Incorporation of 3-nitrotyrosine into the C-terminus of alpha-tubulin is reversible and not detrimental to dividing cells.
The C-terminus of the alpha-chain of tubulin is subject to reversible incorporation of tyrosine by tubulin tyrosine ligase and removal by tubulin carboxypeptidase. Thus, microtubules rich in either tyrosinated or detyrosinated tubulin can coexist in the cell. Substitution of the terminal tyrosine by 3-nitrotyrosine has been claimed to cause microtubule dysfunction and consequent injury of epithelial lung carcinoma A549 cells. Nitrotyrosine is formed in cells by nitration of tyrosine by nitric oxide-derived species. We studied properties of tubulin modified by in vitro nitrotyrosination at the C-terminus of the alpha-subunit, and the consequences for cell functioning. Nitrotyrosinated tubulin was a good substrate of tubulin carboxypeptidase, and showed a similar capability to assemble into microtubules in vitro to that of tyrosinated tubulin. Tubulin of C6 cells cultured in F12K medium in the presence of 500 micro m nitrotyrosine became fully nitrotyrosinated. This nitrotyrosination was shown to be reversible. No changes in morphology, proliferation, or viability were observed during cycles of nitrotyrosination, denitrotyrosination, and re-nitrotyrosination. Similar results were obtained with CHO, COS-7, HeLa, NIH-3T3, NIH-3T3(TTL-), and A549 cells. C6 and A549 cells were subjected to several passages during 45 days or more in the continuous presence of 500 micro m nitrotyrosine without noticeable alteration of morphology, viability, or proliferation. The microtubular networks visualized by immunofluorescence with antibodies to nitrotyrosinated and total tubulin were identical. Furthermore, nitrotyrosination of tubulin in COS cells did not alter the association of tubulin carboxypeptidase with microtubules. Our results demonstrate that substitution of C-terminal tyrosine by 3-nitrotyrosine has no detrimental effect on dividing cells.
Bisig
C Gastón
CG
Centro de Investigaciones en Química Biológica de Córdoba, Departamento de Química Biológica, Universidad Nacional de Córdoba, Argentina.
Purro
Silvia A
SA
Contín
María A
MA
Barra
Héctor S
HS
Arce
Carlos A
CA
D016475:3T3 Cells; D000818:Animals; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002268:Carboxypeptidases / Q000378:metabolism; D002277:Carcinoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D006224:Cricetinae; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D051381:Rats; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology
D014404:Tubulin; C002744:3-nitrotyrosine; D014443:Tyrosine; D002268:Carboxypeptidases; C023212:tyrosyltubulin carboxypeptidase