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

Abstract

Edited by Henrik G. Dohlman 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. Hydrogendeuterium 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.

Temperature does matter—an additional dimension in kinase inhibitor development

“…This could be controlled through conformational changes of the activation segment, which makes direct contact to the substrate and could thus inhibit of favor binding of specific substrates. A similar phenomenon has been described for p38alpha, where changes in the physiological temperature range alter substrate affinity and specificity by subtle conformational changes [7], suggesting this to be a general phenomenon. Thus, while TCMDS-135051 is clearly acting as an antimalaria agent, it may do so by inhibiting the phosphorylation of noncanonical and as of yet unknown targets with equally unknown functionality.…”

“…Similarly, a substrate‐specific change in activity between 33 °C and 39.5 °C has been reported for p38alpha, which is caused by a temperature‐dependent conformational change, in this case in the substrate binding domain, which alters the affinity for different substrates [7]. Such structural rearrangements in close proximity to the substrate and nucleotide binding site switch these kinases on and off or change substrate specificity in a temperature range between 34 °C and 38 °C and will obviously affect the binding of potential inhibitors.…”

“…Recent work shows that the activity and/or specificity of at least two kinase families, p38alpha and CDC2like kinases (CLKs), is highly responsive to temperature changes in the physiological temperature range [7,8]. P38alpha belongs to the family of mitogen-activated kinases [9], whereas CLKs phosphorylate SR proteins, a family of proteins that control pre-mRNA processing, for example, alternative splicing [10].…”

Sensitivity‐Enhanced 13C‐NMR Spectroscopy for Monitoring Multisite Phosphorylation at Physiological Temperature and pH

“…[32] Because this method is not limited by temperature constraints, it may also help in studies focusing on temperature-sensitive activities of kinases involved in circadian rhythm or the consequences of hyperthermia. [33,34] Hence, our improved 13 C-detected NMR approach expands the range of peptides and conditions accessible to NMR phospho-mapping and -monitoring. NMR methods previously published for the characterization of post-translational modifications are based on amide 1 H-detection, which often provides the best sensitivity and possibly higher dimensionality/resolution, using fast acquisition and time-resolved NUS.…”

“…This is of interest, since the abundant Ser/Thr‐Pro motifs are targets of the essential activities of MAP kinases and cyclin‐dependent kinases . Because this method is not limited by temperature constraints, it may also help in studies focusing on temperature‐sensitive activities of kinases involved in circadian rhythm or the consequences of hyperthermia . Hence, our improved 13 C‐detected NMR approach expands the range of peptides and conditions accessible to NMR phospho‐mapping and ‐monitoring.…”