scholarly journals A novel role of farnesylation in targeting a mitotic checkpoint protein, human Spindly, to kinetochores

2015 ◽  
Vol 208 (7) ◽  
pp. 881-896 ◽  
Author(s):  
Devinderjit K. Moudgil ◽  
Nathan Westcott ◽  
Jakub K. Famulski ◽  
Kinjal Patel ◽  
Dawn Macdonald ◽  
...  
Keyword(s):  
Mitotic Checkpoint ◽  
Cysteine Residue ◽  
Farnesyl Transferase ◽  
Checkpoint Protein ◽  
Protein Protein Interaction ◽  
Domain Mapping ◽  
Mitotic Checkpoint Protein ◽  
Dynactin Complex

Kinetochore (KT) localization of mitotic checkpoint proteins is essential for their function during mitosis. hSpindly KT localization is dependent on the RZZ complex and hSpindly recruits the dynein–dynactin complex to KTs during mitosis, but the mechanism of hSpindly KT recruitment is unknown. Through domain-mapping studies we characterized the KT localization domain of hSpindly and discovered it undergoes farnesylation at the C-terminal cysteine residue. The N-terminal 293 residues of hSpindly are dispensable for its KT localization. Inhibition of farnesylation using a farnesyl transferase inhibitor (FTI) abrogated hSpindly KT localization without affecting RZZ complex, CENP-E, and CENP-F KT localization. We showed that hSpindly is farnesylated in vivo and farnesylation is essential for its interaction with the RZZ complex and hence KT localization. FTI treatment and hSpindly knockdown displayed the same mitotic phenotypes, indicating that hSpindly is a key FTI target in mitosis. Our data show a novel role of lipidation in targeting a checkpoint protein to KTs through protein–protein interaction.

scholarly journals Bub1 mediates cell death in response to chromosome missegregation and acts to suppress spontaneous tumorigenesis

2007 ◽  
Vol 179 (2) ◽  
pp. 255-267 ◽  
Author(s):  
Karthik Jeganathan ◽  
Liviu Malureanu ◽  
Darren J. Baker ◽  
Susan C. Abraham ◽  
Jan M. van Deursen
Keyword(s):  
Cell Death ◽  
Chromosome Segregation ◽  
Physiological Role ◽  
Mitotic Checkpoint ◽  
Critical Threshold ◽  
Wild Type ◽  
Checkpoint Protein ◽  
Chromosome Missegregation ◽  
Mitotic Checkpoint Protein

The physiological role of the mitotic checkpoint protein Bub1 is unknown. To study this role, we generated a series of mutant mice with a gradient of reduced Bub1 expression using wild-type, hypomorphic, and knockout alleles. Bub1 hypomorphic mice are viable, fertile, and overtly normal despite weakened mitotic checkpoint activity and high percentages of aneuploid cells. Bub1 haploinsufficient mice, which have a milder reduction in Bub1 protein than Bub1 hypomorphic mice, also exhibit reduced checkpoint activity and increased aneuploidy, but to a lesser extent. Although cells from Bub1 hypomorphic and haploinsufficient mice have similar rates of chromosome missegregation, cell death after an aberrant separation decreases dramatically with declining Bub1 levels. Importantly, Bub1 hypomorphic mice are highly susceptible to spontaneous tumors, whereas Bub1 haploinsufficient mice are not. These findings demonstrate that loss of Bub1 below a critical threshold drives spontaneous tumorigenesis and suggest that in addition to ensuring proper chromosome segregation, Bub1 is important for mediating cell death when chromosomes missegregate.

scholarly journals Role of the kinetochore/cell cycle checkpoint protein ZW10 in interphase cytoplasmic dynein function

2006 ◽  
Vol 172 (5) ◽  
pp. 655-662 ◽  
Author(s):  
Dileep Varma ◽  
Denis L. Dujardin ◽  
Stephanie A. Stehman ◽  
Richard B. Vallee
Keyword(s):  
Cell Cycle ◽  
Leading Edge ◽  
Cytoplasmic Dynein ◽  
Mitotic Checkpoint ◽  
Cell Cycle Checkpoint ◽  
Dominant Negative ◽  
Checkpoint Protein ◽  
Cycle Checkpoint ◽  
Mitotic Checkpoint Protein

Zeste white 10 (ZW10) is a mitotic checkpoint protein and the anchor for cytoplasmic dynein at mitotic kinetochores, though it is expressed throughout the cell cycle. We find that ZW10 localizes to pericentriolar membranous structures during interphase and cosediments with Golgi membranes. Dominant-negative ZW10, anti-ZW10 antibody, and ZW10 RNA interference (RNAi) caused Golgi dispersal. ZW10 RNAi also dispersed endosomes and lysosomes. Live imaging of Golgi, endosomal, and lysosomal markers after reduced ZW10 expression showed a specific decrease in the frequency of minus end–directed movements. Golgi membrane–associated dynein was markedly decreased, suggesting a role for ZW10 in dynein cargo binding during interphase. We also find ZW10 enriched at the leading edge of migrating fibroblasts, suggesting that ZW10 serves as a general regulator of dynein function throughout the cell cycle.

