Tyrosine nitration of cytoskeletal proteins in physiological conditions : role of nitrated alpha-tubulin in microtubule stability

Whereas the whole emphasis of the earlier work on nitration of proteins was focused on its pathological role, there is an increasing evidence that nitration of tyrosine residues could be a physiological event implicated in numerous biological processes modulated by NO. We have previously showed that nitration of proteins occurs in neuronal differentiation as a part of a normal physiological process and that the cytoskeleton is the main cellular fraction containing nitrotyrosined proteins in differentiating neurons and in adult brain. On the ground that cytoskeleton is crucial in neuronal morphogenesis to determine axonal guidance and branching, here we have investigated the possible role of nitration in controlling cytoskeleton remodelling, in particular microtubule rearrangement and dynamics, that underlines neuronal differentiation. The association of nitrated proteins with the cytoskeletal fraction in differentiating neuronal cells following exposure to microtubule depolymerising treatments was studied suggesting that nitration correlates with the increased microtubule stability underlying the progression of neuronal differentiation. Alpha-Tubulin is one of the major target of nitration during this process. Therefore, in order to asses the role of this posttranslation modification in modulating tubulin assembly and stability, we have undertaken an in vitro study to selectively nitrate purified tubulin. The results show that nitrated tubulin remains competent to assembly and microtubules present an increased stability to depolymerising agents. The same results were obtained by live cell imaging suggesting that nitration could play a novel functional role in the complex and dynamic organization of the cytoskeleton underlying neuritogensis and differentiation.