Microtubule dysfunction by posttranslational nitrotyrosination of alpha-tubulin: A nitric oxide-dependent mechanism of cellular injury

NO(2)Tyr (3-Nitrotyrosine) is a modified amino acid that is formed by nitri c oxide-derived species and has been implicated in the pathology of diverse human diseases. Nitration of active-site tyrosine residues is known to com promise protein structure and function, Although free NO(2)Tyr is produced in abundant concentrations under pathological conditions, its capacity to a lter protein structure and function at the translational or posttranslation al level is unknown. Here, we report that free NO(2)Tyr is transported into mammalian cells and selectively incorporated into the extreme carboxyl ter minus of cu-tubulin via a posttranslational mechanism catalyzed by the enzy me tubulin-tyrosine ligase, In contrast to the enzymatically regulated carb oxyl-terminal tyrosination/detyrosination cycle of alpha-tubulin, incorpora tion of NO(2)Tyr shows apparent irreversibility, Nitro-tyrosination of alph a-tubulin induces alterations in cell morphology, changes in microtubule or ganization, loss of epithelial-barrier function, and intracellular redistri bution of the motor protein cytoplasmic dynein, These observations imply th at posttranslational nitrotyrosination of Lu-tubulin invokes conformational changes, either directly or via allosteric interactions, in the surface ex posed carboxyl terminus of cu-tubulin that compromises the function of this critical domain in regulating microtubule organization and binding of moto r-and microtubule-associated proteins. Collectively, these observations ill ustrate a mechanism whereby free NO(2)Tyr can impact deleteriously on cell function under pathological conditions encompassing reactive nitrogen speci es production. The data also yield further insight into the role that the a lpha-tubulin tyrosination/detyrosination cycle plays in microtubule functio n.