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.
Tedeschi
Gabriella
G
DIPAV, Section of Biochemistry, University of Milan, Milan, Italy. gabriella.tedeschi@unimi.it
Cappelletti
Graziella
G
Negri
Armando
A
Pagliato
Lara
L
Maggioni
Maria G
MG
Maci
Rosalba
R
Ronchi
Severino
S
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D015180:Electrophoresis, Gel, Two-Dimensional; D008969:Molecular Sequence Data; D020932:Nerve Growth Factor / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism*; D016716:PC12 Cells; D011487:Protein Conformation; D020543:Proteome / Q000378:metabolism*; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014404:Tubulin / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D020543:Proteome; D014404:Tubulin; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D020932:Nerve Growth Factor