scholarly journals Requirement of the Dynein-adaptor Spindly for mitotic and post-mitotic functions in Drosophila

2017 ◽  
Author(s):  
Giuliana D. Clemente ◽  
Matthew R. Hannaford ◽  
Jens Januschke ◽  
Eric R. Griffis ◽  
Hans-Arno J. Muller
Keyword(s):  
Germ Line ◽  
Mitotic Checkpoint ◽  
Checkpoint Protein ◽  
C Elegans ◽  
Protein Levels ◽  
Egg Morphology ◽  
Female Germ Line ◽  
And Function ◽  
Mitotic Checkpoint Protein ◽  
Dynactin Complex

AbstractSpindly is a mitotic checkpoint protein originally identified as a specific regulator of Dynein activity at the kinetochore. In metaphase, Spindly recruits the Dynein/Dynactin complex, promoting the establishment of stable kinetochore-microtubule interactions and progression into anaphase. While details of Spindly function in mitosis have been worked out in cultured human cells and in the C. elegans zygote, the function of Spindly within the context of an organism has not yet been addressed. Here we present loss- and gain-of-function studies of Spindly in Drosophila. We investigated the requirements of distinct protein domains for the localisation and function of Spindly. We find that knock-down of Spindly results in a range of mitotic defects in the female germ line and during cleavage divisions in embryogenesis. Overexpression of Spindly in the female germ line is embryonic lethal and results in altered egg morphology. To determine whether Spindly plays a role in post-mitotic cells we altered Spindly protein levels in migrating cells and found that ovarian border cell migration is sensitive to the levels of Spindly protein. Our study uncovers novel functions of the mitotic checkpoint protein Spindly in Drosophila.

scholarly journals Aneuploidy in health, disease, and aging

2013 ◽  
Vol 201 (1) ◽  
pp. 11-21 ◽  
Author(s):  
Robin M. Ricke ◽  
Jan M. van Deursen
Keyword(s):  
Mouse Models ◽  
Chromosome Segregation ◽  
Molecular Mechanisms ◽  
Mitotic Checkpoint ◽  
In Vivo Studies ◽  
Aberrant Chromosome ◽  
Checkpoint Protein ◽  
Age Related ◽  
Mitotic Checkpoint Protein

Aneuploidy, an aberrant number of chromosomes, has been recognized as a feature of human malignancies for over a century, but compelling evidence for causality was largely lacking until mouse models for chromosome number instability were used. These in vivo studies have not only uncovered important new insights into the extremely complex aneuploidy–cancer relationship but also into the molecular mechanisms underlying proper and aberrant chromosome segregation. A series of diverse mouse models for the mitotic checkpoint protein BubR1 has provided evidence for a provocative novel link between aneuploidization and the development of age-related pathologies.

scholarly journals The Vertebrate Mitotic Checkpoint Protein BUBR1 Is an Unusual Pseudokinase

2012 ◽  
Vol 22 (6) ◽  
pp. 1321-1329 ◽  
Author(s):  
Saskia J.E. Suijkerbuijk ◽  
Teunis J.P. van Dam ◽  
G. Elif Karagöz ◽  
Eleonore von Castelmur ◽  
Nina C. Hubner ◽  
...  
Keyword(s):  
Mitotic Checkpoint ◽  
Checkpoint Protein ◽  
Mitotic Checkpoint Protein

scholarly journals Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Jonathan L. Portman ◽  
Qiongying Huang ◽  
Michelle L. Reniere ◽  
Anthony T. Iavarone ◽  
Daniel A. Portnoy
Keyword(s):  
Protein Function ◽  
Inhibitory Effect ◽  
Cysteine Residue ◽  
Infection Model ◽  
Listeriolysin O ◽  
Content Type ◽  
Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

scholarly journals A Conserved Residue in the Yeast Bem1p SH3 Domain Maintains the High Level of Binding Specificity Required for Function

2011 ◽  
Vol 286 (22) ◽  
pp. 19470-19477 ◽  
Author(s):  
Maryna Gorelik ◽  
Karen Stanger ◽  
Alan R. Davidson
Keyword(s):  
Sequence Similarity ◽  
Consensus Sequence ◽  
Binding Specificity ◽  
Biologically Relevant ◽  
Protein Protein Interaction ◽  
Nonspecific Interactions ◽  
High Level ◽  
Conserved Residue

The yeast Bem1p SH3b and Nbp2p SH3 domains are unusual because they bind to peptides containing the same consensus sequence, yet they perform different functions and display low sequence similarity. In this work, by analyzing the interactions of these domains with six biologically relevant peptides containing the consensus sequence, they are shown to possess finely tuned and distinct binding specificities. We also identify a residue in the Bem1p SH3b domain that inhibits binding, yet is highly conserved for the purpose of preventing nonspecific interactions. Substitution of this residue results in a marked reduction of in vivo function that is caused by titration of the domain away from its proper targets through nonspecific interactions with other proteins. This work provides a clear illustration of the importance of intrinsic binding specificity for the function of protein-protein interaction modules, and the key role of “negative” interactions in determining the specificity of a domain.

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