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Novel PKC?-mediated phosphorylation site(s) on cofilin and their potential role in terminating histamine release.

ABSTRACT: Using specific inhibitors, kinase-negative mutants, and small interfering RNA against protein kinase C? (PKC?) or PKC?I, we find that PKC?I positively regulates degranulation in rat basophilic leukemia-2H3 cells, whereas PKC? negatively regulates degranulation. Mass spectrometric and mutagenic analyses reveal that PKC? phosphorylates cofilin at Ser-23 and/or Ser-24 during degranulation. Overexpression of a nonphosphorylatable form (S23,24A), but not that of a mutant-mimicking phosphorylated form (S23,24E), increases degranulation. Furthermore, the S23,24A mutant binds to F-actin and retains its depolymerizing and/or cleavage activity; conversely, the S23,24E mutant is unable to sever actin filaments, resulting in F-actin polymerization. In addition, the S23,24E mutant preferentially binds to the 14-3-3? protein. Fluorescence-activated cell sorting analysis with fluorescein isothiocyanate-phalloidin and simultaneous observation of degranulation, PKC translocation, and actin polymerization reveals that during degranulation, actin polymerization is dependent on PKC? activity. These results indicate that a novel PKC?-mediated phosphorylation event regulates cofilin by inhibiting its ability to depolymerize F-actin and bind to 14-3-3?, thereby promoting F-actin polymerization, which is necessary for cessation of degranulation.

SUBMITTER: Sakuma M

PROVIDER: S-EPMC3442417 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Novel PKCα-mediated phosphorylation site(s) on cofilin and their potential role in terminating histamine release.

Sakuma Megumi M Shirai Yasuhito Y Yoshino Ken-ichi K Kuramasu Maho M Nakamura Tomofumi T Yanagita Toshihiko T Mizuno Kensaku K Hide Izumi I Nakata Yoshihiro Y Saito Naoaki N

Molecular biology of the cell 20120801 18

Using specific inhibitors, kinase-negative mutants, and small interfering RNA against protein kinase Cα (PKCα) or PKCβI, we find that PKCβI positively regulates degranulation in rat basophilic leukemia-2H3 cells, whereas PKCα negatively regulates degranulation. Mass spectrometric and mutagenic analyses reveal that PKCα phosphorylates cofilin at Ser-23 and/or Ser-24 during degranulation. Overexpression of a nonphosphorylatable form (S23,24A), but not that of a mutant-mimicking phosphorylated form ...[more]

PMID: 22855535

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Project description:Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a key enzyme for the metabolism of many toxic aldehydes such as acetaldehyde, derived from alcohol drinking, and 4HNE, an oxidative stress-derived lipid peroxidation aldehyde. Post-translational enhancement of ALDH2 activity can be achieved by serine/threonine phosphorylation by epsilon protein kinase C (?PKC). Elevated ALDH2 is beneficial in reducing injury following myocardial infarction, stroke and other oxidative stress and aldehyde toxicity-related diseases. We have previously identified three ?PKC phosphorylation sites, threonine 185 (T185), serine 279 (S279) and threonine 412 (T412), on ALDH2. Here we further characterized the role and contribution of each phosphorylation site to the enhancement of enzymatic activity by ?PKC.Each individual phosphorylation site was mutated to a negatively charged amino acid, glutamate, to mimic a phosphorylation, or to a non-phosphorylatable amino acid, alanine. ALDH2 enzyme activities and protection against 4HNE inactivation were measured in the presence or absence of ?PKC phosphorylation in vitro. Coevolution of ALDH2 and its ?PKC phosphorylation sites was delineated by multiple sequence alignments among a diverse range of species and within the ALDH multigene family.We identified S279 as a critical ?PKC phosphorylation site in the activation of ALDH2. The critical catalytic site, cysteine 302 (C302) of ALDH2 is susceptible to adduct formation by reactive aldehyde, 4HNE, which readily renders the enzyme inactive. We show that phosphomimetic mutations of T185E, S279E and T412E confer protection of ALDH2 against 4HNE-induced inactivation, indicating that phosphorylation on these three sites by ?PKC likely also protects the enzyme against reactive aldehydes. Finally, we demonstrate that the three ALDH2 phosphorylation sites co-evolved with ?PKC over a wide range of species. Alignment of 18 human ALDH isozymes, indicates that T185 and S279 are unique ALDH2, ?PKC specific phosphorylation sites, while T412 is found in other ALDH isozymes. We further identified three highly conserved serine/threonine residues (T384, T433 and S471) in all 18 ALDH isozymes that may play an important phosphorylation-mediated regulatory role in this important family of detoxifying enzymes.?PKC phosphorylation and its coevolution with ALDH2 play an important role in the regulation and protection of ALDH2 enzyme activity.

Dataset Information

Novel PKC?-mediated phosphorylation site(s) on cofilin and their potential role in terminating histamine release.

Publications

Novel PKCα-mediated phosphorylation site(s) on cofilin and their potential role in terminating histamine release.

Similar Datasets

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