Identification by mass spectrometry of a new α-tubulin isotype expressed in human breast and lung carcinoma cell lines

S. Rao, F. Åberg, E. Nieves, S. B. Horwitz, G. A. Orr

Research output: Contribution to journalArticle

31 Scopus citations

Abstract

The extensive C-terminal molecular heterogeneity of α- and β-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 α-tubulin, and phosphorylation of the βIII 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 (∼50 kDa) was excised and digested with CNBr to release the highly divergent C-terminal tubulin fragments. The masses of the human α- and β-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-α1 and β1. Importantly, we report a previously unknown α isotype present at significant levels in both cell lines. Moreover, the degree of posttranslational modifications to all isotypes was limited. Glutubulin, in which the C-terminal tyrosine of α-tubulin is removed, was not detected. In contrast to mammalian neuronal tubulin which exhibits extensive polyglutamylation, only low-level monoglutamylation of the k-αl and β1 isotypes was observed in these two human cell lines.

Original languageEnglish (US)
Pages (from-to)2096-2103
Number of pages8
JournalBiochemistry
Volume40
Issue number7
DOIs
StatePublished - Feb 20 2001

ASJC Scopus subject areas

  • Biochemistry

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