Affiliation:
1. Department of Molecular Medicine, Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland
Abstract
AbstractProtein 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 PKCα whereas 17β-estradiol activates PKCα and PKCδ; however, neither the binding to PKCs nor the mechanism of action has been established. To determine the domains of PKCα and PKCδ involved in binding of aldosterone and 17β-estradiol, glutathione S-transferase fusion recombinant PKCα and PKCδ mutants were used to perform in vitro binding assays with [3H]aldosterone and [3H]17β-estradiol. 17β-Estradiol bound both PKCα and PKCδ but failed to bind PKC mutants lacking a C2 domain. Similarly, aldosterone bound only PKCα and mutants containing C2 domains. Thus, the C2 domain is critical for binding of these hormones. Binding affinities for aldosterone and 17β-estradiol were between 0.5–1.0 nM. Aldosterone and 17β-estradiol competed for binding to PKCα, 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 PKCα and PKCδ by aldosterone and 17β-estradiol was approximately 0.5 nM. Immunoblot analysis using a phospho-PKC antibody revealed that upon binding, PKCα and PKCδ undergo autophosphorylation with an EC50 in the 0.5–1.0 nm range. 17β-Estradiol activated PKCα and PKCδ in estrogen receptor-positive and -negative breast cancer cells (MCF-7 and HCC-38, respectively), suggesting estrogen receptor expression is not required for 17β-estradiol-induced PKC activation. The present results provide first evidence for direct binding and activation of PKCα and PKCδ by steroid hormones and the molecular mechanisms involved.
Subject
Endocrinology,Molecular Biology,General Medicine
Cited by
38 articles.
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