scholarly journals Microtubule Regulation in Mitosis: Tubulin Phosphorylation by the Cyclin-dependent Kinase Cdk1

2006 ◽  
Vol 17 (3) ◽  
pp. 1041-1050 ◽  
Author(s):  
Anne Fourest-Lieuvin ◽  
Leticia Peris ◽  
Vincent Gache ◽  
Isabel Garcia-Saez ◽  
Céline Juillan-Binard ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Microtubule Dynamics ◽  
Cyclin Dependent Kinase ◽  
Gtp Binding ◽  
Tubulin Binding ◽  
A Site ◽  
Β Tubulin ◽  
In Cells

The activation of the cyclin-depdndent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates β-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of β-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-β3-tubulinS172D/E mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to β-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.

Function of Tubulin Binding Proteins in Vivo

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 69-80 ◽  
Author(s):  
James A Fleming ◽  
Leticia R Vega ◽  
Frank Solomon
Keyword(s):  
Binding Proteins ◽  
Binding Protein ◽  
Yeast Cells ◽  
Gene Products ◽  
Salvage Pathway ◽  
Tubulin Binding ◽  
Mutant Cells ◽  
Β Tubulin

Abstract Overexpression of the β-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant α-tubulin, tub1-724, that produces unstable heterodimer. Here we use RBL2 overexpression to identify mutations in other genes that affect formation or stability of heterodimer. This approach identifies four genes—CIN1, CIN2, CIN4, and PAC2—as affecting heterodimer formation in vivo. The vertebrate homologues of two of these gene products—Cin1p/cofactor D and Pac2p/cofactor E—can catalyze exchange of tubulin polypeptides into preexisting heterodimer in vitro. Previous work suggests that both Cin2p or Cin4p act in concert with Cin1p in yeast, but no role for vertebrate homologues of either has been reported in the in vitro reaction. Results presented here demonstrate that these proteins can promote heterodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant cells causes microtubule disassembly and enhanced formation of Rbl2p-β-tubulin complex, as it does in the α-tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant α-tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential under conditions where the levels of dissociated tubulin polypeptides increase. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of free β-tubulin.

scholarly journals β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

2002 ◽  
Vol 13 (8) ◽  
pp. 2919-2932 ◽  
Author(s):  
Mohan L. Gupta ◽  
Claudia J. Bode ◽  
Douglas A. Thrower ◽  
Chad G. Pearson ◽  
Kathy A. Suprenant ◽  
...  
Keyword(s):  
Essential Gene ◽  
Dynamic Properties ◽  
Microtubule Dynamics ◽  
Cytoplasmic Microtubule ◽  
Bud Emergence ◽  
Spindle Elongation ◽  
Mutant Cells ◽  
Β Tubulin

Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast β-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.

scholarly journals Mammalian end binding proteins control persistent microtubule growth

2009 ◽  
Vol 184 (5) ◽  
pp. 691-706 ◽  
Author(s):  
Yulia Komarova ◽  
Christian O. De Groot ◽  
Ilya Grigoriev ◽  
Susana Montenegro Gouveia ◽  
E. Laura Munteanu ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Microtubule Dynamics ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dimerization Domain ◽  
Negative Effect ◽  
Microtubule Growth ◽  
Tracking Behavior ◽  
In Cells

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.

scholarly journals The Biochemistry and Physiology of Metallic Fluoride: Action, Mechanism, and Implications

2003 ◽  
Vol 14 (2) ◽  
pp. 100-114 ◽  
Author(s):  
Liang Li
Keyword(s):  
Binding Proteins ◽  
Phosphoryl Transfer ◽  
Oral Diseases ◽  
Bone Biology ◽  
Gtp Binding Proteins ◽  
Gtpase Activating Proteins ◽  
Protein Activator ◽  
Gtp Binding

Fluoride is a well-known G protein activator. Activation of heterotrimeric GTP-binding proteins by fluoride requires trace amounts of Al3+ or Be2+ ions. AlFx mimics a γ-phosphate at its transition state in a G α protein and is therefore able to inhibit its GTPase activity. AlFx also forms complexes with small GTP-binding proteins in the presence of their GTPase-activating proteins (GAP). As phosphate analogs, AlFx or BeFx affect the activity of a variety of phosphoryl transfer enzymes. Most of these enzymes are fundamentally important in cell signal transduction or energy metabolism. Al3+ and F- tend to form stable complexes in aqueous solution. The exact structure and concentration of AlFx depend on the pH and the amount of F- and Al3+ in the solution. Humans are exposed to both F and Al. It is possible that Al-F complexes may be formed in vivo, or formed in vitro prior to their intake by humans. Al-F complexes may play physiological or pathological roles in bone biology, fluorosis, neurotoxicity, and oral diseases such as dental caries and periodontal disease. The aim of this review is to discuss the basic chemical, biochemical, and toxicological properties of metallic fluoride, to explore its potential physiological and clinical implications.

scholarly journals Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble αβ-tubulin pool for microtubule dynamics

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Stanley Nithianantham ◽  
Sinh Le ◽  
Elbert Seto ◽  
Weitao Jia ◽  
Julie Leary ◽  
...  
Keyword(s):  
Ternary Complex ◽  
Microtubule Dynamics ◽  
Gtp Hydrolysis ◽  
Tubulin Dimer ◽  
Physical Mechanisms ◽  
Biochemical Assays ◽  
Sequential Binding ◽  
Β Tubulin

Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.

scholarly journals Protection from Free β-Tubulin by the β-Tubulin Binding Protein Rbl2p

