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Direct Binding and Activation of Protein Kinase C Isoforms by Aldosterone and 17ß-Estradiol

Department of Molecular Medicine, Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland

Address all correspondence and requests for reprints to: Rodrigo Alzamora, Molecular Medicine, Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, P.O. Box 9063, Dublin 9, Ireland. E-mail: ralzamora{at}rcsi.ie.

Protein kinase C (PKC) is a signal transduction protein that has been proposed to mediate rapid responses to steroid hormones. Previously, we have shown aldosterone directly activates PKC whereas 17ß-estradiol activates PKC and PKC; however, neither the binding to PKCs nor the mechanism of action has been established. To determine the domains of PKC and PKC involved in binding of aldosterone and 17ß-estradiol, glutathione S-transferase fusion recombinant PKC and PKC mutants were used to perform in vitro binding assays with [³H]aldosterone and [³H]17ß-estradiol. 17ß-Estradiol bound both PKC and PKC but failed to bind PKC mutants lacking a C2 domain. Similarly, aldosterone bound only PKC and mutants containing C2 domains. Thus, the C2 domain is critical for binding of these hormones. Binding affinities for aldosterone and 17ß-estradiol were between 0.5–1.0 nM. Aldosterone and 17ß-estradiol competed for binding to PKC, suggesting they share the same binding site. Phorbol 12,13-dybutyrate did not compete with hormone binding; furthermore, they have an additive effect on PKC activity. EC₅₀ for activation of PKC and PKC by aldosterone and 17ß-estradiol was approximately 0.5 nM. Immunoblot analysis using a phospho-PKC antibody revealed that upon binding, PKC and PKC undergo autophosphorylation with an EC₅₀ in the 0.5–1.0 nM range. 17ß-Estradiol activated PKC and PKC in estrogen receptor-positive and -negative breast cancer cells (MCF-7 and HCC-38, respectively), suggesting estrogen receptor expression is not required for 17ß-estradiol-induced PKC activation. The present results provide first evidence for direct binding and activation of PKC and PKC by steroid hormones and the molecular mechanisms involved.

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Ligands:   Hydrocortisone  |  17β-Estradiol  |  Aldosterone