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

Abstract

Class III fi-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 I3-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 rst glutamate residue, Glu-438, in the C-terminal sequence Tyr-Glu-AspAsp-Glu-Glu-Glu-Ser-Glu-Ala-Gln-Gly-Pro-Lys.Microtubules, assembled from two similar 50-kDa proteins designated a-and f3-tubulin, are involved in a number of important biological processes including segregation of chromosomes during cell division, cell motility, organelle transport, and maintenance of cell shape (1). Brain tubulin exhibits a high degree of heterogeneity. Up to 21 charge variants have been observed by isoelectric focusing (IEF) techniques (2-4).Both a-and 83-tubulins are encoded by small multigene families (5). Since the number of charge variants exceeds the number of tubulin genes by approximately a factor of 2, the remaining isoforms are assumed to result from posttranslational modifications. a-Tubulin is reported to undergo acetylation (6-8), glutamylation (9), and the addition and removal of a C-terminal tyrosine residue (10, 11). Phosphorylation of a single seine residue in one of the five P-tubulin isotypes expressed in brain has also been described (12-16).Class III p8-tubulin, a vertebrate isotype found only in neurons and cells in the mammalian testis (17), undergoes a developmentally regulated increase in heterogeneity in the former but not the latter tissue (18,19). The testis and earliest embryonic rat brain isoforms cofocus on an IEF gel (19). At least six additional isoforms of neuron-specific class III ,B-tubulin are detected in the adult rat or bovine brain.Previous work localized the site of heterogeneity to the C-terminal 20 amino acids (19), a highly acidic region of the protein thought to be involved in the interaction of microtubules with both calcium ions (20) and microtubule-associated proteins (21-23).To further characterize both the nature and location of structural modifications responsible for the observed heterogeneity, class III /3-tubulin from bovine brain was subjected to amino acid sequence analysis by tandem mass spectrometry. We report that the neuron-specific class III /3-tubulin isoforms result in part from phosphorylation at Ser-444 and attachment of at least three and perhaps as many as six glutamic acid molecules to the side chain of the first glutamate residue, Glu-438, in the C-terminal sequence Tyr-GluAsp-Asp-Glu-Glu-Glu-Ser-Glu-Ala-Gln-Gly-Pro-Lys (YED-DEEESEAQGPK). MATERIALS AND METHODSIsolati...

A Ubiquitous β-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

“…On the other hand, some reports have hinted at unique properties for these two isotypes. Of all the ␤-tubulin isotypes, for example, only ␤3 and ␤6 are known to undergo phosphorylation (Diaz-Nido et al, 1990;Alexander et al, 1991;Rudiger and Weber, 1993). In PC12 cells ␤3-tubulin has been described to assemble less well than other isotypes and to have a granular appearance in developing neurites (Asai and Remolona, 1989;Joshi and Cleveland, 1989).…”

Reversible polyglutamylation of alpha- and beta-tubulin and microtubule dynamics in mouse brain neurons.

“…This finding has been extended to class III (a neuron-specific f3-tubulin isotype, denoted here d3') (Alexander et al, 1991) and class II (Redeker et al, 1992;Riidiger et al, 1992) 0-tubulin. Polyglutamylated tubulin is very abundant in nerve cells and is also found, at basal levels, in other cell types (Edde et al, 1990;Wolff et al, 1992).…”

“…Indeed, glutamylation is a polymodification, and several tubulin species bearing one to at least six negatively-charged glutamyl units were characterized (Edde et al, 1990;Alexander et al, 1991;Redeker et al, 1992;Rudiger et al, 1992). Because the GT335 mAb recognizes indifferently all of these species (Wolff et al, 1992), it discriminates between glutamylated and nonglutamylated forms but cannot reveal a shortening or extension of the polyglutamyl chain.…”

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

“…However, tubulin, a highly conserved molecule, contains no significant hydrophobic domains (Cleveland and Sullivan, 1985). Furthermore, known posttranslational modifications of tubulin [e.g., tyrosination (Raybin and Flavin, 1977;Deanin et al, 1980), acetylation (L'Hernault and Rosenbaum, 1985;LeDizet and Piperno, 1991), glutamylation (Edde et al, 1990;Alexander et al, 1991), and phosphorylation (Gard and Kirschner, 1985;Alexander et al, 1991;Peters et al, 1996)] cannot account for membrane association. This has led to the suggestion that the putative membrane tubulin may actually be contaminating cytoplasmic tubulin.…”