Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Jun 1;88(11):4685–4689. doi: 10.1073/pnas.88.11.4685

Characterization of posttranslational modifications in neuron-specific class III beta-tubulin by mass spectrometry.

J E Alexander 1, D F Hunt 1, M K Lee 1, J Shabanowitz 1, H Michel 1, S C Berlin 1, T L MacDonald 1, R J Sundberg 1, L I Rebhun 1, A Frankfurter 1
PMCID: PMC51730  PMID: 2052551

Abstract

Class III beta-tubulin, isolated from adult bovine brain, is resolved into at least seven charge variants on isoelectric focusing gels. To identify the posttranslational modifications responsible for this heterogeneity, a mixture of brain tubulins was treated with cyanogen bromide and the C-terminal fragments from the class III beta-tubulin isoforms were then isolated by binding them to the monoclonal antibody TuJ1. Combined use of tandem mass spectrometry and both subtractive and automated Edman degradation chemistry on the isolated peptides indicates that many of the isoforms differ by phosphorylation at Ser-444 plus attachment of one to six glutamic acid molecules to the side chain of the first glutamate residue, Glu-438, in the C-terminal sequence Tyr-Glu-Asp-Asp-Glu-Glu-Glu-Ser-glu-Ala-Gln-Gly-Pro-Lys.

