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

Area of Science:

• Molecular biology
• Cellular signaling
• Biophysics

Background:

• Body temperature influences acute lung injury severity, with hyperthermia worsening and hypothermia mitigating the condition.
• p38 mitogen-activated protein kinase (MAPK) inhibitors blunt this temperature dependence, suggesting a role for p38 MAPK isoforms.
• p38α is proinflammatory, while p38β is cytoprotective, indicating isoform-specific functions in temperature-related lung injury.

Purpose of the Study:

• To investigate the temperature dependence of p38 MAPK activation, substrate interaction, and tertiary structure.
• To elucidate the specific roles of p38α and p38β isoforms in temperature-mediated cellular responses.
• To understand how temperature affects the interaction between p38α and its substrate MAPK-activated kinase-2 (MK2).

Main Methods:

• HeLa cells were incubated at varying temperatures (33 °C, 37 °C, 39.5 °C).
• In vitro kinase assays were used to measure substrate phosphorylation by p38α and p38β.
• Surface plasmon resonance (SPR) assessed p38α:substrate binding affinity.
• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyzed temperature-dependent conformational changes in p38α and p38β.

Main Results:

• Incubating cells at 39.5 °C modestly stimulated p38 activation but did not alter TNFα-induced activation.
• MK2 phosphorylation by activated p38α was 14.5-fold greater at 39.5 °C compared to 33 °C.
• HDX-MS revealed temperature-dependent conformational changes in p38α near the MK2 binding site, but not in p38β.
• p38α:substrate binding affinity showed temperature dependence mirroring substrate phosphorylation.

Conclusions:

• The proinflammatory p38α isoform exhibits significant temperature-dependent changes in its structure and function, particularly in its interaction with MK2.
• These temperature-dependent alterations in p38α may contribute to the observed worsening of lung injury during fever.
• The cytoprotective p38β isoform does not show similar temperature-dependent conformational changes, highlighting isoform-specific thermal sensitivity.