Direct binding and activation of protein kinase C isoforms by aldosterone and 17beta-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 PKCalpha whereas 17beta-estradiol activates PKCalpha and PKCdelta; however, neither the binding to PKCs nor the mechanism of action has been established. To determine the domains of PKCalpha and PKCdelta involved in binding of aldosterone and 17beta-estradiol, glutathione S-transferase fusion recombinant PKCalpha and PKCdelta mutants were used to perform in vitro binding assays with [(3)H]aldosterone and [(3)H]17beta-estradiol. 17beta-Estradiol bound both PKCalpha and PKCdelta but failed to bind PKC mutants lacking a C2 domain. Similarly, aldosterone bound only PKCalpha and mutants containing C2 domains. Thus, the C2 domain is critical for binding of these hormones. Binding affinities for aldosterone and 17beta-estradiol were between 0.5-1.0 nM. Aldosterone and 17beta-estradiol competed for binding to PKCalpha, 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. EC(50) for activation of PKCalpha and PKCdelta by aldosterone and 17beta-estradiol was approximately 0.5 nM. Immunoblot analysis using a phospho-PKC antibody revealed that upon binding, PKCalpha and PKCdelta undergo autophosphorylation with an EC(50) in the 0.5-1.0 nm range. 17beta-Estradiol activated PKCalpha and PKCdelta in estrogen receptor-positive and -negative breast cancer cells (MCF-7 and HCC-38, respectively), suggesting estrogen receptor expression is not required for 17beta-estradiol-induced PKC activation. The present results provide first evidence for direct binding and activation of PKCalpha and PKCdelta by steroid hormones and the molecular mechanisms involved.
Department of Molecular Medicine, Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, P.O. Box 9063, Dublin 9, Ireland. ralzamora@rcsi.ie
Rodrigo
R
Alzamora
Laura R
LR
Brown
Brian J
BJ
Harvey
D000450:Aldosterone / Q000737:chemistry*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004952:Esters / Q000737:chemistry; D004958:Estradiol / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013256:Steroids / Q000737:chemistry / Q000378:metabolism
D004952:Esters; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D013256:Steroids; D000450:Aldosterone; D004958:Estradiol; D005982:Glutathione Transferase; D011493:Protein Kinase C