Gigaxonin interacts with tubulin folding cofactor B and controls its degradation through the ubiquitin-proteasome pathway

Gigaxonin is mutated in human giant axonal neuropathy (GAN), an autosomal recessive neurodegenerative disorder. The presence of generalized cytoskeletal abnormalities , including few microtubules and accumulated intermediate filaments (IFs), in GAN suggests an essential role of gigaxonin in cytoskeletal organization and dynamics. However, the molecular mechanisms underlying the cytoskeletal pathology remain to be elucidated. Over the years, the ubiquitin-proteasome system (UPS) of intracellular protein degradation has been implicated in the control of many fundamental cellular processes. Defects in this system seem to be directly linked to the development of human diseases, including cancers and neurodegenerative diseases . Here, we show that gigaxonin controls protein degradation of tubulin folding cofactor B (TBCB) , a function disrupted by GAN-associated mutations. The substantial TBCB protein accumulation caused by impaired UPS may be a causative factor of cytoskeletal pathology in GAN. Our study provides important insight into pathogenesis of neurodegenerative diseases associated with cytoskeletal abnormalities.     

Domain analysis of the tubulin cofactor system: a model for tubulin folding and dimerization

Background  

The correct folding and dimerization of tubulins, before their addition to the microtubular structure, needs a group of conserved proteins called cofactors A to E. The biochemical analysis of cofactors gave an insight to their general functions, however not much is known about the domain structure and detailed, molecular function of these proteins.     

HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo

Microtubules are cylindrical cytoskeletal structures found in almost all eukaryotic cell types which are involved in a great variety of cellular processes. Reversible acetylation on the epsilon-amino group of alpha-tubulin Lys40 marks stabilized microtubule structures and may contribute to regulating microtubule dynamics. Yet, the enzymes catalysing this acetylation/deacetylation have remained unidentified until recently. Here we report that beta-tubulin interacts with histone deacetylase-6 (HDAC-6) in a yeast two-hybrid assay and in vitro. We find that HDAC-6 is a micro tubule-associated protein capable of deacetylating alpha-tubulin in vivo and in vitro. HDAC-6's microtubule binding and deacetylation functions both depend on the hdac domains. Overexpression of HDAC-6 in mammalian cells leads to tubulin hypoacetylation. In contrast, inhibition of HDAC-6 function by two independent mechanisms--pharmacological (HDAC inhibitors) or genetic (targeted inactivation of HDAC-6 in embryonic stem cells)--leads to hyperacetylation of tubulin and microtubules. Taken together, our data provide evidence that HDAC-6 might act as a dual deacetylase for tubulin and histones, and suggest the possibility that acetylated non-histone proteins might represent novel targets for pharmacological therapy by HDAC inhibitors.     

Dysferlin interacts with histone deacetylase 6 and increases alpha-tubulin acetylation

Dysferlin is a multi-C2 domain transmembrane protein involved in a plethora of cellular functions, most notably in skeletal muscle membrane repair, but also in myogenesis, cellular adhesion and intercellular calcium signaling. We previously showed that dysferlin interacts with alpha-tubulin and microtubules in muscle cells. Microtubules are heavily reorganized during myogenesis to sustain growth and elongation of the nascent muscle fiber. Microtubule function is regulated by post-translational modifications, such as acetylation of its alpha-tubulin subunit, which is modulated by the histone deacetylase 6 (HDAC6) enzyme. In this study, we identified HDAC6 as a novel dysferlin-binding partner. Dysferlin prevents HDAC6 from deacetylating alpha-tubulin by physically binding to both the enzyme, via its C2D domain, and to the substrate, alpha-tubulin, via its C2A and C2B domains. We further show that dysferlin expression promotes alpha-tubulin acetylation, as well as increased microtubule resistance to, and recovery from, Nocodazole- and cold-induced depolymerization. By selectively inhibiting HDAC6 using Tubastatin A, we demonstrate that myotube formation was impaired when alpha-tubulin was hyperacetylated early in the myogenic process; however, myotube elongation occurred when alpha-tubulin was hyperacetylated in myotubes. This study suggests a novel role for dysferlin in myogenesis and identifies HDAC6 as a novel dysferlin-interacting protein.     

p21-activated kinase 1 regulates microtubule dynamics by phosphorylating tubulin cofactor B

p21-activated kinase 1 (Pak1) induces cytoskeleton reorganization in part by regulating microtubule dynamics through an elusive mechanism. Using a yeast two-hybrid screen, we identified tubulin cofactor B (TCoB) (a cofactor in the assembly of the alpha/beta-tubulin heterodimers) as an interacting substrate of Pak1. Pak1 directly phosphorylated TCoB in vitro and in vivo on serines 65 and 128 and colocalized with TCoB on newly polymerized microtubules and on centrosomes. TCoB interacted with the GTPase-binding domain of Pak1 and activated Pak1 in vitro and in vivo. In contrast to wild-type TCoB, an S65A, S128A double mutant and knock-down of the endogenous TCoB or Pak1 reduced microtubule polymerization, suggesting that Pak1 phosphorylation is necessary for normal TCoB function. Overexpression of TCoB dramatically increased the number of gamma-tubulin-containing microtubule-organizing centers, a phenotype reminiscent of cells overexpressing Pak1. TCoB was overexpressed and phosphorylated in breast tumors. These findings reveal a novel role for TCoB and Pak1 in regulating microtubule dynamics.     

ZNRF1 interacts with tubulin and regulates cell morphogenesis

The ubiquitin-proteasome system has been implicated in neuronal degeneration and regeneration. We demonstrated that overexpression of ZNRF1, which has been identified as a crucial molecule in nerve regeneration, causes morphological changes such as neurite-like elongation. Molecular dissections showed that both the RING finger domain and zinc finger domain are required for morphological changes. Furthermore, we identified β-tubulin type 2 (Tubb2) as a ZNRF1-binding protein by yeast two-hybrid screening. In vivo binding assay showed that ZNRF1 interacts with Tubb2 and immunofluorescent staining suggests that ZNRF1 is colocalized with Tubb2. These results suggest that ZNRF1 mediates regulation of neuritogenesis via interaction with tubulin.     

The Mr 78,000 intermediate chain of Chlamydomonas outer arm dynein interacts with alpha-tubulin in situ

We have used the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to examine protein-protein associations within purified outer arm dynein and axonemes from Chlamydomonas flagella. When axonemes were treated with 0.5-1 mM EDC in either the presence or absence of ATP/vanadate, a polypeptide band of Mr 127,000 recognized by monoclonal antibody 1878A (specific for the Mr 78,000 intermediate chain (IC78) of outer arm dynein) was generated. This conjugate was not obtained when purified dynein was treated with EDC. Further immunological analysis demonstrated that this complex also contained alpha- (but not beta-) tubulin. These results indicate that IC78 interacts with alpha-tubulin in situ in an ATP-insensitive manner. Identification of this interface between dynein and tubulin suggests that IC78, which probably is located at the base of the dynein particle (King, S. M., and Witman, G. B. (1990) J. Biol. Chem. 265, 19807-19811), contributes to the structural attachment of the dynein arms to the A-tubules of the outer doublet microtubules. Analysis of the cross-linked products from the purified dynein revealed several additional interactions involving the intermediate chains; these adducts provide further evidence for an intermediate chain/light chain complex within dynein and confirm that IC78 and IC69 associate directly.     

4-Hydroxynonenal interacts with tubulin by reacting with its functional -SH groups

4-Hydroxynonenal, which is one of the most important products of lipid peroxidation, alters microtubular organization and structure in 3T3 fibroblasts. Changes in cell shape and the disappearance of microtubules are observed by immunofluorescence after incubation with the aldehyde, and the colchicine binding activity of tubulin from 3T3 cells is modified. Moreover, the aldehyde determines a decrease in the ability of purified tubulin to polymerize and to bind colchicine. These effects may be related to the interaction of the aldehyde with functional -SH groups of tubulin which are necessary for protein integrity and functions. Indeed, the addition of cysteine protects against the damaging effects of the aldehyde.     

Parkin-co-regulated gene (PACRG) product interacts with tubulin and microtubules

Parkin-co-regulated gene (PACRG) is a gene that shares a bidirectional promoter with Parkinson's disease-related Parkin/Park2 gene. Recently, the PACRG gene product was implicated in the function of flagella. However, its exact function remains unknown. Here, I assessed the interaction between PACRG and tubulin. Co-sedimentation experiments revealed that PACRG directly binds to microtubules and alpha/beta-tubulin heterodimers with high affinity. Microscopic studies showed that PACRG bundles microtubules and forms branched aggregates with unpolymerized tubulin dimers. The amino acid sequence of the microtubule-binding region of PACRG is highly conserved among various organisms, suggesting that tubulin binding is a basic property of PACRG.     

Familial amyotrophic lateral sclerosis-linked mutant SOD1 aberrantly interacts with tubulin

Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause 20-25% of familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 causes motor neuron degeneration through toxic gain-of-function(s). However, the direct molecular targets of mutant SOD1, underlying its toxicity, are not fully understood. In this study, we found that α/β-tubulin is one of the major mutant SOD1-interacting proteins, but that wild-type SOD1 does not interact with it. The interaction between tubulin and mutant SOD1 was detected in the spinal cords of mutant G93A SOD1 transgenic mice before the onset of symptoms. Tubulin interacted with amino acid residues 1-23 and 116-153 of SOD1. Overexpression of mutant SOD1 resulted in the accumulation of tubulin in detergent-insoluble fractions. In a cell-free system, mutant SOD1 modulated tubulin polymerization, while wild-type SOD1 did not. Since tightly regulated microtubule dynamics is essential for neurons to remain viable, α/β-tubulin could be an important direct target of mutant SOD1.     

