Characterization of nitroproteome in neuron-like PC12 cells differentiated with nerve growth factor: Identification of two nitration sites in alpha-tubulin

Nitric oxide (NO) is a precursor of reactive nitrating species, peroxynitrite and nitrogen dioxide above all, that modify proteins to generate oxidized species such as 3-nitrotyrosine which has been used as an hallmark of peroxynitrite-mediated oxidative stress on proteins. In the last few years however a growing number of evidences indicates that NO regulates also a myriad of physiologic responses by modifying tyrosine residues. Looking for the molecular event triggered by nitric oxide in nerve growth factor induced neuronal differentiation, we have recently reported that in differentiating PC12 cells the cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins and alpha-tubulin results to be the major target. In the present work we focus on the investigation of the sites of tyrosine nitration in alpha-tubulin purified by 2D electrophoresis following anti alpha-tubulin immunoprecipitation of protein extract of NGF-treated PC12 cells. By using western blot and MALDI-TOF analysis we show for the first time, both in vivo and in vitro, that nitration can occur on alpha-tubulin at sites different from the C-terminus and we positively identify Tyr 161 and Tyr 357 as two specific amino acids endogenously nitrated.