Dataset Information

PKC?-Mediated PDK1 Phosphorylation Enhances T Cell Activation by Increasing PDK1 Stability.

ABSTRACT: PDK1 is essential for T cell receptor (TCR)-mediated activation of NF-?B, and PDK1-induced phosphorylation of PKC? is important for TCR-induced NF-?B activation. However, inverse regulation of PDK1 by PKC? during T cell activation has not been investigated. In this study, we found that PKC? is involved in human PDK1 phosphorylation and that its kinase activity is crucial for human PDK1 phosphorylation. Mass spectrometry analysis of wild-type PKC? or of kinase-inactive form of PKC? revealed that PKC? induced phosphorylation of human PDK1 at Ser-64. This PKC?-induced PDK1 phosphorylation positively regulated T cell activation and TCR-induced NF-?B activation. Moreover, phosphorylation of human PDK1 at Ser-64 increased the stability of human PDK1 protein. These results suggest that Ser-64 is an important phosphorylation site that is part of a positive feedback loop for human PDK1-PKC?-mediated T cell activation.

SUBMITTER: Kang JA

PROVIDER: S-EPMC5303887 | biostudies-other | 2017 Jan

REPOSITORIES: biostudies-other

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PKCθ-Mediated PDK1 Phosphorylation Enhances T Cell Activation by Increasing PDK1 Stability.

Kang Jung-Ah JA Choi Hyunwoo H Yang Taewoo T Cho Steve K SK Park Zee-Yong ZY Park Sung-Gyoo SG

Molecules and cells 20170126 1

PDK1 is essential for T cell receptor (TCR)-mediated activation of NF-κB, and PDK1-induced phosphorylation of PKCθ is important for TCR-induced NF-κB activation. However, inverse regulation of PDK1 by PKCθ during T cell activation has not been investigated. In this study, we found that PKCθ is involved in human PDK1 phosphorylation and that its kinase activity is crucial for human PDK1 phosphorylation. Mass spectrometry analysis of wild-type PKCθ or of kinase-inactive form of PKCθ revealed that ...[more]

PMID: 28152304

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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

PKC?-Mediated PDK1 Phosphorylation Enhances T Cell Activation by Increasing PDK1 Stability.

Publications

PKCθ-Mediated PDK1 Phosphorylation Enhances T Cell Activation by Increasing PDK1 Stability.

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