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A temperature-dependent conformational shift in p38? MAPK substrate-binding region associated with changes in substrate phosphorylation profile.

ABSTRACT: Febrile-range hyperthermia worsens and hypothermia mitigates lung injury, and temperature dependence of lung injury is blunted by inhibitors of p38 mitogen-activated protein kinase (MAPK). Of the two predominant p38 isoforms, p38? is proinflammatory and p38? is cytoprotective. Here, we analyzed the temperature dependence of p38 MAPK activation, substrate interaction, and tertiary structure. Incubating HeLa cells at 39.5 °C stimulated modest p38 activation, but did not alter tumor necrosis factor-? (TNF?)-induced p38 activation. In in vitro kinase assays containing activated p38? and MAPK-activated kinase-2 (MK2), MK2 phosphorylation was 14.5-fold greater at 39.5 °C than at 33 °C. By comparison, we observed only 3.1- and 1.9-fold differences for activating transcription factor-2 (ATF2) and signal transducer and activator of transcription-1? (STAT1?) and a 7.7-fold difference for p38? phosphorylation of MK2. The temperature dependence of p38?:substrate binding affinity, as measured by surface plasmon resonance, paralleled substrate phosphorylation. Hydrogen-deuterium exchange MS (HDX-MS) of p38? performed at 33, 37, and 39.5 °C indicated temperature-dependent conformational changes in an ? helix near the common docking and glutamate:aspartate substrate-binding domains at the known binding site for MK2. In contrast, HDX-MS analysis of p38? did not detect significant temperature-dependent conformational changes in this region. We observed no conformational changes in the catalytic domain of either isoform and no corresponding temperature dependence in the C-terminal p38?-interacting region of MK2. Because MK2 participates in the pathogenesis of lung injury, the observed changes in the structure and function of proinflammatory p38? may contribute to the temperature dependence of acute lung injury.

SUBMITTER: Deredge D

PROVIDER: S-EPMC6709627 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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A temperature-dependent conformational shift in p38α MAPK substrate-binding region associated with changes in substrate phosphorylation profile.

Deredge Daniel D Wintrode Patrick L PL Tulapurkar Mohan E ME Nagarsekar Ashish A Zhang Yinghua Y Weber David J DJ Shapiro Paul P Hasday Jeffrey D JD

The Journal of biological chemistry 20190618 34

Febrile-range hyperthermia worsens and hypothermia mitigates lung injury, and temperature dependence of lung injury is blunted by inhibitors of p38 mitogen-activated protein kinase (MAPK). Of the two predominant p38 isoforms, p38α is proinflammatory and p38β is cytoprotective. Here, we analyzed the temperature dependence of p38 MAPK activation, substrate interaction, and tertiary structure. Incubating HeLa cells at 39.5 °C stimulated modest p38 activation, but did not alter tumor necrosis factor ...[more]

PMID: 31213525

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Project description:UnlabelledBackgroundThe p38 mitogen-activated protein kinases (MAPK) family plays pivotal roles in skeletal muscle metabolism. Recent evidence revealed that p38? and p38? exert paradoxical effects on muscle protein homeostasis. However, it is unknown why p38?, but not p38?, is capable of mediating muscle catabolism via selective activation of the C/EBP? that upregulates atrogin1/MAFbx.MethodsTryptic phosphopeptide mapping was carried out to identify p38?- and p38?-mediated phosphorylation sites in C/EBP?. Chromosome immunoprecipitation (ChIP) assay was used to evaluate p38? and p38? effect on C/EBP? binding to the atrogin1/MAFbx promoter. Overexpression or siRNA-mediated gene knockdown of p38? and p38?, and site-directed mutagenesis or knockout of C/EBP?, were used to analyze the roles of these kinases in muscle catabolism in C2C12 myotubes and mice.ResultsCellular expression of constitutively active p38? or p38? resulted in phosphorylation of C/EBP? at multiple serine and threonine residues; however, only p38? phosphorylated Thr-188, which had been known to be critical to the DNA-binding activity of C/EBP?. Only p38?, but not p38?, activated C/EBP?-binding to the atrogin1/MAFbx promoter. A C/EBP? mutant in which Thr-188 was replaced by alanine acted as a dominant-negative inhibitor of atrogin1/MAFbx upregulation induced by either p38? or Lewis lung carcinoma (LLC) cell-conditioned medium (LCM). In addition, knockdown of p38? specifically inhibited C/EBP? activation and atrogin1/MAFbx upregulation induced by LCM. Finally, expression of active p38? in mouse tibialis anterior specifically induced C/EBP? phosphorylation at Thr-188, atrogin1/MAFbx upregulation and muscle mass loss, which were blocked in C/EBP?-null mice.ConclusionsThe ? and ? isoforms of p38 MAPK are capable of recognizing distinct phosphorylation sites in a substrate. The unique capacity of p38? in mediating muscle catabolism is due to its capability in phosphorylating Thr-188 of C/EBP?.

Project description:BackgroundRenal interstitial fibrosis (RIF) is a common pathway to end-stage renal disease regardless of the initial etiology. Currently, the molecular mechanisms for RIF remains not fully elucidated. Nuclear receptor subfamily 4 group A member 1(Nr4a1), a member of the NR4A subfamily of nuclear receptors, is a ligand-activated transcription factor. The role of Nr4a1 in RIF remains largely unknown.MethodsIn this study, we determined the role and action mechanism of Nr4a1 in RIF. We used unilateral ureteral obstruction (UUO) mice and transforming growth factor (TGF)-β1-treated human renal proximal tubular epithelial cells (HK-2 cells) as in vivo and in vitro models of RIF. A specific Nr4a1 agonist Cytosporone B (Csn-B) was applied to activate Nr4a1 both in vivo and in vitro, and Nr4a1 small interfering RNA was applied in vitro. Renal pathological changes were evaluated by hematoxylin and eosin and Masson staining, and the expression of fibrotic proteins including fibronectin (Fn) and collagen-I (Col-I), and phosphorylated p38 MAPK was measure by immunohistochemical staining and western blot analysis.ResultsThe results showed that Nr4a1 was upregulated in UUO mouse kidneys, and was positively correlated with the degree of interstitial kidney injury and the levels of fibrotic proteins. Csn-B treatment aggravated UUO-induced renal interstitial fibrosis, and induced p38 MAPK phosphorylation. In vitro, TGF-β induced Nr4a1 expression, and Nr4a1 downregulation prevented TGF-β1-induced expression of Fn and Col-I and the activation of p38 MAPK. Csn-B induced fibrotic proteins expression and p38 MAPK phosphorylation, and moreover Csn-B induced fibrotic proteins expression was abrogated by treatment with p38 MAPK inhibitor SB203580. We provided further evidence that Csn-B treatment promoted cytoplasmic accumulation of Nr4a1.ConclusionThe findings in the present study indicate that Nr4a1 promotes renal fibrosis potentially through activating p38 MAPK kinase.

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A temperature-dependent conformational shift in p38? MAPK substrate-binding region associated with changes in substrate phosphorylation profile.

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A temperature-dependent conformational shift in p38α MAPK substrate-binding region associated with changes in substrate phosphorylation profile.

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