Premium
Characterization of nitroproteome in neuron‐like PC12 cells differentiated with nerve growth factor: Identification of two nitration sites in α‐tubulin
Author(s) -
Tedeschi Gabriella,
Cappelletti Graziella,
Negri Armando,
Pagliato Lara,
Maggioni Maria G.,
Maci Rosalba,
Ronchi Severino
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401208
Subject(s) - tubulin , peroxynitrite , tyrosine , nerve growth factor , microtubule , chemistry , biochemistry , nitrotyrosine , microbiology and biotechnology , immunoprecipitation , reactive nitrogen species , biology , oxidative stress , nitric oxide synthase , enzyme , gene , receptor , superoxide
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 α‐tubulin is the major target. In the present work, we focus on the investigation of the sites of tyrosine nitration in α‐tubulin purified by two‐dimensional gel electrophoresis following anti‐α‐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 α‐tubulin at sites other than the C ‐terminus and we positively identify Tyr 161 and Tyr 357 as two specific amino acids endogenously nitrated.