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