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.
Alzamora
Rodrigo
R
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
Brown
Laura R
LR
Harvey
Brian J
BJ
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