Differential binding regulation of microtubule-associated proteins MAP1A, MAP1B, and MAP2 by tubulin polyglutamylation.

The major neuronal post-translational modification of tubulin, polyglutamylation, can act as a molecular potentiometer to modulate microtubule-associated proteins (MAPs) binding as a function of the polyglutamyl chain length. The relative affinity of Tau, MAP2, and kinesin has been shown to be optimal for tubulin modified by approximately 3 glutamyl units. Using blot overlay assays, we have tested the ability of polyglutamylation to modulate the interaction of two other structural MAPs, MAP1A and MAP1B, with tubulin. MAP1A and MAP2 display distinct behavior in terms of tubulin binding; they do not compete with each other, even when the polyglutamyl chains of tubulin are removed, indicating that they have distinct binding sites on tubulin. Binding of MAP1A and MAP1B to tubulin is also controlled by polyglutamylation and, although the modulation of MAP1B binding resembles that of MAP2, we found that polyglutamylation can exert a different mode of regulation toward MAP1A. Interestingly, although the affinity of the other MAPs tested so far decreases sharply for tubulins carrying long polyglutamyl chains, the affinity of MAP1A for these tubulins is maintained at a significant level. This differential regulation exerted by polyglutamylation toward different MAPs might facilitate their selective recruitment into distinct microtubule populations, hence modulating their functional properties.

Polyglutamylation of atlantic cod tubulin: immunochemical localization and possible role in pigment granule transport.

In higher organisms, there is a large variety of tubulin isoforms, due to multiple tubulin genes and extensive post-translational modification. The properties of microtubules may be modulated by their tubulin isoform composition. Polyglutamylation is a post-translational modification that is thought to influence binding of both structural microtubule associated proteins (MAPs) and mechano-chemical motors to tubulin. The present study investigates the role of tubulin polyglutamylation in a vesicle transporting system, cod (Gadus morhua) melanophores. We did this by microinjecting an antibody against polyglutamylated tubulin into these cells. To put our results into perspective, and to be able to judge their universal application, we characterized cod tubulin polyglutamylation by Western blotting technique, and compared it to what is known from mammals. We found high levels of polyglutamylation in tissues and cell types whose functions are highly dependent on interactions between microtubules and motor proteins. Microinjection of the anti-polyglutamylation antibody GT335 into cultured melanophores interfered with pigment granule dispersion, while dynein-dependent aggregation was unaffected. Additional experiments showed that GT335-injected cells were able to aggregate pigment even when actin filaments were depolymerized, indicating that the maintained ability of pigment aggregation in these cells was indeed microtubule-based and did not depend upon actin filaments. The results indicate that dynein and the kinesin-like dispersing motor protein in cod melanophores bind to tubulin on slightly different sites, and perhaps depend differentially on polyglutamylation for their interaction with microtubules. The binding site of the dispersing motor may bind directly to the polyglutamate chain, or more closely than dynein.

Tubulin polyglutamylase: isozymic variants and regulation during the cell cycle in HeLa cells.

Polyglutamylation is a posttranslational modification of tubulin that is very common in neurons and ciliated or flagellated cells. It was proposed to regulate the binding of microtubule associated proteins (MAPs) and molecular motors as a function of the length of the polyglutamyl side-chain. Though much less common, this modification of tubulin also occurs in proliferating cells like HeLa cells where it is associated with centrioles and with the mitotic spindle. Recently, we partially purified tubulin polyglutamylase from mouse brain and described its enzymatic properties. In this work, we focused on tubulin polyglutamylase activity from HeLa cells. Our results support the existence of a tubulin polyglutamylase family composed of several isozymic variants specific for alpha- or beta-tubulin subunits. In the latter case, the specificity probably also concerns the different beta-tubulin isotypes. Interestingly, we found that tubulin polyglutamylase activity is regulated in a cell cycle dependent manner and peaks in G(2)-phase while the level of glutamylated tubulin peaks in mitosis. Consistent results were obtained by treating the cells with hydroxyurea, nocodazole or taxotere. In particular, in mitotic cells, tubulin polyglutamylase activity was always low while glutamylation level was high. Finally, tubulin polyglutamylase activity and the level of glutamylated tubulin appeared to be inversely related. This paradox suggests a complex regulation of both tubulin polyglutamylase and the reverse deglutamylase activity.

