Yeast Chfr homologs retard cell cycle at G<sub>1</sub>and G<sub>2</sub>/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination

Loring, Greta L.; Christensen, Kathryn C.; Gerber, Scott A.; Brenner, Charles

doi:10.4161/cc.7.1.5113

Cited by 30 publications

(46 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, this highly overexpressed nuclear Chfr was degraded during the cell cycle in an autoubiquitination-dependent manner and caused a G1 cell cycle arrest. Notably, overexpression of the yeast homolog of Chfr in Saccharomyces cerevisiae can induce both G1 and G2 arrests (Loring et al, 2008). At lower levels, exogenous Chfr behaved similar to the endogenous protein, with both proteins stable throughout the cell cycle and localizing to the cytoplasm during interphase, and to the spindle during mitosis.…”

Section: Discussionmentioning

confidence: 99%

Chfr interacts and colocalizes with TCTP to the mitotic spindle

et al. 2008

View full text Add to dashboard Cite

Chfr is a checkpoint protein that plays an important function in cell cycle progression and tumor suppression, although its exact role and regulation are unclear. Previous studies have utilized overexpression of Chfr to determine the signaling pathway of this protein in vivo. In this study, we demonstrate, by using three different antibodies against Chfr, that the endogenous and highly overexpressed ectopic Chfr protein is localized and regulated differently in cells. Endogenous and lowly expressed ectopic Chfr are cytoplasmic and localize to the spindle during mitosis. Higher expression of ectopic Chfr correlates with a shift in the localization of this protein to the nucleus/PML bodies, and with a block of cell proliferation. In addition, endogenous and lowly expressed ectopic Chfr is stable throughout the cell cycle, whereas when highly expressed, ectopic Chfr is actively degraded during S-G2/ M phases in an autoubiquitination and proteasome-dependent manner. A two-hybrid screen identified TCTP as a possible Chfr-interacting partner. Biochemical analysis with the endogenous proteins confirmed this interaction and identified b-tubulin as an additional partner for Chfr, supporting the mitotic spindle localization of Chfr. The Chfr-TCTP interaction was stable throughout the cell cycle, but it could be diminished by the complete depolymerization of the microtubules, providing a possible mechanism where Chfr could be the sensor that detects microtubule disruption and then activates the prophase checkpoint.

show abstract

Section: Discussionmentioning

confidence: 99%

Chfr interacts and colocalizes with TCTP to the mitotic spindle

et al. 2008

View full text Add to dashboard Cite

show abstract

“…Because the loss of Chfr ubiquitin ligase activity in FLAG-Chfr ⌬RF or FLAG-Chfr I306A might affect cell-cycle progression due to loss of stability and failure to degrade extrinsic target substrates, we attempted to produce a mutant Chfr construct that did not contain target sites for auto-ubiquitylation but still retained its ligase activity. Previous studies proposed that a Chfr fragment comprising amino acids 281-375, which contains the entire RING finger E3 ligase domain, may target sites in flanking regions for polyubiquitination (26). We, therefore, generated three mutant constructs in which putative auto-ubiquitylation target lysine residues were replaced with alanine: FLAG-Chfr K2A, containing K384A and K393A substitutions; FLAG-Chfr K3A, containing three Lys-to-Ala substitutions (K257A/K258A/K259A); and FLAG-Chfr K5A, containing all five substitutions (Fig.…”

Section: Substitution Of Alanine For Putative Auto-ubiquitylation Tarmentioning

confidence: 99%

The Auto-ubiquitylation of E3 Ubiquitin-protein Ligase Chfr at G2 Phase Is Required for Accumulation of Polo-like Kinase 1 and Mitotic Entry in Mammalian Cells

Kim

Park

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G 2 /M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G 2 /M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G 2 phase, but not at S phase, were ϳ2-3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G 2 phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G 2 /M transition.

show abstract

“…The Dma1 and Dma2 proteins are paralogous E3 ubiquitin ligases that share 58% identity in their primary sequence. They carry a C-terminal RING finger domain that can catalyze both K48-and K63-linked ubiquitin chains (Loring et al 2008) and a central FHA domain that is thought to bind Thr-phosphorylated proteins (Durocher et al 2000). So far, Dma1 and Dma2 have been redundantly involved in spindle positioning, septin organization, and vacuole inheritance (Fraschini et al 2004;Merlini et al 2012;Chahwan et al 2013;Yau et al 2014).…”

mentioning

confidence: 99%

Control of Formin Distribution and Actin Cable Assembly by the E3 Ubiquitin Ligases Dma1 and Dma2

