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Conserved modular domains team up to latch-open active protein kinase C?.

ABSTRACT: Signaling proteins comprised of modular domains have evolved along with multicellularity as a method to facilitate increasing intracellular bandwidth. The effects of intramolecular interactions between modular domains within the context of native proteins have been largely unexplored. Here we examine intra- and intermolecular interactions in the multidomain signaling protein, protein kinase C? (PKC?). We identify three interactions between two activated PKC molecules that synergistically stabilize a nanomolar affinity homodimer. Disruption of the homodimer results in a loss of PKC-mediated ERK1/2 phosphorylation, whereas disruption of the auto-inhibited state promotes the homodimer and prolongs PKC membrane localization. These observations support a novel regulatory mechanism wherein homodimerization dictates the equilibrium between the auto-inhibited and active states of PKC by sequestering auto-inhibitory interactions. Our findings underscore the physiological importance of context-dependent modular domain interactions in cell signaling.

SUBMITTER: Swanson CJ

PROVIDER: S-EPMC4067214 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Conserved modular domains team up to latch-open active protein kinase Cα.

Swanson Carter J CJ Ritt Michael M Wang William W Lang Michael J MJ Narayan Arvind A Tesmer John J JJ Westfall Margaret M Sivaramakrishnan Sivaraj S

The Journal of biological chemistry 20140430 25

Signaling proteins comprised of modular domains have evolved along with multicellularity as a method to facilitate increasing intracellular bandwidth. The effects of intramolecular interactions between modular domains within the context of native proteins have been largely unexplored. Here we examine intra- and intermolecular interactions in the multidomain signaling protein, protein kinase Cα (PKCα). We identify three interactions between two activated PKC molecules that synergistically stabili ...[more]

PMID: 24790081

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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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Conserved modular domains team up to latch-open active protein kinase C?.

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Conserved modular domains team up to latch-open active protein kinase Cα.

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