Identification by Mass Spectrometry of a New -Tubulin Isotype Expressed in Human Breast and Lung Carcinoma Cell Lines
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- 作者：Srinivasa Rao ; Fredrik Å ; berg ; Edward Nieves ; Susan Band Horwitz ; and George A. Orr
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：February 20, 2001
- 年：2001
- 卷：40
- 期：7
- 页码：2096 - 2103
- 全文大小：89K
- 年卷期：v.40,no.7(February 20, 2001)
- ISSN：1520-4995
文摘
The extensive C-terminal molecular heterogeneity of 
- and 
-tubulin is a consequence ofmultiple isotypes, the products of distinct genes, that undergo several posttranslational modifications.These include polyglutamylation and polyglycylation of both subunits, reversible tyrosination and removalof the penultimate glutamate from -tubulin, and phosphorylation of the III isotype. A mass spectrometry-based method has been developed for the analysis of the C-terminal diversity of tubulin from human celllines. Total cell extracts are resolved by SDS-PAGE and transferred to nitrocellulose, and the region ofthe blot corresponding to tubulin (~50 kDa) was excised and digested with CNBr to release the highlydivergent C-terminal tubulin fragments. The masses of the human - and -tubulin CNBr-derived C-terminalpeptides are all in the 1500-4000 Da mass range and can be analyzed directly by MALDI-TOF massspectrometry in the negative ion mode without significant interference from other released peptides. Inthis 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 linesare k-1 and 1. Importantly, we report a previously unknown isotype present at significant levels inboth cell lines. Moreover, the degree of posttranslational modifications to all isotypes was limited. Glu-tubulin, in which the C-terminal tyrosine of -tubulin is removed, was not detected. In contrast tomammalian neuronal tubulin which exhibits extensive polyglutamylation, only low-level monoglutamylationof the k-1 and 1 isotypes was observed in these two human cell lines.
- and -tubulin is a consequence ofmultiple isotypes, the products of distinct genes, that undergo several posttranslational modifications.These include polyglutamylation and polyglycylation of both subunits, reversible tyrosination and removalof the penultimate glutamate from -tubulin, and phosphorylation of the III isotype. A mass spectrometry-based method has been developed for the analysis of the C-terminal diversity of tubulin from human celllines. Total cell extracts are resolved by SDS-PAGE and transferred to nitrocellulose, and the region ofthe blot corresponding to tubulin (~50 kDa) was excised and digested with CNBr to release the highlydivergent C-terminal tubulin fragments. The masses of the human - and -tubulin CNBr-derived C-terminalpeptides are all in the 1500-4000 Da mass range and can be analyzed directly by MALDI-TOF massspectrometry in the negative ion mode without significant interference from other released peptides. Inthis 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 linesare k-1 and 1. Importantly, we report a previously unknown isotype present at significant levels inboth cell lines. Moreover, the degree of posttranslational modifications to all isotypes was limited. Glu-tubulin, in which the C-terminal tyrosine of -tubulin is removed, was not detected. In contrast tomammalian neuronal tubulin which exhibits extensive polyglutamylation, only low-level monoglutamylationof the k-1 and 1 isotypes was observed in these two human cell lines.