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Phosphorylation of Nox1 regulates association with NoxA1 activation domain.

ABSTRACT: RATIONALE:Activation of Nox1 initiates redox-dependent signaling events crucial in the pathogenesis of vascular disease. Selective targeting of Nox1 is an attractive potential therapy, but requires a better understanding of the molecular modifications controlling its activation. OBJECTIVE:To determine whether posttranslational modifications of Nox1 regulate its activity in vascular cells. METHODS AND RESULTS:We first found evidence that Nox1 is phosphorylated in multiple models of vascular disease. Next, studies using mass spectroscopy and a pharmacological inhibitor demonstrated that protein kinase C-beta1 mediates phosphorylation of Nox1 in response to tumor necrosis factor-?. siRNA-mediated silencing of protein kinase C-beta1 abolished tumor necrosis factor-?-mediated reactive oxygen species production and vascular smooth muscle cell migration. Site-directed mutagenesis and isothermal titration calorimetry indicated that protein kinase C-beta1 phosphorylates Nox1 at threonine 429. Moreover, Nox1 threonine 429 phosphorylation facilitated the association of Nox1 with the NoxA1 activation domain and was necessary for NADPH oxidase complex assembly, reactive oxygen species production, and vascular smooth muscle cell migration. CONCLUSIONS:We conclude that protein kinase C-beta1 phosphorylation of threonine 429 regulates activation of Nox1 NADPH oxidase.

SUBMITTER: Streeter J

PROVIDER: S-EPMC5025877 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Phosphorylation of Nox1 regulates association with NoxA1 activation domain.

Streeter Jennifer J Schickling Brandon M BM Jiang Shuxia S Stanic Bojana B Thiel William H WH Gakhar Lokesh L Houtman Jon C D JC Miller Francis J FJ

Circulation research 20140916 11

<h4>Rationale</h4>Activation of Nox1 initiates redox-dependent signaling events crucial in the pathogenesis of vascular disease. Selective targeting of Nox1 is an attractive potential therapy, but requires a better understanding of the molecular modifications controlling its activation.<h4>Objective</h4>To determine whether posttranslational modifications of Nox1 regulate its activity in vascular cells.<h4>Methods and results</h4>We first found evidence that Nox1 is phosphorylated in multiple mo ...[more]

PMID: 25228390

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Project description:Recent work indicates that the LKB1 tumour suppressor protein kinase, which is mutated in Peutz-Jeghers cancer syndrome, phosphorylates and activates a group of protein kinases that are related to AMPK (AMP-activated protein kinase). Ten of the 14 AMPK-related protein kinases activated by LKB1, including SIK (salt-induced kinase), MARK (microtubule-affinity-regulating kinase) and BRSK (brain-specific kinase) isoforms, possess a ubiquitin-associated (UBA) domain immediately C-terminal to the kinase catalytic domain. These are the only protein kinases in the human genome known to possess a UBA domain, but their roles in regulating AMPK-related kinases are unknown. We have investigated the roles that the UBA domain may play in regulating these enzymes. Limited proteolysis of MARK2 revealed that the kinase and UBA domains were contained within a fragment that was resistant to trypsin proteolysis. SAXS (small-angle X-ray scattering) analysis of inactive and active LKB1-phosphorylated MARK2 revealed that activation of MARK2 is accompanied by a significant conformational change that alters the orientation of the UBA domain with respect to the catalytic domain. Our results indicate that none of the UBA domains found in AMPK-related kinases interact with polyubiquitin or other ubiquitin-like molecules. Instead, the UBA domains appear to play an essential conformational role and are required for the LKB1-mediated phosphorylation and activation of AMPK-related kinases. This is based on the findings that mutation or removal of the UBA domains of several AMPK-related kinases, including isoforms of MARK, SIK and BRSK, markedly impaired the catalytic activity and LKB1-mediated phosphorylation of these enzymes. We also provide evidence that the UBA domains do not function as LKB1-STRAD (STE20-related adaptor)-MO25 (mouse protein 25) docking/interacting sites and that mutations in the UBA domain of SIK suppressed the ability of SIK to localize within punctate regions of the nucleus. Taken together, these findings suggest that the UBA domains of AMPK-related kinases play an important role in regulating the conformation, activation and localization of these enzymes.

Project description:Chk2 is a critical regulator of the cellular DNA damage repair response. Activation of Chk2 in response to IR-induced damage is initiated by phosphorylation of the Chk2 SQ/TQ cluster domain at Ser(19), Ser(33), Ser(35), and Thr(68). This precedes autophosphorylation of Thr(383)/Thr(387) in the T-loop region of the kinase domain an event that is a prerequisite for efficient kinase activity. We conducted an in-depth analysis of phosphorylation within the T-loop region (residues 366-406). We report four novel phosphorylation sites at Ser(372), Thr(378), Thr(389), and Tyr(390). Substitution mutation Y390F was defective for kinase function. The substitution mutation T378A ablated the IR induction of kinase activity. Interestingly, the substitution mutation T389A demonstrated a 6-fold increase in kinase activity when compared with wild-type Chk2. In addition, phosphorylation at Thr(389) was a prerequisite to phosphorylation at Thr(387) but not at Thr(383). Quantitative mass spectrometry analysis revealed IR-induced phosphorylation and subcellular distribution of Chk2 phosphorylated species. We observed IR-induced increase in phosphorylation at Ser(379), Thr(389), and Thr(383)/Thr(389). Phosphorylation at Tyr(390) was dramatically reduced following IR. Exposure to IR was also associated with changes in the ratio of chromatin/nuclear localization. IR-induced increase in chromatin localization was associated with phosphorylation at Thr(372), Thr(379), Thr(383), Thr(389), Thr(383)/Thr(387), and Thr(383)/Thr(389). Chk2 hyper-phosphorylated species at Thr(383)/Thr(387)/Thr(389) and Thr(383)/Thr(387)/Thr(389)/Tyr(390) relocalized from almost exclusively chromatin to predominately nuclear expression, suggesting a role for phosphorylation in regulation of chromatin targeting and egress. The differential impact of T-loop phosphorylation on Chk2 ubiquitylation suggests a co-dependence of these modifications. The results demonstrate that a complex interdependent network of phosphorylation events within the T-loop exchange region regulates dimerization/autophosphorylation, kinase activation, and chromatin targeting/egress of Chk2.

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Phosphorylation of Nox1 regulates association with NoxA1 activation domain.

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Phosphorylation of Nox1 regulates association with NoxA1 activation domain.

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