Full text

PDF

Images in this article

4686Image
on p.4686
4688Image
on p.4688
4688Image
on p.4688
4689Image
on p.4689

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barra H. S., Arce C. A., Rodríguez J. A., Caputto R. Some common properties of the protein that incorporates tyrosine as a single unit and the microtubule proteins. Biochem Biophys Res Commun. 1974 Oct 23;60(4):1384–1390. doi: 10.1016/0006-291x(74)90351-9. [DOI] [PubMed] [Google Scholar]
  2. Biemann K. Contributions of mass spectrometry to peptide and protein structure. Biomed Environ Mass Spectrom. 1988 Oct;16(1-12):99–111. doi: 10.1002/bms.1200160119. [DOI] [PubMed] [Google Scholar]
  3. Black M. M., Keyser P. Acetylation of alpha-tubulin in cultured neurons and the induction of alpha-tubulin acetylation in PC12 cells by treatment with nerve growth factor. J Neurosci. 1987 Jun;7(6):1833–1842. doi: 10.1523/JNEUROSCI.07-06-01833.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cichowicz D. J., Shane B. Mammalian folylpoly-gamma-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme. Biochemistry. 1987 Jan 27;26(2):504–512. doi: 10.1021/bi00376a024. [DOI] [PubMed] [Google Scholar]
  5. Eddé B., Denoulet P., de Néchaud B., Koulakoff A., Berwald-Netter Y., Gros F. Posttranslational modifications of tubulin in cultured mouse brain neurons and astroglia. Biol Cell. 1989;65(2):109–117. doi: 10.1111/j.1768-322x.1989.tb00779.x. [DOI] [PubMed] [Google Scholar]
  6. Eddé B., Rossier J., Le Caer J. P., Desbruyères E., Gros F., Denoulet P. Posttranslational glutamylation of alpha-tubulin. Science. 1990 Jan 5;247(4938):83–85. doi: 10.1126/science.1967194. [DOI] [PubMed] [Google Scholar]
  7. Eipper B. A. Properties of rat brain tubulin. J Biol Chem. 1974 Mar 10;249(5):1407–1416. [PubMed] [Google Scholar]
  8. Erickson A. K., Payne D. M., Martino P. A., Rossomando A. J., Shabanowitz J., Weber M. J., Hunt D. F., Sturgill T. W. Identification by mass spectrometry of threonine 97 in bovine myelin basic protein as a specific phosphorylation site for mitogen-activated protein kinase. J Biol Chem. 1990 Nov 15;265(32):19728–19735. [PubMed] [Google Scholar]
  9. Ferone R., Hanlon M. H., Singer S. C., Hunt D. F. alpha-Carboxyl-linked glutamates in the folylpolyglutamates of Escherichia coli. J Biol Chem. 1986 Dec 15;261(35):16356–16362. [PubMed] [Google Scholar]
  10. Field D. J., Collins R. A., Lee J. C. Heterogeneity of vertebrate brain tubulins. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4041–4045. doi: 10.1073/pnas.81.13.4041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Forgue S. T., Dahl J. L. Rat brain tubulin: subunit heterogeneity and phosphorylation. J Neurochem. 1979 Mar;32(3):1015–1025. doi: 10.1111/j.1471-4159.1979.tb04588.x. [DOI] [PubMed] [Google Scholar]
  12. Gard D. L., Kirschner M. W. A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line. J Cell Biol. 1985 Mar;100(3):764–774. doi: 10.1083/jcb.100.3.764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Griffin P. R., Kumar S., Shabanowitz J., Charbonneau H., Namkung P. C., Walsh K. A., Hunt D. F., Petra P. H. The amino acid sequence of the sex steroid-binding protein of rabbit serum. J Biol Chem. 1989 Nov 15;264(32):19066–19075. [PubMed] [Google Scholar]
  14. Hunt D. F., Shabanowitz J., Yates J. R., 3rd, Zhu N. Z., Russell D. H., Castro M. E. Tandem quadrupole Fourier-transform mass spectrometry of oligopeptides and small proteins. Proc Natl Acad Sci U S A. 1987 Feb;84(3):620–623. doi: 10.1073/pnas.84.3.620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hunt D. F., Yates J. R., 3rd, Shabanowitz J., Winston S., Hauer C. R. Protein sequencing by tandem mass spectrometry. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6233–6237. doi: 10.1073/pnas.83.17.6233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. L'Hernault S. W., Rosenbaum J. L. Chlamydomonas alpha-tubulin is posttranslationally modified by acetylation on the epsilon-amino group of a lysine. Biochemistry. 1985 Jan 15;24(2):473–478. doi: 10.1021/bi00323a034. [DOI] [PubMed] [Google Scholar]
  17. Lee J. C., Field D. J., George H. J., Head J. Biochemical and chemical properties of tubulin subspecies. Ann N Y Acad Sci. 1986;466:111–128. doi: 10.1111/j.1749-6632.1986.tb38388.x. [DOI] [PubMed] [Google Scholar]
  18. Lee M. K., Rebhun L. I., Frankfurter A. Posttranslational modification of class III beta-tubulin. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7195–7199. doi: 10.1073/pnas.87.18.7195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee M. K., Tuttle J. B., Rebhun L. I., Cleveland D. W., Frankfurter A. The expression and posttranslational modification of a neuron-specific beta-tubulin isotype during chick embryogenesis. Cell Motil Cytoskeleton. 1990;17(2):118–132. doi: 10.1002/cm.970170207. [DOI] [PubMed] [Google Scholar]
  20. Lewis S. A., Cowan N. J. Complex regulation and functional versatility of mammalian alpha- and beta-tubulin isotypes during the differentiation of testis and muscle cells. J Cell Biol. 1988 Jun;106(6):2023–2033. doi: 10.1083/jcb.106.6.2023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ludueña R. F., Zimmermann H. P., Little M. Identification of the phosphorylated beta-tubulin isotype in differentiated neuroblastoma cells. FEBS Lett. 1988 Mar 28;230(1-2):142–146. doi: 10.1016/0014-5793(88)80658-6. [DOI] [PubMed] [Google Scholar]
  22. Piperno G., Fuller M. T. Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol. 1985 Dec;101(6):2085–2094. doi: 10.1083/jcb.101.6.2085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Raybin D., Flavin M. An enzyme tyrosylating alpha-tubulin and its role in microtubule assembly. Biochem Biophys Res Commun. 1975 Aug 4;65(3):1088–1095. doi: 10.1016/s0006-291x(75)80497-9. [DOI] [PubMed] [Google Scholar]
  24. Serrano L., Avila J., Maccioni R. B. Controlled proteolysis of tubulin by subtilisin: localization of the site for MAP2 interaction. Biochemistry. 1984 Sep 25;23(20):4675–4681. doi: 10.1021/bi00315a024. [DOI] [PubMed] [Google Scholar]
  25. Serrano L., de la Torre J., Maccioni R. B., Avila J. Involvement of the carboxyl-terminal domain of tubulin in the regulation of its assembly. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5989–5993. doi: 10.1073/pnas.81.19.5989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smith R. D., Loo J. A., Edmonds C. G., Barinaga C. J., Udseth H. R. New developments in biochemical mass spectrometry: electrospray ionization. Anal Chem. 1990 May 1;62(9):882–899. doi: 10.1021/ac00208a002. [DOI] [PubMed] [Google Scholar]
  27. Sullivan K. F., Cleveland D. W. Identification of conserved isotype-defining variable region sequences for four vertebrate beta tubulin polypeptide classes. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4327–4331. doi: 10.1073/pnas.83.12.4327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sullivan K. F. Structure and utilization of tubulin isotypes. Annu Rev Cell Biol. 1988;4:687–716. doi: 10.1146/annurev.cb.04.110188.003351. [DOI] [PubMed] [Google Scholar]
  29. Vera J. C., Rivas C. I., Maccioni R. B. Biochemical dissection of the role of the one-kilodalton carboxyl-terminal moiety of tubulin in its assembly into microtubules. Biochemistry. 1989 Jan 10;28(1):333–339. doi: 10.1021/bi00427a045. [DOI] [PubMed] [Google Scholar]
  30. Williams R. C., Jr, Lee J. C. Preparation of tubulin from brain. Methods Enzymol. 1982;85(Pt B):376–385. doi: 10.1016/0076-6879(82)85038-6. [DOI] [PubMed] [Google Scholar]
  31. Wolff A., Denoulet P., Jeantet C. High level of tubulin microheterogeneity in the mouse brain. Neurosci Lett. 1982 Aug 31;31(3):323–328. doi: 10.1016/0304-3940(82)90041-6. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES