms of were the first to be cloned [1] and to be characterized bioprotein kinase C (PKC) in mediating cellular responseschemically [5]. (PKCP consistsof two forms that are subsequent to activationof that enzyme.We have ad- alternatively spliced variants of a single gene.) PKCa, -D, and dressed this issue by the use of antisense technology. -7are all phospholipid-, diacylglycerol-,and calcium-depend-. (PKCP consistsof two forms that are subsequent to activationof that enzyme.We have ad- alternatively spliced variants of a single gene.) PKCa, -D, and dressed this issue by the use of antisense technology. We have stably transfected Madin-Darby caninekidney cells with antisense PKCa, PKC/3,orbothPKCa and -6 cDNAs. The transfected cDNA was integrated and expressed. The more recently described 6, t, 5; q, and L PKC isoforms are dependent on phospholipid and diacylglycerolfor theiractivation, but lacking the putative calcium binding site expressed in the regulatory region of PKCa, -P and -7,they arecalcium-independachidonatereleasewas notsignificantlydifferent from ent andshow distinct in vitro substrate specificities [6]. An- to implicate a particular form of PKC are its translocation tisense-inhibited expression oPfKC isoforms may pro- and/or phorbol ester-induced down-regulation The down-regulation paradigm has thecomplicating factor of “activation” of the kinase being a prereq-

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