SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities.

Verma, Rati; Feldman, R. M. Renny; Deshaies, Raymond J.

doi:10.1091/mbc.8.8.1427

Cited by 160 publications

(167 citation statements)

References 39 publications

(65 reference statements)

Supporting

Mentioning

164

Contrasting

Order By: Relevance

“…Presumably, this unphosphorylated Cdc6 can then reload onto chromatin just as if it were Cdc6*. In our studies, wild-type Cdc6 was only able to reload when Cdc6 expression was sufficiently high to cause an abnormal bud morphology suggestive of inhibition of CDK activity (Nugroho and Mendenhall, 1994;Elsasser et al, 1996;Verma et al, 1997). Expression of Cdc6* had relatively little effect on bud morphology, presumably because Cdc6* does not bind Cdc28 very well (Wolf et al, 1999;Mimura et al, 2004;Supplementary Figure 10) and so cannot inhibit it.…”

Section: Discussionmentioning

confidence: 66%

Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

Honey

Futcher

2007

MBoC

View full text Add to dashboard Cite

The Saccharomyces cerevisiae Cdc6 protein is crucial for DNA replication. In the absence of cyclin-dependent kinase (CDK) activity, Cdc6 binds to replication origins, and loads Mcm proteins. In the presence of CDK activity, Cdc6 does not bind to origins, and this helps prevent rereplication. CDK activity affects Cdc6 function by multiple mechanisms: CDK activity affects transcription of CDC6, degradation of Cdc6, nuclear import of Cdc6, and binding of Cdc6 to Clb2. Here we examine some of these mechanisms individually. We find that when Cdc6 is forced into the nucleus during late G1 or S, it will not substantially reload onto chromatin no matter whether its CDK sites are present or not. In contrast, at a G2/M nocodazole arrest, Cdc6 will reload onto chromatin if and only if its CDK sites have been removed. Trace amounts of nonphosphorylatable Cdc6 are dominant lethal in strains bearing nonphosphorylatable Orc2 and Orc6, apparently because of rereplication. This synthetic dominant lethality occurs even in strains with wild-type MCM genes. Nonphosphorylatable Cdc6, or Orc2 and Orc6, sensitize cells to rereplication caused by overexpression of various replication initiation proteins such as Dpb11 and Sld2.

show abstract

Section: Discussionmentioning

confidence: 66%

Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

Honey

Futcher

2007

MBoC

View full text Add to dashboard Cite

show abstract

“…The conservation of the p27-binding domain is surprising, given that S. cerevisiae does not contain a p27 homolog detectable by BLAST search. The Clb-Cdc28p kinase inhibitor Sic1p has been proposed to bind to Clb5p via a C-terminal sequence with some distant similarity to the cyclin A-interacting regions of p27 and other cyclin A targets (15,39). We tested the ability of Sic1p to bind to Clb5p with and without a functional p27-binding domain.…”

Section: Resultsmentioning

confidence: 99%

“…3) is consistent with this idea, although such an interaction is clearly not required for binding and kinase inhibition. Candidate RXL-like sequences in an important C-terminal region of Sic1p have been described (15,39).…”

Section: Specific Cyclin Requirements For the Budding Yeast Cell Cyclementioning

confidence: 99%

Conservation and Function of a Potential Substrate-Binding Domain in the Yeast Clb5 B-Type Cyclin