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.

Estramustine resistance correlates with tau over-expression in human prostatic carcinoma cells.

Estramustine (EM) is an anti-microtubule drug used in the treatment of hormone-refractory advanced prostate cancer. Since microtubules are the targets for EM cytotoxicity, we investigated the effects of EM on the microtubule-associated protein tau to determine what role it may play in drug resistance. We have compared tau expression in human prostate cancer cells (DU145) and an EM-resistant derived cell line (E4). Reverse transcriptase polymerase chain reaction has established that tau is expressed in both cell lines but increased 1.9-fold in E4 compared with DU145 cells. This result was confirmed at the protein level by Western blotting. Tau is a phosphoprotein, most of its reported phosphorylation sites being serine or threonine residues. We have shown, however, that tau is also phosphorylated at tyrosine residues in DU145 cells and that the phosphotyrosine level of tau is significantly increased in E4 cells. Moreover, DU145 cells exposed to short term micromolar drug concentrations enter a phase of microtubule depolymerization, display an increased level of tau phosphorylation and follow a pattern similar to that observed in EM-resistant E4 cells. EM is therefore able to induce a very rapid change in the posttranslational state of tau. Our results show that the acquisition of EM resistance in E4 cells, which is accompanied by changes at the tubulin level, is also associated with important changes in tau expression and phosphorylation.

Tubulin polyglutamylase: partial purification and enzymatic properties.

In this work, we report on a novel enzyme, tubulin polyglutamylase, which catalyzes the posttranslational formation of polyglutamyl side chains onto alpha- and beta-tubulin. The length of the polyglutamyl side chain regulates the interaction between tubulin and various microtubule-associated proteins. We first developed an in vitro glutamylation assay. Activity measured in brain, a tissue particularly enriched with glutamylated tubulin, decreases during postnatal development. Thus, brains from 3-day-old mice were chosen as the starting material, and the enzyme was purified approximately 1000-fold. Its Mr was estimated to be 360K and its sedimentation coefficient 10 s. The enzyme catalyzes the MgATP-dependent addition of l-glutamate onto tubulin subunits. Microtubules are much better substrates than unpolymerized tubulin, and the reaction is very specific for glutamate, other amino acids or glutamate analogues not being substrates. Moreover, glutamyl units are added sequentially onto tubulin, leading to progressive elongation of the polyglutamyl side chains. Side chains of one to six or seven glutamyl units were obtained with microtubules, whereas much longer side chains (up to 15-20 units) were formed with unpolymerized tubulin. Interestingly, such very long polyglutamyl side chains were recently detected in some situations in vivo.

Association of estramustine resistance in human prostatic carcinoma cells with modified patterns of tubulin expression.

Estramustine (EM) is an antimicrotubule drug used in the treatment of hormone refractory advanced prostate cancer. To investigate the mechanism of resistance to EM, we compared its effects on human prostate cancer cells (DU145) and an estramustine-resistant derived cell line (E4). Immunofluorescence demonstrated that EM caused depolymerization of microtubules and blocked cells in mitosis in DU145 cells, with less effect in E4 cells. Using tubulin isotype-specific antibodies, a threefold increase in betaIII and approximately twofold increase in betaI + II isotype in E4 cells compared to DU145 cells were observed. A most interesting observation concerned an increase in the posttranslational modification of alpha-tubulin of both polyglutamylation and acetylation in the E4 cells. Significant to this observation, using direct EM photoaffinity labeling of tubulin, drug binding to the most acidic posttranslationally modified forms of alpha-tubulin was shown to be minimal. Taken together, these results indicate that the modification of the tubulin expression pattern may be responsible for estramustine resistance by both lowering the amount of drug bound to microtubules and inducing more stable microtubules.

Interaction of kinesin motor domains with alpha- and beta-tubulin subunits at a tau-independent binding site. Regulation by polyglutamylation.

