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 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 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 Regulation of the p53 Family Proteins by the Ubiquitin Proteasomal Pathway

2019 ◽  
Vol 21 (1) ◽  
pp. 261 ◽  
Author(s):  
Scott Bang ◽  
Sandeep Kaur ◽  
Manabu Kurokawa
Keyword(s):  
Genetic Defect ◽  
Tumor Development ◽  
Tumor Suppressor P53 ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
P53 Family ◽  
E3 Ligases ◽  
Damage Response

The tumor suppressor p53 and its homologues, p63 and p73, play a pivotal role in the regulation of the DNA damage response, cellular homeostasis, development, aging, and metabolism. A number of mouse studies have shown that a genetic defect in the p53 family could lead to spontaneous tumor development, embryonic lethality, or severe tissue abnormality, indicating that the activity of the p53 family must be tightly regulated to maintain normal cellular functions. While the p53 family members are regulated at the level of gene expression as well as post-translational modification, they are also controlled at the level of protein stability through the ubiquitin proteasomal pathway. Over the last 20 years, many ubiquitin E3 ligases have been discovered that directly promote protein degradation of p53, p63, and p73 in vitro and in vivo. Here, we provide an overview of such E3 ligases and discuss their roles and functions.

scholarly journals p21-Activated Kinase 1 Regulates Microtubule Dynamics by Phosphorylating Tubulin Cofactor B

2005 ◽  
Vol 25 (9) ◽  
pp. 3726-3736 ◽  
Author(s):  
Ratna K. Vadlamudi ◽  
Christopher J. Barnes ◽  
Suresh Rayala ◽  
Feng Li ◽  
Seetharaman Balasenthil ◽  
...  
Keyword(s):  
Microtubule Dynamics ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Microtubule Polymerization ◽  
Cytoskeleton Reorganization ◽  
P21 Activated Kinase ◽  
Gtpase Binding Domain ◽  
Two Hybrid

ABSTRACT 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 α/β-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 γ-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.

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.

Microtubular organization visualized by immunofluorescence microscopy during erythrocytic schizogony in Plasmodium falciparum and investigation of post-translational modifications of parasite tubulin

Parasitology ◽  
1993 ◽  
Vol 106 (3) ◽  
pp. 223-232 ◽  
Author(s):  
M. Read ◽  
T. Sherwin ◽  
S. P. Holloway ◽  
K. Gull ◽  
J. E. Hyde
Keyword(s):  
Plasmodium Falciparum ◽  
Immunofluorescence Microscopy ◽  
Nuclear Body ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Large Numbers ◽  
Spindle Development ◽  
Microtubular Structures

SUMMARYWe describe a novel procedure for the immunofluorescent investigation of Plasmodium falciparum. This has allowed us to visualize clearly microtubular structures and their changing conformation through the erythrocytic cell-cycle, to the stage of cytodifferentiation leading to merozoite release. The images of spindle development we observed, together with an analysis of nuclear body numbers in large numbers of parasites, indicate that there is an apparent asynchrony in chromosomal multiplication within a single parasite. Using antibodies specific for post-translational modification of α- tubulin, we also demonstrate that the C-terminal tyrosine-containing epitope of P. falciparum α-tubulin I is similar to that of other organisms. Lysine-40 in the same molecule, a target for highly specific in vivo acetylation in some organisms, is unmodified in the blood stages we examined here. After in vitro acetylation of this residue, however, the epitope to which it contributes was recognized by antibody, showing that the conformation of this part of the molecule is also conserved, despite a lack of primary sequence homology immediately downstream of the target lysine residue.

scholarly journals Post-translational modification of α-dystroglycan is not critical for lymphocytic choriomeningitis virus receptor function in vivo

2008 ◽  
Vol 89 (11) ◽  
pp. 2713-2722 ◽  
Author(s):  
Mauro Imperiali ◽  
Roman Spörri ◽  
Jane Hewitt ◽  
Annette Oxenius
Keyword(s):  
Lymphocytic Choriomeningitis ◽  
Lymphocytic Choriomeningitis Virus ◽  
Cellular Receptor ◽  
Receptor Function ◽  
Post Translational Modification ◽  
Rare Type ◽  
Post Translational Modifications ◽  
Virus Receptor

