A chemical genetic approach reveals that p38alpha MAPK activation by diphosphorylation aggravates myocardial infarction and is prevented by the direct binding of SB203580.
The use of nonselective pharmacological inhibitors has resulted in controversy regarding the mechanism and consequences of p38 activation during myocardial infarction. Classic p38 inhibitors such as SB203580 rely on a critical "gatekeeper" threonine residue for binding. We addressed these controversies by using mice in which the p38alpha alleles were targeted to cause substitution of the gatekeeper residue and resistance to inhibition. In homozygous drug-resistant compared with wild-type hearts, SB203580 failed to inhibit the activating phosphorylation of p38 or to reduce the infarction caused by myocardial ischemia. However, BIRB796, a p38 inhibitor not reliant on the gatekeeper for binding, similarly reduced p38-activating phosphorylation and infarction in both wild-type and knock-in mice, thereby excluding a nonspecific inhibitor-dependent phenotype resulting from the targeting strategy. Furthermore, the activation during myocardial ischemia involved phosphorylation of both the threonine and tyrosine residues in the activation loop of p38 despite the phosphorylation of the threonine alone being sufficient to create the epitope for dual phosphospecific antibody binding. Finally, SB203580 failed to reduce infarction in heterozygous drug-resistant hearts, suggesting that near complete inhibition of p38alpha kinase activity is necessary to elicit protection. These results indicate that, during myocardial ischemia, p38alpha (i) is the dominant-active p38 isoform, (ii) contributes to infarction, (iii) is responsible for the cardioprotective effect of SB203580, and (iv) is activated by a mechanism consistent with autodiphosphorylation despite this necessitating the phosphorylation of a tyrosine residue by an archetypal serine/threonine kinase.
Kumphune
Sarawut
S
King's College London British Heart Foundation Centre, Cardiovascular Division, The Rayne Institute and StThomas' Hospital, London SE1 7EH, United Kingdom.
Bassi
Rekha
R
Jacquet
Sebastien
S
Sicard
Pierre
P
Clark
James E
JE
Verma
Sharwari
S
Avkiran
Metin
M
O'Keefe
Stephen J
SJ
Marber
Michael S
MS
D000818:Animals; D004789:Enzyme Activation; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D020935:MAP Kinase Signaling System; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008954:Models, Biological; D009203:Myocardial Infarction / Q000378:metabolism*; D017202:Myocardial Ischemia; D010766:Phosphorylation; D020033:Protein Isoforms; D011725:Pyridines / Q000494:pharmacology*; D014443:Tyrosine / Q000737:chemistry; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D007093:Imidazoles; D020033:Protein Isoforms; D011725:Pyridines; D014443:Tyrosine; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580