Interaction of rat kinesin and Drosophila nonclaret disjunctional motor domains with tubulin was studied by a blot overlay assay. Either plus-end or minus-end-directed motor domain binds at the same extent to both alpha- and beta-tubulin subunits, suggesting that kinesin binding is an intrinsic property of each tubulin subunit and that motor directionality cannot be related to a preferential interaction with a given tubulin subunit. Binding features of dimeric versus monomeric rat kinesin heads suggest that dimerization could drive conformational changes to enhance binding to tubulin. Competition experiments have indicated that kinesin interacts with tubulin at a Tau-independent binding site. Complementary experiments have shown that kinesin does not interact with the same efficiency with the different tubulin isoforms. Masking the polyglutamyl chains with a specific monoclonal antibody leads to a complete inhibition of kinesin binding. These results are consistent with a model in which polyglutamylation of tubulin regulates kinesin binding through progressive conformational changes of the whole carboxyl-terminal domain of tubulin as a function of the polyglutamyl chain length, thus modulating the affinity of tubulin for kinesin and Tau as well. These results indicate that microtubules, through tubulin polymorphism, do have the ability to control microtubule-associated protein binding.

[Microtubules: functional polymorphisms of tubulin and associated proteins (structural and motor MAP's)]. / Les microtubules: polymorphismes fonctionnels de la tubuline et des protéines associées (MAP's structurales et motrices).

In neuronal cells, microtubules are built from a very large number of alpha- and beta-tubulin variants. This diversity is due to the expression of a multigene family and to a combination of several original posttranslational modifications. Similarly, structural and motor microtubule-associated proteins, which regulate the assembly of microtubules, the modeling of their network and the mediation of their functions, are also very heterogeneous. As a consequence, mixing of these two protein polymorphisms leads to the formation of functionally-distinct microtubules. We have shown that polyglutamylation, the major posttranslational modification of neuronal tubulin, was used as a progressive regulator in the binding of structural and motor microtubule-associated proteins, in modulating gradually the conformation of the tubulin carboxy-terminal domain, playing thus a crucial role in microtubule dynamics.

The network organization and the phosphorylation of cytokeratins are concomitantly modified by forskolin in the enterocyte-like differentiated Caco-2 cell line.

Confluent Caco-2 cells, originating from a human colon carcinoma, display morphological and functional characteristics of differentiated enterocytes such as the presence of a polarized monolayer covered by an apical brush border that express several hydrolases. The adaptation of these cells to grow in the continuous presence of forskolin, a drug known to stimulate adenylyl cyclase permanently, has been previously shown to result in a decreased apical expression of hydrolases and in morphological alterations including the disappearance of intercellular spaces and shortening of microvilli. In the present work we have analyzed the possibility that cytoskeletal proteins may be the target of forskolin in living Caco-2 cells. We show that forskolin initiates dramatic changes in the spatial organization of the cytokeratin network that correlate with an increased phosphorylation of cytokeratin molecules, whereas microtubules, microfilaments and vimentin remain mainly unaffected. Indirect immunofluorescence studies show that the cytokeratin network is redistributed from the cell periphery to the cytoplasm. Biochemical experiments indicate that forskolin doesn't interfere with the cytokeratin profile, since the three cytokeratins normally found in intestine (CK 8, CK 18, CK 19) are similarly expressed in both control and forskolin-Caco-2 cells. Analysis of 32P-labeled cytokeratin extracted from the two cell populations demonstrates that forskolin quantitatively increases the phosphorylation of type I cytokeratin (CK 18 and CK 19), whereas the phosphorylation of type II cytokeratin (CK 8) is altered both quantitatively and qualitatively with the emergence of a new phosphorylation site. These results provide a new cell system in which it is possible to control the subcellular distribution of cytokeratin by changing their phosphorylation status and therefore to study their potential cellular functions.

Polyglutamylation of tubulin as a progressive regulator of in vitro interactions between the microtubule-associated protein Tau and tubulin.

The multiple functions of microtubules are mediated by various structural and motor microtubule-associated proteins (MAPs). To harmonize these functions in different places of a single cell, the key problem is to regulate the interactions of these proteins with microtubules. The chemical diversity of tubulin isoforms, which constitute the microtubule wall, could represent a molecular basis for this control. Using an in vitro assay of ligand blotting, we found that the microtubule-associated protein Tau interacts differentially with the diverse posttranslationally-modified isotubulins: its binding is mainly restricted to moderately-modified alpha- and beta-tubulin isoforms. We obtained evidence that the recently-discovered polyglutamylation, which consists of the sequential, posttranslational addition of one to six glutamyl units to both alpha- and beta-tubulin subunits, regulates the binding of Tau as a function of its chain length. The relative affinity of Tau, very low for unmodified tubulin, increases progressively for isotubulins carrying from one to three glutamyl units, reaches an optimal value, and then decreases progressively when the polygutamyl chain lengthens up to six residues. Our results suggest that the unmodified C-terminus of tubulin exerts a constitutive inhibition on Tau binding, probably by locking the MAP-binding site, and that this inhibition could be first released and then restored as the polyglutamyl chain grows. As the posttranslational chain does not appear to interact directly with Tau, it is thought that the growth of this chain from one to six glutamyl units causes a progressive, conformational shift in the structure of the C-terminal domain of tubulin, thus leading to the observed modulation of affinity.

Developmental regulation of polyglutamylated alpha- and beta-tubulin in mouse brain neurons.

Polyglutamylation is an important posttranslational modification of tubulin that is very active in nerve cells, where it accounts for the main factor responsible for tubulin heterogeneity. In the present work, we have analyzed quantitative and qualitative changes in glutamylated alpha- and beta-tubulin occurring during neuronal differentiation in culture. Glutamylated alpha- and beta-tubulin both markedly accumulate during this process with a time course remarkably similar to that observed in vivo during brain development. However, the characteristics of the glutamylation of the two subunits are not exactly the same. Glutamylated alpha-tubulin is already abundant in very young neurons and displays, at this stage, a wide range of its degree of glutamylation (1 to 6 glutamyl units present in the lateral polyglutamyl chain), which remains unchanged during the entire period of the culture. Glutamylated beta-tubulin is present at very low levels in young neurons and its accumulation during differentiation is accompanied by a progressive increase in its degree of glutamylation from 2 to 6 glutamyl units. Posttranslational incorporation of [3H]glutamate into alpha- and beta-tubulin decreases during differentiation, as well as the rate of the reverse deglutamylation reaction, suggesting that accumulation of glutamylated tubulin is accompanied by a decrease in the turnover of glutamyl units onto tubulin. Neuronal differentiation is also accompanied by an increase of other posttranslationally modified forms of tubulin, including acetylated and non-tyrosinatable alpha-tubulin, which can occur in combination with polyglutamylation and contributes to increase the complexity of tubulin in mature neurons.

Production and characterization of polyclonal anti-idiotypic anti-TRH antibodies: application to the study of pituitary TRH receptor.

cDNA encoding the thyrotropin-releasing hormone receptor (TRH-R) was recently cloned in rat pituitary prolactin cells and in mouse thyrotropes. The molecular weights of the protein sequences obtained are 46.6 and 44.5 kD. However, TRH-R has not yet been purified to homogeneity and specific anti-TRH-R antibody could not yet be obtained by classical biochemical methods. We thus attempted to obtain antibodies specific for TRH-R using an anti-idiotypic approach. Rabbits of the same allotype were immunized using Igs (Ab1) extracted from rabbit polyclonal anti-TRH immune serum. Anti-idiotypic rabbit polyclonal anti-anti-TRH antibodies (Ab2) were obtained, as shown by their ability to inhibit the formation of TRH-anti-TRH complexes in a radioimmunoassay system. One of them, the polyclonal Ab2 R38/B12, was tested for its ability to recognize the TRH-R in rat pituitary, tumor-derived, GH3/B6 prolactin-secreting cells. Immunoreactive material was immunocytochemically detected in fixed and saponin-permeabilized GH3/B6 cells. The immunostaining was localized at the plasma membrane and on intracellular structures. It was not observed using non-anti-TRH Ab2 and was abolished in the presence of excess TRH. Furthermore, binding of [125I]R38/B12 on fixed and saponin-permeabilized GH3/B6 cells was partially inhibited by excess TRH. By immunoblot analyses of Triton X-114 cell extracts performed under reducing or nonreducing conditions, the polyclonal R38/B12 Igs revealed two main protein species of approximately 98 and approximately 76 kD as well as several proteins < or = 46 kD. In the presence of excess TRH, the approximately 98- and approximately 42-kD bands were abolished, whereas the intensity of the other bands was faintly attenuated only. The approximately 98-kD protein was also revealed in a two-dimensional PAGE analysis. Nevertheless, the effects of R38/B12 Igs on [3H]TRH binding by GH3/B6 cells and on basal or TRH-induced prolactin secretion were not markedly different from those elicited by control Ab2. These data suggest that we have characterized Ab2 antibodies which recognize a molecular entity that might be related to the TRH-R in GH3B6 cells.

