Curcumin Inhibits Protein Kinase Cα 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 PKCα, but not PKCε in CHO-K1 cells (Pany, S. (2016) Biochemistry 55, 2135–43). To understand which domain(s) of PKCα is/are 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 PKCα and PKCε. Phorbol ester-induced membrane translocation studies using confocal microscopy and immune-blotting revealed that curcumin inhibited phorbol ester induced membrane translocation of PKCε mutants, in which εC1 domain was replaced with αC1, but not the PKCα mutant in which αC1 was replaced with εC1 domain, suggesting that αC1 is a determinant for curcumin’s inhibitory effect. Further, curcumin inhibited membrane translocation of PKCε mutants, in which εC1A and εC1B domain were replaced with αC1A and αC1B domains, respectively, indicating the role of both αC1A and αC1B domains in curcumin’s inhibitory effects. Phorbol 13-acetate inhibited curcumin binding to αC1A and αC1B with IC50 values of 6.27 μM and 4.47 μM, respectively. Molecular docking and molecular dynamics studies also supported higher affinity of curcumin for αC1B than αC1A. The C2 domain swapped mutants were inactive in phorbol ester induced membrane translocation. These results indicate that curcumin binds to the C1 domain of PKCα and highlight the importance of this domain in achieving PKC isoform selectivity.

Anti-Malignant Ascites Effect of Total Diterpenoids from Euphorbiae ebracteolatae Radix Is Attributable to Alterations of Aquaporins via Inhibiting PKC Activity in the Kidney

“…The PKC family has included several isoforms [ 26 ]. After activation, the PKCs translocate from the cytosol to the plasma membrane, bind to diacylglycerol (DAG), and subsequently induce a series of physiological changes [ 27 ]. PKC translocation from the cytosol to the plasma membrane is an indicator of PKC activation [ 28 ].…”

Resveratrol inhibits phorbol ester-induced membrane translocation of presynaptic Munc13-1

“…The pellets were washed twice with 70% acetone, and re-dissolved in 40 μl of Laemmli sample buffer [51]. Cell fractions were subjected to SDS-PAGE and immunoblot analysis as described earlier [52]. The anti-rabbit GFP (1:1000); anti-rabbit β-actin (1:2000); and anti-rabbit HRP-conjugated (1:5000) (Cell Signaling Technology, Inc. Danvers, MA) were used for immunoblot analysis.…”

“…Confocal microscopic analysis was carried out using Leica SP8 confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL) with 60x, NA 1.42 oil objective as reported earlier [52]. Munc13-1 transfected HT22 cells were either treated or co-treated with TPA and resveratrol on glass coverslips (VWR, Atlanta, GA) for 15 min.…”

“…Coverslips were mounted in mounting medium (Vector Laboratories, Inc., Burlingame, CA). ImageJ (http://rsb.info.nih.gov/ij/) was used to measure the sub-cellular distribution of Munc13-1 protein [52]. …”

“…The mean intensity of fluorescence of Munc13-1 in cytosol and plasma membrane in Tuj1-positive neurons were measured, as well as area of the cytosol and plasma membrane were quantified using LAS X software (Leica Microsystem INC., USA). Data were expressed as ratio of mean intensity of Munc13-1 in plasma membrane and cytosol per μm 2 area [48, 52]. …”