2002 ◽  
Vol 22 (1) ◽  
pp. 138-147 ◽  
Author(s):  
Katharine C. Abruzzi ◽  
Adelle Smith ◽  
William Chen ◽  
Frank Solomon
Keyword(s):  
Binding Protein ◽  
In Vitro Assay ◽  
In Vitro Studies ◽  
Microtubule Assembly ◽  
Vitro Assay ◽  
Tubulin Binding ◽  
And Function ◽  
Β Tubulin ◽  
In Cells

ABSTRACT Free β-tubulin not in heterodimers with α-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free β-tubulin. This study focused specifically on the function of Rbl2p, which, like α-tubulin, can rescue cells from free β-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of β-tubulin relative to α-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of β-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to β-tubulin. The data suggest that Rbl2p binds transiently to free β-tubulin, which then passes into an aggregated form that is not toxic.

scholarly journals An α-Tubulin Mutant Destabilizes the Heterodimer: Phenotypic Consequences and Interactions with Tubulin-binding Proteins

1998 ◽  
Vol 9 (9) ◽  
pp. 2349-2360 ◽  
Author(s):  
Leticia R. Vega ◽  
James Fleming ◽  
Frank Solomon
Keyword(s):  
Binding Proteins ◽  
Microtubule Assembly ◽  
Wild Type ◽  
Cellular Regulation ◽  
Tubulin Binding ◽  
Cold Sensitive ◽  
Mutant Cells ◽  
Dependent Processes ◽  
Β Tubulin

Many effectors of microtubule assembly in vitro enhance the polymerization of subunits. However, several Saccharomyces cerevisiae genes that affect cellular microtubule-dependent processes appear to act at other steps in assembly and to affect polymerization only indirectly. Here we use a mutant α-tubulin to probe cellular regulation of microtubule assembly.tub1-724 mutant cells arrest at low temperature with no assembled microtubules. The results of several assays reported here demonstrate that the heterodimer formed between Tub1-724p and β-tubulin is less stable than wild-type heterodimer. The unstable heterodimer explains several conditional phenotypes conferred by the mutation. These include the lethality of tub1-724haploid cells when the β-tubulin–binding protein Rbl2p is either overexpressed or absent. It also explains why theTUB1/tub1-724 heterozygotes are cold sensitive for growth and why overexpression of Rbl2p rescues that conditional lethality. Both haploid and heterozygous tub1-724 cells are inviable when another microtubule effector, PAC2, is overexpressed. These effects are explained by the ability of Pac2p to bind α-tubulin, a complex we demonstrate directly. The results suggest that tubulin-binding proteins can participate in equilibria between the heterodimer and its components.

scholarly journals SUMOylation of α-tubulin is a novel modification regulating microtubule dynamics

2021 ◽  
Author(s):  
Wenfeng Feng ◽  
Rong Liu ◽  
Xuan Xie ◽  
Lei Diao ◽  
Nannan Gao ◽  
...  
Keyword(s):  
Microtubule Dynamics ◽  
In Vitro Experiments ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Post Translational Modifications ◽  
Neurite Extension ◽  
Microtubule Polymerization ◽  
Β Tubulin

Abstract Microtubules are regulated by a number of known post-translational modifications on α/β-tubulin to fulfill diverse cellular functions. Here, we showed that SUMOylation is a novel post-translational modification on α-tubulin in vivo and in vitro. The SUMOylation on α-tubulin mainly occurred at Lys 96 (K96), K166, and K304 of soluble α-tubulin and could be removed by SUMO-specific peptidase 1. In vitro experiments showed that tubulin SUMOylation could reduce inter-protofilament interaction, promote microtubule catastrophe, and impede microtubule polymerization. In cells, mutation of the SUMOylation sites on α-tubulin reduced catastrophe frequency and increased the proportion of polymerized α-tubulin, while upregulation of SUMOylation with fusion of SUMO1 reduced α-tubulin assembly into microtubules. Additionally, overexpression of SUMOylation-deficient α-tubulin attenuated the neurite extension in Neuro-2a cells. Thus, SUMOylation on α-tubulin represents a new player in the regulation of microtubule properties.

scholarly journals Cdk2-dependent phosphorylation of p21 regulates the role of Cdk2 in cisplatin cytotoxicity

2011 ◽  
Vol 300 (5) ◽  
pp. F1171-F1179 ◽  
Author(s):  
Rawad Hodeify ◽  
Adel Tarcsafalvi ◽  
Judit Megyesi ◽  
Robert L. Safirstein ◽  
Peter M. Price
Keyword(s):  
Mass Spectrometry ◽  
Cell Death ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Cdk2 Activity ◽  
Adenoviral Transduction ◽  
A Site

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. We found that an 18-kDa protein identified by mass spectrometry as p21WAF1/Cip1 was phosphorylated by Cdk2 starting 12 h after cisplatin exposure. The analysis showed it was phosphorylated at serine 78, a site not previously identified. The adenoviral transduction of p21 before cisplatin exposure protects from cytotoxicity by inhibiting Cdk2. Although cisplatin causes induction of endogenous p21, the protection is inefficient. We hypothesized that phosphorylation of p21 at serine 78 could affect its role as a Cdk inhibitor, and thereby lessen its ability to protect from cisplatin cytotoxicity. To investigate the effect of serine 78 phosphorylation on p21 activity, we replaced serine 78 with aspartic acid, creating the phosphomimic p21S78D. Mutant p21S78D was an inefficient inhibitor of Cdk2 and was inefficient at protecting TKPTS cells from cisplatin-induced cell death. We conclude that phosphorylation of p21 by Cdk2 limits the effectiveness of p21 to inhibit Cdk2, which is the mechanism for continued cisplatin cytotoxicity even after the induction of a protective protein.

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