Cross

Jacobson

2000

Molecular and Cellular Biology

View full text Add to dashboard Cite

Cyclin A contains a region implicated in binding to the p27 inhibitor and to substrates. There is strong evolutionary conservation of surface residues contributing to this region in many cyclins, including yeast B-type cyclins, despite the absence of a yeast p27 homolog. The yeast S-phase B-type cyclin Clb5p interacted with mammalian p27 in a two-hybrid assay. This interaction was disrupted by mutations designed to disrupt hydrophobic interactions (hpm mutation) or hydrogen bonding (Q241A mutation) based on the cyclin A-p27 crystal structure. In contrast, mutation of the Clb5p p27-binding domain only slightly reduced binding and inhibition by the Sic1p Clb-Cdc28p kinase inhibitor. Mutations disrupting the p27-binding domain strongly reduced Clb5p biological activity in diverse assays without reducing Clb5p-associated kinase activity. An analogous hpm mutation in the mitotic cyclin Clb2p reduced mitotic function, but in some assays this mutation increased the ability of Clb2p to perform functions normally restricted to Clb5p. These results support the idea of a modular, structurally conserved cyclin domain involved in substrate targeting.Cyclin-dependent kinases (Cdks) drive key events in the eukaryotic cell cycle. Most eukaryotes contain multiple cyclin genes, expressed at different times in the cell cycle and appearing to control distinct cell cycle events. It has been proposed that simple oscillation of a single generic Cdk activity could account for cell cycle regulation (23,35). However, recent observations (reviewed in reference 25) suggest that such models do not take into account strong differences in the efficiency with which different cyclins carry out different biological roles (for example, see references 8, 13, and 18). The molecular basis for such cyclin specificity remains unclear. We proposed recently that a candidate substrate "docking" site characterized in cyclin A (5, 29) might be evolutionarily conserved in many cyclins (8). Here we show that this docking site is functionally conserved in the yeast B-type cyclin Clb5p, both for the ability to bind specific proteins (the mammalian inhibitors p21 and p27) and for biological function in a range of assays presumably requiring interaction with diverse Clb5p substrates. These findings suggest that evolution of this region on the cyclin surface occurred during primordial eukaryotic evolution (predating the separation of yeast and metazoans) and probably arose as a substrate-binding domain rather than as a domain for binding inhibitors. In eukaryotic systems with multiple cyclins due to gene duplication, this region could then diverge to give different binding specificity to different cyclins, resulting in sharp differences in the biological efficiency of driving specific cell cycle events. MATERIALS AND METHODSYeast strains. A clb3,4,5,6 GAL::CLB5 strain in the W303 background (K3418 [31]; provided by K. Nasmyth) was converted to ura3 by selecting hisG-URA3-hisG popouts on 5-fluoroorotic acid (5-FOA), to make K3418-1. ϫ1924, a diploid clb3,4,5,6 pGA...

show abstract

“…The E3 ubiquitin ligase involved in promoting G1?S transition is referred to SCF complex, consisting of Skp1, Cdc53/cullin, F box proteins (Cdc4, Grr1, etc) King et al, 1996;Skowyra et al, 1997). Through F-box protein Cdc4, SCF promotes ubiquitination and degradation of SIC1, a negative regulator of G1 progression, leading to activation of Cdc28P/cyclin B that trigger the initiation of DNA synthesis Verma et al, 1997). On the other hand, through F box protein Grr1, SCF induces ubiquitination of G1 cyclin CLN1/2 to limit their accumulation (Deshaies et al, 1995; Hadwiger et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation

Swaroop¹,

Wang²,

Miller³

et al. 2000

Oncogene

View full text Add to dashboard Cite

In an attempt to understand the signaling pathway mediating redox-induced apoptosis, we cloned SAG, an evolutionarily conserved zinc RING ®nger gene that, when overexpressed, protects cells from apoptosis induced by redox agents. Here we report functional characterization of SAG by the use of yeast genetics approach. Targeted disruption of ySAG, yeast homolog of human SAG, and subsequent tetrad analysis revealed that ySAG is required for yeast viability. Complementation experiment showed that the lethal phenotype induced by the ySAG deletion is fully rescued by wildtype SAG, but not by several hSAG mutants. Complementation experiment has also con®rmed that ySAG is essential for normal vegetative growth, rather than being required for sporulation. Furthermore, cell death induced by SAG deletion was accompanied by cell enlargement and abnormal cell cycle pro®ling with an increased DNA content. Importantly, SAG was found to be the second family member of Rbx (RING box protein) or ROC (Regulator of cullins) or Hrt that is a component of SCF E3 ubiquitin ligase. Indeed, like ROC1/Rbx1/Hrt1, SAG binds to Cul1 and SAG-Cul1 complex has ubiquitin ligase activity to promote poly-ubiquitination of E2/ Cdc34. This ligase activity is required for complementation of death phenotype induced by ySAG disruption. Finally, chip pro®ling of the entire yeast genome revealed induction of several G1/S as well as G2/M checkpoint control genes upon SAG withdrawal. Thus, SAG appears to control cell cycle progression in yeast by promoting ubiquitination and degradation of cell cycle regulatory proteins.

show abstract

SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities.

Cited by 160 publications

References 39 publications

Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

Conservation and Function of a Potential Substrate-Binding Domain in the Yeast Clb5 B-Type Cyclin

Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation

Contact Info

Product

Resources

About