Irradiation by electrons of tubulin solution in vitro causes tubulin assembly in absence of a magnesium cofactor

It was shown that irradiation of a tubulin solution (5 x 10(-6) mol/l) with electrons makes tubulin assemble to microtubules. The response of the system was monitored by the femtosecond laser technique. The assembly under these conditions occurs without Mg2+ (magnesium cofactor) and GTP. At 730 (the first harmonic) and 365 nm (the second harmonic), a rise in signal intensity occurs during the first 60-70 ns followed by the onset of tubulin assembly to microtubules, which was registered by the methods of spectrophotometry and electron spectroscopy. Theoretically this effect can be explained by the appearance of hydrated electrons in the solvated tubulin shell. Hydrated electrons are mostly in a long-living polaron state, which can be considered as an ensemble of quasi-dipoles of the electron-H3O+ type. The interaction of quasi-dipoles with the weak internal electrostatic field of tubulin leads, due to nonlinear effects, to a manifold rise in the intensity of the electrostatic field of the solvated shell of initially nonpolymerized tubulin chains. Finally, the increased field makes separate tubulin chains aggregate to microtubules. The effect observed is identical to the action of Mg2+ on the GTP site of beta-tubulin, which transfers it to a slightly perturbed triplet state.     

CacyBP/SIP interacts with tubulin in neuroblastoma NB2a cells and induces formation of globular tubulin assemblies

CacyBP/SIP, originally identified as a S100A6 (calcyclin) target, was later shown to interact with some other members of the S100 family as well as with Siah-1 and Skp1 proteins. Recently, it has been shown that CacyBP/SIP is up-regulated during differentiation of cardiomyocytes. In this work we show that the level of CacyBP/SIP is higher in differentiated neuroblastoma NB2a cells than in undifferentiated ones and that in cells overexpressing CacyBP/SIP the level of GAP-43, a marker of differentiation, was increased. Since the process of differentiation is accompanied by an extensive rearrangement of microtubules, we examined whether CacyBP/SIP interacted with tubulin. By applying cross-linking experiments we found that these two proteins bind directly. The dissociation constant of the tubulin-CacyBP/SIP complex determined by the surface plasmon resonance technique is 1.57 x 10(-7 )M which suggests that the interaction is tight. The interaction and co-localization of CacyBP/SIP and tubulin was also demonstrated by co-immunoprecipitation, affinity chromatography and immunofluorescence methods. Light scattering measurements and electron microscopy studies revealed that CacyBP/SIP, but not its homologue, Sgt1, increased tubulin oligomerization. Altogether, our results suggest that CacyBP/SIP, via its interaction with tubulin, might contribute to the differentiation of neuroblastoma NB2a cells.     

Phenotypic consequences of tubulin overproduction in Saccharomyces cerevisiae: differences between alpha-tubulin and beta-tubulin.

Overexpression of alpha- and beta-tubulin genes in Saccharomyces cerevisiae, separately or together, leads to accumulation of large excesses of each of the polypeptides and arrest of cell division. However, other consequences of overexpression of these genes differ in several ways. As shown previously (D. Burke, P. Gasdaska, and L. Hartwell, Mol. Cell. Biol. 9:1049-1059, 1989), overexpression of beta-tubulin leads, at early times, to loss of microtubule structures and loss of viability. Eventually, the excess beta-tubulin forms abnormal structures. We show here that, in contrast, overexpression of alpha-tubulin led to none of these phenotypes and in fact could suppress each of the phenotypes associated with beta-tubulin accumulation. Truncated forms of beta-tubulin that were not competent to carry out microtubule functions also failed to elicit the beta-tubulin-specific phenotypes when overexpressed. The data support the hypothesis that beta-tubulin in excess over alpha-tubulin is uniquely toxic, perhaps because it interferes with normal microtubule assembly.     

Multiple forms of tubulin in Polytomella and Chlamydomonas: evidence for a precursor of flagellar alpha-tubulin

The quadriflagellate alga polytomella agilis contains several α-tubulins with distinct isoelectric points (McKeithan, T.W., and J.L. Rosenbaum, 1981, J. Cell Biol., 91:352-360). While α-3 is the major component in flagella, α-1 predominates in cytoskeletal microtubules. For determination of whether the differences in α- tubulins are due to distinct genes or to posttranslational modification of a common α-tubulin precursor, poly A+ RNA was isolated from deflagellated and control (nonregenerating) cells and translated in vitro. Approximately twice as much α-1 was synthesized using RNA from deflagellated as compared to control cells; however, there was no detectable synthesis in vitro of α-3 in either. These results suggest that α -3 tubulin is formed in vivo by posttranslational modification of a form co- migrating with, and possibly identical to, cytoskeletal α-tubulin. In the related alga chlamydomonas reinhardii deflagellation greatly stimulates synthesis of tubulin and tubulin mRNA. As in polytomella, the principal α-tubulin synthesized both in vivo and in vitro following deflagellation in chlamydomonas is more basic than the major α-tubulin and appears to correspond to α-1 tubulin in polytomella. The conversion of α-1 to α-3 receives additional support from in vivo labeling and pulse-chase experiments. In addition, in both polytomella and chlamydomonas some conversion of α-1 to α-3 appears to occur even when protein synthesis is inhibited.