Inhibition of Membrane Lipid-independent Protein Kinase Cα Activity by Phorbol Esters, Diacylglycerols, and Bryostatin-1

Simon J Slater,Frank J Taddeo,Anthony Mazurek,Brigid A Stagliano,Shawn K Milano,Mary Beth Kelly,Cojen Ho,Christopher D Stubbs

doi:10.1074/jbc.273.36.23160

Simon J Slater, Frank J Taddeo + Show 6 more

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https://doi.org/10.1074/jbc.273.36.23160

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Abstract

The activity of membrane-associated protein kinase C (PKC) has previously been shown to be regulated by two discrete high and low affinity binding regions for diacylglycerols and phorbol esters (Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631). PKC is also known to interact with both cytoskeletal and nuclear proteins; however, less is known concerning the mode of activation of this non-membrane form of PKC. By using the fluorescent phorbol ester, sapintoxin D (SAPD), PKCalpha, alone, was found to possess both low and high affinity phorbol ester-binding sites, showing that interaction with these sites does not require association with the membrane. Importantly, a fusion protein containing the isolated C1A/C1B (C1) domain of PKCalpha also bound SAPD with low and high affinity, indicating that the sites may be confined to this domain rather than residing elsewhere on the enzyme molecule. Both high and low affinity interactions with native PKCalpha were enhanced by protamine sulfate, which activates the enzyme without requiring Ca2+ or membrane lipids. However, this "non-membrane" PKC activity was inhibited by the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA) and also by the fluorescent analog, SAPD, opposite to its effect on membrane-associated PKCalpha. Bryostatin-1 and the soluble diacylglycerol, 1-oleoyl-2-acetylglycerol, both potent activators of membrane-associated PKC, also competed for both low and high affinity SAPD binding and inhibited protamine sulfate-induced activity. Furthermore, the inactive phorbol ester analog 4alpha-TPA (4alpha-12-O-tetradecanoylphorbol-13-acetate) also inhibited non-membrane-associated PKC. In keeping with these observations, although TPA could displace high affinity SAPD binding from both forms of the enzyme, 4alpha-TPA was only effective at displacing high affinity SAPD binding from non-membrane-associated PKC. 4alpha-TPA also displaced SAPD from the isolated C1 domain. These results show that although high and low affinity phorbol ester-binding sites are found on non-membrane-associated PKC, the phorbol ester binding properties change significantly upon association with membranes.

Highlights

Protein kinase C (PKC)1 constitutes a group of isozymes that are central in cellular signaling pathways that regulate numerous cellular processes, including cell growth, differentiation, and metabolism [1]
In order to determine if these binding site(s) “pre-exist” on lipid-independent PKC␣ or whether they are exposed upon membrane association, phorbol ester binding to non-membrane PKC␣ was studied
Binding of phorbol esters, which are commonly classed as activators of membrane-associated PKC, resulted in a potent inhibition of lipid-independent enzyme activity induced by protein-protein interactions with the arginine-rich protein, protamine sulfate

Summary

Introduction

Protein kinase C (PKC) constitutes a group of isozymes that are central in cellular signaling pathways that regulate numerous cellular processes, including cell growth, differentiation, and metabolism [1]. Interaction with arginine-rich proteins such as protamine sulfate relieves the Ca2ϩ and phospholipid requirements for PKC activity, it is not known whether this nonmembrane PKC activity is modulated by phorbol esters and/or diacylglycerols in a similar manner to the membrane-associated enzyme. Evidence supporting this possibility was provided recently by the finding that PKC␦ is capable of binding phorbol esters with low affinity in the absence of membrane lipids [22] and that these compounds can induce binding of PKC⑀ to fila-. The non-membrane, phorbol-induced interaction of PKC with F-actin departs from the original definition of RACKs that were described as proteins that bind PKC that has been activated by membrane association [25]

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