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

摘要: Febrile-range hyperthermia worsens and hypothermia mitigates lung injury, temperature dependence of injury is blunted by inhibitors p38 mitogen-activated protein kinase (MAPK). Of the two predominant isoforms, p38α proinflammatory p38β cytoprotective. Here, we analyzed MAPK activation, substrate interaction, tertiary structure. Incubating HeLa cells at 39.5 °C stimulated modest but did not alter tumor necrosis factor-α (TNFα)-induced activation. In in vitro assays containing activated MAPK-activated kinase-2 (MK2), MK2 phosphorylation was 14.5-fold greater than 33 °C. By comparison, observed only 3.1- 1.9-fold differences for activating transcription factor-2 (ATF2) signal transducer activator transcription-1α (STAT1α) a 7.7-fold difference MK2. The p38α:substrate binding affinity, as measured surface plasmon resonance, paralleled phosphorylation. Hydrogen–deuterium exchange MS (HDX-MS) performed 33, 37, indicated temperature-dependent conformational changes an α helix near common docking glutamate:aspartate substrate-binding domains known site contrast, HDX-MS analysis detect significant this region. We no catalytic domain either isoform corresponding C-terminal p38α-interacting region Because participates pathogenesis structure function may contribute to acute injury.