Direct binding and activation of protein kinase C isoforms by aldosterone and 17β-estradiol

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 alpha whereas 17 beta-estradiol activates PKC alpha and PKC delta; however, neither the binding to PKCs nor the mechanism of action has been established. To determine the domains of PKC alpha and PKC delta involved in binding of aldosterone and 17 beta-estradiol, glutathione S-transferase fusion recombinant PKC alpha and PKC delta mutants were used to perform in vitro binding assays with [ H-3] aldosterone and [ H-3] 17 beta-estradiol. 17 beta-Estradiol bound both PKC alpha and PKC delta but failed to bind PKC mutants lacking a C2 domain. Similarly, aldosterone bound only PKC alpha and mutants containing C2 domains. Thus, the C2 domain is critical for binding of these hormones. Binding affinities for aldosterone and 17 beta-estradiol were between 0.5-1.0 nM. Aldosterone and 17 beta-estradiol competed for binding to PKC alpha, 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 alpha and PKC delta by aldosterone and 17 beta-estradiol was approximately 0.5 nM. Immunoblot analysis using a phospho-PKC antibody revealed that upon binding, PKC alpha and PKC delta undergo autophosphorylation with an EC50 in the 0.5-1.0 nM range. 17 beta-Estradiol activated PKC alpha and PKC delta in estrogen receptor-positive and -negative breast cancer cells ( MCF-7 and HCC-38, respectively), suggesting estrogen receptor expression is not required for 17 beta-estradiol-induced PKC activation. The present results provide first evidence for direct binding and activation of PKC alpha and PKC delta by steroid hormones and the molecular mechanisms involved.