α-Dystroglycan (α-DG) is a ubiquitously expressed molecule that has been identified as a cellular receptor for lymphocytic choriomeningitis virus (LCMV) and other arenaviruses. Recently, it was demonstrated that LCMV receptor function is critically dependent on post-translational modifications, namely glycosylation. In particular, it was shown that O-mannosylation, a rare type of mammalian O-linked glycosylation, is important in determining the binding of LCMV to its cellular receptor. All studies carried out so far showed a dependence on glycosylation in LCMV receptor function in vitro. This work extended these studies to two in vivo models of α-DG hypoglycosylation. The results confirm earlier findings on the in vitro dependence of carbohydrate modifications in LCMV receptor function. However, experiments in animal models showed that this dependence was only very weak in vivo. It is likely that alternative receptors or alternative entry pathways may account for this attenuated in vivo phenotype.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

scholarly journals TMOD-24. GENERATION AND CHARACTERIZATION OF A NOVEL TRANSGENIC IDO REPORTER MOUSE FOR IDO POSTTRANSLATIONAL MODIFICATION ANALYSIS IN SITU

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii233-ii233
Author(s):  
April Bell ◽  
Lijie Zhai ◽  
Erik Ladomersky ◽  
Kristen Lauing ◽  
Lakshmi Bollu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Cell ◽  
Central Nervous System Tumor ◽  
Post Translational Modifications ◽  
Reporter Mouse ◽  
C Terminus ◽  
Human Gbm

Abstract Glioblastoma (GBM) is the most common and aggressive primary central nervous system tumor in adults with a median survival of 14.6 months. GBM is a potently immunosuppressive cancer due in-part to the prolific expression of immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO). Tumor cell IDO facilitates the intratumoral accumulation of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Although immunosuppressive IDO activity is canonically characterized by the conversion of tryptophan into kynurenine, we have utilized transgenic and syngeneic mouse models and mutant glioma lines to demonstrate that tumor cell IDO increases Treg accumulation independent of tryptophan metabolism. Here, we address the gap in our understanding of IDO signaling activity in vivo. Subcutaneously-engrafted human GBM expressing human IDO-GFP cDNA was isolated from immunodeficient humanized NSG-SGM3 mice. The tumor was immunoprecipitated for the GFP tag using GFP-TRAP followed by mass spectrometry which revealed a novel methylation site on a lysine residue at amino acid 373 in the IDO C-terminus region. Western blot analysis of IDO protein also revealed the presence of tyrosine phosphorylation. Additionally, we recently created a new transgenic IDO reporter mouse model whereby endogenous IDO is fused to GFP via a T2A linker (IDO→GFP). This model allows for the isolation of IDO+ cells in real-time and without causing cell death, thereby creating the opportunity for downstream molecular analysis of in situ-isolated GFP+ cells. Collectively, our work suggests that IDO non-enzyme activity may involve the post-translational modifications we recently identified. As IDO activity may differ between in vitro and in vivo modeling systems, we will use the new IDO→GFP reporter mouse model for an improved mechanistic understanding of how immunosuppressive IDO facilitates Treg accumulation in vivo.

scholarly journals ADP-ribosylation of integrin α7 modulates the binding of integrin α7β1 to laminin

2004 ◽  
Vol 385 (1) ◽  
pp. 309-317 ◽  
Author(s):  
Zhefeng ZHAO ◽  
Joanna GRUSZCZYNSKA-BIEGALA ◽  
Anna ZOLKIEWSKA
Keyword(s):  
C2c12 Myotubes ◽  
Post Translational Modification ◽  
Integrin Β1 ◽  
Adp Ribosylation ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
C2c12 Skeletal Muscle Cells ◽  
Adp Ribosyltransferase

The extracellular domain of integrin α7 is ADP-ribosylated by an arginine-specific ecto-ADP-ribosyltransferase after adding exogenous NAD+ to intact C2C12 skeletal muscle cells. The effect of ADP-ribosylation on the structure or function of integrin α7β1 has not been explored. In the present study, we show that ADP-ribosylation of integrin α7 takes place exclusively in differentiated myotubes and that this post-translational modification modulates the affinity of α7β1 dimer for its ligand, laminin. ADP-ribosylation in the 37-kDa ‘stalk’ region of α7 that takes place at micromolar NAD+ concentrations increases the binding of the α7β1 dimer to laminin. Increased in vitro binding of integrin α7β1 to laminin after ADP-ribosylation of the 37-kDa fragment of α7 requires the presence of Mn2+ and it is not observed in the presence of Mg2+. In contrast, ADP-ribosylation of the 63-kDa N-terminal region comprising the ligand-binding site of α7 that occurs at approx. 100 μM NAD+ inhibits the binding of integrin α7β1 to laminin. Furthermore, incubation of C2C12 myotubes with NAD+ increases the expression of an epitope on integrin β1 subunit recognized by monoclonal antibody 9EG7. We discuss our results based on the current models of integrin activation. We also hypothesize that ADP-ribosylation may represent a mechanism of regulation of integrin α7β1 function in myofibres in vivo when the continuity of the membrane is compromised and NAD+ is available as a substrate for ecto-ADP-ribosylation.

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