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MAP Kinase p38alpha but Not p38beta Exhibits Temperature-Dependent Conformational and Functional Changes Within the Clinical Temperature Range that Are Relevant to Acute Lung Injury
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A2969 - MAP Kinase p38alpha but Not p38beta Exhibits Temperature-Dependent Conformational and Functional Changes Within the Clinical Temperature Range that Are Relevant to Acute Lung Injury
Author Block: J. D. Hasday1, M. Tulapurkar2, D. Deredge3, P. Wintrod3, P. Shapiro3; 1Medicine/Pulmonary and Critical Care, University of Maryland, Baltimore, MD, United States, 2Univ of Maryland School of Medicine, Baltimore, MD, United States, 3Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, United States.
Rationale: We previously showed that febrile-range hyperthermia worsens and therapeutic hypothermia improves models of lung injury and implicated p38 as a central temperature-dependent signaling pathway. Previous work suggests that p38alpha is the proinflammatory p38 isoform and p38beta is cytoprotective. In this study, we analyzed the effects of temperature shifts on auto-activation, substrate-specific kinase activity, and tertiary structure of p38alpha and beta.
Methods: All recombinant proteins were expressed in our laboratory. Auto-activation of p38 was measured by incubating recombinant p38alpha or beta with ATP at 33°C-40°C°C and immunoblotting using antibodies for activated (phosphorylated) p38. Kinase activity for three different substrates, MK2, ATF2, and Stat-1alpha was measured using standard in vitro kinase assays conducted at 33°C, 37°C, or 39.5°C with recombinant dual phosphorylated p38alpha or beta, substrate protein, and [32P]ATP and analyzing substrate phosphorylation by phosphorimaging. Conformational changes in p38alpha and beta proteins between 33°C, 37°C, and 39.5°C was analyzed by hydrogen-deuterium exchange mass spectrometry (HDX).
Results: p38alpha but not beta underwent auto-phosphorylation at an increasing rate as temperature was increased between 38°C and 40°C. Phosphorylation of MK2 and ATF2 by p38alpha was 8.5- and 2.8-fold greater at 39.5°C than 33°C compared with only 3-fold and 2-fold differences for p38beta. Phosphorylation of stat-1alpha by both p38 isoforms was similar at all three temperatures. HDX showed temperature-dependent conformational changes in an MK2 binding region of p38alpha but not beta, but not in the catalytic domain of either isoform.
Conclusion: We showed temperature-dependence of structure and function of the proinflammatory p38 isoform, including increased phosphorylation of MK2, a substrate required for endothelial paracellular pathway opening and leukocyte migration. These results provide a mechanistic explanation for the deleterious effects of febrile-range hyperthemia and salutary effects of hypothermia on lung injury and support clinical studies of therapeutic hypothermia for ARDS.
Author Block: J. D. Hasday1, M. Tulapurkar2, D. Deredge3, P. Wintrod3, P. Shapiro3; 1Medicine/Pulmonary and Critical Care, University of Maryland, Baltimore, MD, United States, 2Univ of Maryland School of Medicine, Baltimore, MD, United States, 3Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, United States.
Rationale: We previously showed that febrile-range hyperthermia worsens and therapeutic hypothermia improves models of lung injury and implicated p38 as a central temperature-dependent signaling pathway. Previous work suggests that p38alpha is the proinflammatory p38 isoform and p38beta is cytoprotective. In this study, we analyzed the effects of temperature shifts on auto-activation, substrate-specific kinase activity, and tertiary structure of p38alpha and beta.
Methods: All recombinant proteins were expressed in our laboratory. Auto-activation of p38 was measured by incubating recombinant p38alpha or beta with ATP at 33°C-40°C°C and immunoblotting using antibodies for activated (phosphorylated) p38. Kinase activity for three different substrates, MK2, ATF2, and Stat-1alpha was measured using standard in vitro kinase assays conducted at 33°C, 37°C, or 39.5°C with recombinant dual phosphorylated p38alpha or beta, substrate protein, and [32P]ATP and analyzing substrate phosphorylation by phosphorimaging. Conformational changes in p38alpha and beta proteins between 33°C, 37°C, and 39.5°C was analyzed by hydrogen-deuterium exchange mass spectrometry (HDX).
Results: p38alpha but not beta underwent auto-phosphorylation at an increasing rate as temperature was increased between 38°C and 40°C. Phosphorylation of MK2 and ATF2 by p38alpha was 8.5- and 2.8-fold greater at 39.5°C than 33°C compared with only 3-fold and 2-fold differences for p38beta. Phosphorylation of stat-1alpha by both p38 isoforms was similar at all three temperatures. HDX showed temperature-dependent conformational changes in an MK2 binding region of p38alpha but not beta, but not in the catalytic domain of either isoform.
Conclusion: We showed temperature-dependence of structure and function of the proinflammatory p38 isoform, including increased phosphorylation of MK2, a substrate required for endothelial paracellular pathway opening and leukocyte migration. These results provide a mechanistic explanation for the deleterious effects of febrile-range hyperthemia and salutary effects of hypothermia on lung injury and support clinical studies of therapeutic hypothermia for ARDS.
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