Structure of the polyglutamyl chain of tubulin: occurrence of alpha and gamma linkages between glutamyl units revealed by monoreactive polyclonal antibodies.

Polyglutamylation, a posttranslational modification which consists of the sequential addition of one to six glutamyl units in the carboxy-terminal domain of both tubulin subunits, is a major event in neurons. Its structure has been investigated by using monoreactive polyclonal antibodies directed against distinct glutamylation motifs, ie alpha- and gamma-linkages between glutamyl units. It is shown that, beside alpha-linkages previously characterized, gamma-linkages also occur in glutamyl chains of brain tubulin. The co-existence of these two basic motifs leads to a conception of the polyglutamyl chain with a very sophisticated structure which could, through its complexity, help the microtubule to reach its structure and fulfil its functions.

Differential distribution of glutamylated tubulin during spermatogenesis in mammalian testis.

The distribution of glutamylated tubulin has been analyzed in mammalian testis using the specific mAb GT335 by immunoelectron microscopy and immunoblotting. In spermatozoa of various species, immunogold labeling showed the presence of glutamylated tubulin in all of the microtubules of axoneme and centrioles, whereas the microtubule network of the spermatid manchette was unlabeled. In earlier germ cells, centriole was the only microtubule structure to be labeled. A similar distribution was observed using the anti-acetylated tubulin antibody (6-11B-1), confirming previous results of Hermo et al. [Anat. Rec. 229:31-50, 1991]. However, among testicular somatic cells, microtubules of some Sertoli cell branches were not acetylated but glutamylated. 2-D PAGE of mouse and hamster sperm extracts showed a high level of alpha and beta-tubulin heterogeneity, comparable to that found in brain. Immunoblotting with GT335 revealed a large amount of glutamylated tubulin resolved into numerous alpha as well as beta-tubulin isoforms. This suggests that the major testis-specific tubulin isotypes (m alpha 3/7 and m beta 3) are also glutamylatable. These results show a subcellular sorting of posttranslationally modified tubulin isoforms in spermatids, glutamylation being associated with the most stable microtubule structures.

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

The relationship between microtubule dynamics and polyglutamylation of tubulin was investigated in young differentiating mouse brain neurons. Selective posttranslational labeling with [3H]glutamate and immunoblotting with a specific monoclonal antibody (GT335) enabled us to analyze polyglutamylation of both alpha and beta subunits. Nocodazole markedly inhibited incorporation of [3H]glutamate into alpha- and beta-tubulin, whereas taxol had no effect for alpha-tubulin and a stimulating effect for beta-tubulin. These results strongly suggest that microtubule polymers are the preferred substrate for polyglutamylation. Chase experiments revealed the existence of a reversal reaction that, in the case of alpha-tubulin, was not affected by microtubule drugs, suggesting that deglutamylation of this subunit can occur on both polymers and soluble tubulin. Evidence was obtained that deglutamylation of alpha-tubulin operates following two distinct rates depending on the length of the polyglutamyl chain, the distal units (4th-6th) being removed rapidly whereas the proximal ones (1st-3rd) appearing much more resistant to deglutamylation. Partition of glutamylated alpha-tubulin isoforms was also correlated with the length of the polyglutamyl chain. Forms bearing four to six units were recovered specifically in the polymeric fraction, whereas those bearing one to three units were distributed evenly between polymeric and soluble fractions. It thus appears that the slow rate component of the deglutamylation reaction offers to neurons the possibility to maintain a basal level of glutamylated alpha-tubulin in the soluble pool independently of microtubule dynamics. Finally, some differences observed in the glutamylation of alpha- and beta-tubulin suggest that distinct enzymes are involved.

