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, which modify proteins to generate oxidized species such as 3-nitrotyrosine that has been used as a hallmark of peroxynitrite-mediated oxidative stress on proteins. In the last few years however, a growing body of evidence indicates that NO also regulates a myriad of physiologic responses by modifying tyrosine residues. Looking for the molecular event triggered by NO in nerve growth factor (NGF)-induced neuronal differentiation, we recently reported that in differentiating PC12 cells, the cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and alpha-tubulin is the major target. In the present work, we focus on the investigation of the sites of tyrosine nitration in alpha-tubulin purified by two-dimensional gel electrophoresis following anti-alpha-tubulin immunoprecipitation of protein extract from NGF-treated PC12 cells. Using Western blotting and matrix-assisted laser desorption/ionization-time of flight analysis, we show for the first time, both in vivo and in vitro, that nitration can occur on alpha-tubulin at sites other than the C-terminus and we positively identify Tyr 161 and Tyr 357 as two specific amino acids endogenously nitrated.