Mitogen-activated protein kinase phosphorylation in kidneys of beta(S) sickle cell mice

Previous studies in beta(S) sickle cell mice demonstrated renal immunostain ing for nitrotyrosine, which is putative evidence of peroxynitrite (ONOO-) formation. ONOO- is known to nitrate tyrosine residues of various enzymes, thereby interfering with phosphorylation and inactivating them. The present study examined the state of phosphorylation of mitogen-activated protein ( MAP) kinase signal transduction enzymes, i.e., p38, c-Jun NH2-terminal kina se (JNK), and extracellular signal-regulated kinase (ERK). Western blot per formed with antibodies directed against specific phosphorylated threonine/ tyrosine residues of these enzymes demonstrated reduced phosphorylation of renal p38 and a trend toward reduced phosphorylation of ERK. In contrast, p hosphorylation of renal JNK was markedly increased compared with normal mic e. The abundance of MAP kinase phosphatase-1 (MKP-1), a key upstream enzyme that modulates phosphorylation of MAP kinases, was not different in beta(S ) versus normal mice. To determine whether nitration of tyrosine by ONOO- w as responsible for reduced phosphorylation of p38 and ERK, mercaptoethylgua nidine (MEG), a compound known to reduce inducible isoform of nitric oxide synthase activity and to scavenge ONOO-, was administered to B-S mice for 5 d. MEG was found to restore phosphorylation of D38 and ERK toward normal l evels. These observations provide evidence that ONOO- (or closely related r eaction products of NO) contributes to dephosphorylation of p38 and ERK, an d presumably reduces activity of these enzymes. The increased phosphorylati on of JNK, which suggests activation of this signaling pathway by extracell ular stress signals, may play a role in apoptosis in the kidneys of these m ice. The changes in phosphorylation of MAP kinase pathways found in this st udy could have important consequences for regulation of nuclear transcripti on factors, and thus renal function and pathology in sickle cell kidneys.