Identification by mass spectrometry of a new alpha-tubulin isotype expressed in human breast and lung carcinoma cell lines.
The extensive C-terminal molecular heterogeneity of alpha- and beta-tubulin is a consequence of multiple isotypes, the products of distinct genes, that undergo several posttranslational modifications. These include polyglutamylation and polyglycylation of both subunits, reversible tyrosination and removal of the penultimate glutamate from alpha-tubulin, and phosphorylation of the beta III isotype. A mass spectrometry-based method has been developed for the analysis of the C-terminal diversity of tubulin from human cell lines. Total cell extracts are resolved by SDS--PAGE and transferred to nitrocellulose, and the region of the blot corresponding to tubulin (approximately 50 kDa) was excised and digested with CNBr to release the highly divergent C-terminal tubulin fragments. The masses of the human alpha- and beta-tubulin CNBr-derived C-terminal peptides are all in the 1500--4000 Da mass range and can be analyzed directly by MALDI-TOF mass spectrometry in the negative ion mode without significant interference from other released peptides. In this study, the tubulin isotype diversity in MDA-MB-231, a human breast carcinoma cell line, and A549, a human non-small lung cancer cell line, is reported. The major tubulin isotypes present in both cell lines are k-alpha 1 and beta 1. Importantly, we report a previously unknown alpha isotype present at significant levels in both cell lines. Moreover, the degree of posttranslational modifications to all isotypes was limited. Glu-tubulin, in which the C-terminal tyrosine of alpha-tubulin is removed, was not detected. In contrast to mammalian neuronal tubulin which exhibits extensive polyglutamylation, only low-level monoglutamylation of the k-alpha 1 and beta 1 isotypes was observed in these two human cell lines.
Rao
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S
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Aberg
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F
Nieves
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Band Horwitz
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S
Orr
G A
GA
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D001943:Breast Neoplasms / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D003488:Cyanogen Bromide; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification; D010802:Phylogeny; D020033:Protein Isoforms / Q000096:biosynthesis / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014404:Tubulin / Q000096:biosynthesis* / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; D020033:Protein Isoforms; D014404:Tubulin; D018698:Glutamic Acid; D003488:Cyanogen Bromide