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Curcumin Inhibits Protein Kinase C? Activity by Binding to Its C1 Domain.

ABSTRACT: Curcumin is a polyphenolic nutraceutical that acts on multiple biological targets, including protein kinase C (PKC). PKC is a family of serine/threonine kinases central to intracellular signal transduction. We have recently shown that curcumin selectively inhibits PKC?, but not PKC?, in CHO-K1 cells [Pany, S. (2016) Biochemistry 55, 2135-2143]. To understand which domain(s) of PKC? is responsible for curcumin binding and inhibitory activity, we made several domain-swapped mutants in which the C1 (combination of C1A and C1B) and C2 domains are swapped between PKC? and PKC?. Phorbol ester-induced membrane translocation studies using confocal microscopy and immunoblotting revealed that curcumin inhibited phorbol ester-induced membrane translocation of PKC? mutants, in which the ?C1 domain was replaced with ?C1, but not the PKC? mutant in which ?C1 was replaced with the ?C1 domain, suggesting that ?C1 is a determinant for curcumin's inhibitory effect. In addition, curcumin inhibited membrane translocation of PKC? mutants, in which the ?C1A and ?C1B domains were replaced with the ?C1A and ?C1B domains, respectively, indicating the role of both ?C1A and ?C1B domains in curcumin's inhibitory effects. Phorbol 13-acetate inhibited the binding of curcumin to ?C1A and ?C1B with IC50 values of 6.27 and 4.47 ?M, respectively. Molecular docking and molecular dynamics studies also supported the higher affinity of curcumin for ?C1B than for ?C1A. The C2 domain-swapped mutants were inactive in phorbol ester-induced membrane translocation. These results indicate that curcumin binds to the C1 domain of PKC? and highlight the importance of this domain in achieving PKC isoform selectivity.

SUBMITTER: Pany S

PROVIDER: S-EPMC5657492 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Curcumin Inhibits Protein Kinase Cα Activity by Binding to Its C1 Domain.

Pany Satyabrata S You Youngki Y Das Joydip J

Biochemistry 20161102 45

Curcumin is a polyphenolic nutraceutical that acts on multiple biological targets, including protein kinase C (PKC). PKC is a family of serine/threonine kinases central to intracellular signal transduction. We have recently shown that curcumin selectively inhibits PKCα, but not PKCε, in CHO-K1 cells [Pany, S. (2016) Biochemistry 55, 2135-2143]. To understand which domain(s) of PKCα is responsible for curcumin binding and inhibitory activity, we made several domain-swapped mutants in which the C1 ...[more]

PMID: 27776404

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Project description:Because human prostate-distributed UDP-glucuronosyltransferase (UGT) 2B15 metabolizes 5α-dihydrotestosterone (DHT) and 3α-androstane-5α,17β-diol metabolite, we sought to determine whether 2B15 requires regulated phosphorylation similar to UGTs already analyzed. Reversible down-regulation of 2B15-transfected COS-1 cells following curcumin treatment and irreversible inhibition by calphostin C, bisindolylmaleimide, or röttlerin treatment versus activation by phorbol 12-myristate 13-acetate indicated that 2B15 undergoes PKC phosphorylation. Mutation of three predicted PKC and two tyrosine kinase sites in 2B15 caused 70-100 and 80-90% inactivation, respectively. Anti-UGT-1168 antibody trapped 2B15-His-containing co-immunoprecipitates of PKCα in 130-140- and >150-kDa complexes by gradient SDS-PAGE analysis. Complexes bound to WT 2B15-His remained intact during electrophoresis, whereas 2B15-His mutants at phosphorylation sites differentially dissociated. PKCα siRNA treatment inactivated >50% of COS-1 cell-expressed 2B15. In contrast, treatment of 2B15-transfected COS-1 cells with the Src-specific activator 1,25-dihydroxyvitamin D(3) enhanced activity; treatment with the Src-specific PP2 inhibitor or Src siRNA inhibited >50% of the activity. Solubilized 2B15-His-transfected Src-free fibroblasts subjected to in vitro [γ-(33)P]ATP-dependent phosphorylation by PKCα and/or Src, affinity purification, and SDS gel analysis revealed 2-fold more radiolabeling of 55-58-kDa 2B15-His by PKCα than by Src; labeling was additive for combined kinases. Collectively, the evidence indicates that 2B15 requires regulated phosphorylation by both PKCα and Src, which is consistent with the complexity of synthesis and metabolism of its major substrate, DHT. Whether basal cells import or synthesize testosterone for transport to luminal cells for reduction to DHT by 5α-steroid reductase 2, comparatively low-activity luminal cell 2B15 undergoes a complex pattern of regulated phosphorylation necessary to maintain homeostatic DHT levels to support occupation of the androgen receptor for prostate-specific functions.

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Curcumin Inhibits Protein Kinase C? Activity by Binding to Its C1 Domain.

Publications

Curcumin Inhibits Protein Kinase Cα Activity by Binding to Its C1 Domain.

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