Distribution of glutamylated alpha and beta-tubulin in mouse tissues using a specific monoclonal antibody, GT335.

A monoclonal antibody (GT335) directed against polyglutamylated tubulin was obtained by immunization with a synthetic peptide which mimics the structure of the polyglutamylated site of alpha-tubulin. This peptide corresponds to the C-terminal sequence Glu441-Gly448 and was chemically modified by the addition of two glutamyl units at Glu445. The specificity of GT335 was assayed by direct and competitive enzyme-linked immunosorbent assay (ELISA) against tubulin and several synthetic peptides differing either by the structure of the added polyglutamyl chain or by their amino acid sequence. Further characterization was carried out by immunoblotting detection after one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The epitope appears to be formed by at least two constituents: a basic motif of monoglutamylation which is retained in the polyglutamylated forms independent of their degree of glutamylation, and some elements of the polypeptide chain close to the site of glutamylation. Given the specificity of GT335 and the delineation of its epitope, our results indicate that, in addition to alpha and beta' (class III)-tubulin, other beta-tubulin isotypes are also glutamylated. This antibody has been used to analyze the cell and tissue distributions of glutamylated tubulin. In mouse brain extracts, GT335 reacts strongly with alpha-tubulin and, to a lesser extent, with beta' (class III) and beta-tubulin. The same reactivity is also observed with cultured neurons whereas astroglial cells exhibit only low levels of glutamylated tubulin. In non-nervous mouse tissues such as spleen, lung or testis, glutamylation was shown to involve only beta-tubulin, but at far lower levels than in brain.

Heterogeneity of Tau proteins during mouse brain development and differentiation of cultured neurons.

Tau microtubule-associated proteins constitute a group of developmentally regulated neuronal proteins. Using the high-resolution two-dimensional polyacrylamide gel electrophoresis system, we have resolved more than 60 distinct Tau isoforms in the adult mouse brain. Tau protein heterogeneity increases drastically during the second week of brain development. In neuronal primary cell cultures, some of these developmental changes can be observed. The increase of Tau heterogeneity in culture is more limited and reaches a plateau after a period corresponding to the second week of development. Most, if not all, of the vast Tau heterogeneity can be attributed to intensive post-translational phosphorylation, which may affect the structure of the proteins.

Polyglutamylated alpha-tubulin can enter the tyrosination/detyrosination cycle.

We have previously identified a major modification of neuronal alpha-tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated alpha-tubulin [Eddé, B., Rossier, J., Le Caer, J.P., Desbruyères, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated alpha-tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six alpha-tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of alpha-tubulin, the structure of this region was investigated. [3H]tyrosinated tubulin was mixed with a large excess of unlabeled mouse brain tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of alpha-tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated alpha-tubulin. A quantitative analysis showed that the different tyrosinated forms of alpha-tubulin represent a minor (13%) fraction of the total alpha-tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)

A predominant basic alpha-tubulin isoform present in prophase Xenopus oocyte decreases during meiotic maturation.

Xenopus oocytes are blocked in prophase of the first meiotic division. During the G2/M transition drastic changes occur both in the cytoskeletal organization and in the capacity of tubulin to polymerize. Posttranslational modification of tubulin isoforms might be one of the factors that control the dynamic properties of microtubules. We have therefore analysed, by two-dimensional polyacrylamide gel electrophoresis, the isotubulins purified from Xenopus oocytes, and we show that tubulin is resolved into at least four alpha-isoforms and four beta-isoforms. We have identified a basic alpha (alpha b)-tubulin isoform which is specific to prophase arrested oocyte and that progressively disappears during meiotic maturation; its decrease is initiated when the nuclear envelope breaks down and is controlled by the nucleus. Using 35S methionine labelled oocytes we demonstrate that the disappearance of the alpha b isotubulin results from both an arrest of its biosynthesis after maturation, and from posttranslational modification which induces a shift of this alpha-isoform to a more acidic pI. Moreover, in vitro experiments using 35S prelabelled tubulin purified from prophase oocytes show that metaphase extracts containing MPF activity are able to induce the acidification of the alpha b-isoform, suggesting that the observed posttranslational modification might be regulated by p34cdc2. However, the nature of this modification remains to be elucidated.