Protein phosphatase 1alpha is tyrosine-phosphorylated and inactivated by peroxynitrite in erythrocytes through the src family kinase fgr.
Protein serine/threonine phosphorylation is a significant component of the intracellular signal that together with tyrosine phosphorylation regulates several processes, including cell-cycle progression, muscle contraction, transcription, and neuronal signaling. Cross-talk between phosphoserine/threonine- and phosphotyrosine-mediated pathways is not yet well understood. In this study we found that peroxynitrite, a physiological oxidant formed by the fast radical-radical reaction between the nitric oxide and the superoxide anion, induced tyrosine phosphorylation of the serine/threonine protein phosphatase 1alpha (PP1alpha) in human erythrocytes through activation of src family kinases. We have previously shown in mouse red cells that upregulation of the src kinase fgr phosphorylates PP1alpha, acting as an upstream negative regulator of PP1alpha, and downregulates K-Cl cotransport. Here we found that PP1alpha is a selective substrate of peroxynitrite-activated fgr and that tyrosine phosphorylation of PP1alpha corresponds to an inhibition of its enzymatic activity. Despite fgr activation and PP1alpha downregulation, peroxynitrite stimulated in a dose-dependent fashion the function of the K-Cl cotransporter. In an attempt to understand the mechanism of K-Cl cotransport activation, we found that the effect of peroxynitrite is completely reversed by dithriothreitol, suggesting that peroxynitrite acts as an oxidizing agent by an SH-dependent and PP1alpha-independent mechanism. These findings highlight a novel function of peroxynitrite in regulating the intracellular signal transduction pathways involving serine/threonine phosphorylation and the functional role of proteins that are targets of these phosphatases.
Mallozzi
Cinzia
C
Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena, 299-00161 Rome, Italy. mallozzi@iss.it
De Franceschi
Lucia
L
Brugnara
Carlo
C
Di Stasi
A M Michela
AM
D015536:Down-Regulation; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck; D027981:Symporters / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*; D000096922:K Cl- Cotransporters
D008981:Molsidomine; D030421:Peroxynitrous Acid; D010749:Phosphoprotein Phosphatases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051572:Proto-Oncogene Proteins c-hck; D027981:Symporters; D014443:Tyrosine; D019061:src-Family Kinases; D000096922:K Cl- Cotransporters; C002385:linsidomine; C496631:HCK protein, human; C053102:proto-oncogene proteins c-fgr