Juanes¹,

Piatti²

2016

Genetics

View full text Add to dashboard Cite

Formins are widespread actin-polymerizing proteins that play pivotal roles in a number of processes, such as cell polarity, morphogenesis, cytokinesis, and cell migration. In agreement with their crucial function, formins are prone to a variety of regulatory mechanisms that include autoinhibition, post-translational modifications, and interaction with formin modulators. Furthermore, activation and function of formins is intimately linked to their ability to interact with membranes. In the budding yeast Saccharomyces cerevisiae, the two formins Bni1 and Bnr1 play both separate and overlapping functions in the organization of the actin cytoskeleton. In addition, they are controlled by both common and different regulatory mechanisms. Here we show that proper localization of both formins requires the redundant E3 ubiquitin ligases Dma1 and Dma2, which were previously involved in spindle positioning and septin organization. In dma1 dma2 double mutants, formin distribution at polarity sites is impaired, thus causing defects in the organization of the actin cable network and hypersensitivity to the actin depolymerizer latrunculin B. Expression of a hyperactive variant of Bni1 (Bni1-V360D) rescues these defects and partially restores proper spindle positioning in the mutant, suggesting that the failure of dma1 dma2 mutant cells to position the spindle is partly due to faulty formin activity. Strikingly, Dma1/2 interact physically with both formins, while their ubiquitin-ligase activity is required for formin function and polarized localization. Thus, ubiquitylation of formin or a formin interactor(s) could promote formin binding to membrane and its ability to nucleate actin. Altogether, our data highlight a novel level of formin regulation that further expands our knowledge of the complex and multilayered controls of these key cytoskeleton organizers. KEYWORDS actin; formin; ubiquitylation; budding yeast T HE ability to polarize is a fundamental property of all types of cells, being crucial for numerous cellular processes such as proliferation, differentiation, and morphogenesis. Indeed, dysregulation of cell polarity can underlie developmental disorders and cancers (Wodarz and Nathke 2007). Cell polarization is strictly linked to the reorganization of the cytoskeleton and in particular of the actin network, whose dynamics must be tightly controlled for polarized processes to occur properly.The unicellular budding yeast Saccharomyces cerevisiae divides asymmetrically and undergoes highly polarized cell growth throughout its life cycle. Most aspects of polarized growth in budding yeast arise from a precise arrangement of the cortical actin cytoskeleton during the cell cycle. Three main actin structures can be found in yeast cells: (i) actin patches, which are sites of active endocytosis, (ii) actin cables, which serve as tracks for polarized secretion and segregation of organelles, and (iii) the contractile acto-myosin ring, which is involved in cytokinesis (Adams and Pringle 1984;Kilmartin and Adams 1984;Bi et ...

show abstract

Yeast Chfr homologs retard cell cycle at G₁and G₂/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination

Cited by 30 publications

References 58 publications

Chfr interacts and colocalizes with TCTP to the mitotic spindle

Chfr interacts and colocalizes with TCTP to the mitotic spindle

The Auto-ubiquitylation of E3 Ubiquitin-protein Ligase Chfr at G2 Phase Is Required for Accumulation of Polo-like Kinase 1 and Mitotic Entry in Mammalian Cells

Control of Formin Distribution and Actin Cable Assembly by the E3 Ubiquitin Ligases Dma1 and Dma2

Contact Info

Product

Resources

About

Yeast Chfr homologs retard cell cycle at G1and G2/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination

Cited by 30 publications

References 58 publications

Chfr interacts and colocalizes with TCTP to the mitotic spindle

Chfr interacts and colocalizes with TCTP to the mitotic spindle

The Auto-ubiquitylation of E3 Ubiquitin-protein Ligase Chfr at G2 Phase Is Required for Accumulation of Polo-like Kinase 1 and Mitotic Entry in Mammalian Cells

Control of Formin Distribution and Actin Cable Assembly by the E3 Ubiquitin Ligases Dma1 and Dma2

Contact Info

Product

Resources

About

Yeast Chfr homologs retard cell cycle at G₁and G₂/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination