scholarly journals Saccharomyces cerevisiae Ime2 phosphorylates Sic1 at multiple PXS/T sites but is insufficient to trigger Sic1 degradation

2006 ◽  
Vol 399 (1) ◽  
pp. 151-160 ◽  
Author(s):  
Chantelle Sedgwick ◽  
Matthew Rawluk ◽  
James Decesare ◽  
Sheetal Raithatha ◽  
James Wohlschlegel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phase Boundary ◽  
S Phase ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase ◽  
Proliferating Cells ◽  
Initiation Of Dna Replication ◽  
Cdk Phosphorylation

The initiation of DNA replication in Saccharomyces cerevisiae depends upon the destruction of the Clb–Cdc28 inhibitor Sic1. In proliferating cells Cln–Cdc28 complexes phosphorylate Sic1, which stimulates binding of Sic1 to SCFCdc4 and triggers its proteosome mediated destruction. During sporulation cyclins are not expressed, yet Sic1 is still destroyed at the G1-/S-phase boundary. The Cdk (cyclin dependent kinase) sites are also required for Sic1 destruction during sporulation. Sic1 that is devoid of Cdk phosphorylation sites displays increased stability and decreased phosphorylation in vivo. In addition, we found that Sic1 was modified by ubiquitin in sporulating cells and that SCFCdc4 was required for this modification. The meiosis-specific kinase Ime2 has been proposed to promote Sic1 destruction by phosphorylating Sic1 in sporulating cells. We found that Ime2 phosphorylates Sic1 at multiple sites in vitro. However, only a subset of these sites corresponds to Cdk sites. The identification of multiple sites phosphorylated by Ime2 has allowed us to propose a motif for phosphorylation by Ime2 (PXS/T) where serine or threonine acts as a phospho-acceptor. Although Ime2 phosphorylates Sic1 at multiple sites in vitro, the modified Sic1 fails to bind to SCFCdc4. In addition, the expression of Ime2 in G1 arrested haploid cells does not promote the destruction of Sic1. These data support a model where Ime2 is necessary but not sufficient to promote Sic1 destruction during sporulation.

scholarly journals Phosphorylation of Sic1, a Cyclin-dependent Kinase (Cdk) Inhibitor, by Cdk Including Pho85 Kinase Is Required for Its Prompt Degradation

1998 ◽  
Vol 9 (9) ◽  
pp. 2393-2405 ◽  
Author(s):  
Masafumi Nishizawa ◽  
Masaoki Kawasumi ◽  
Marie Fujino ◽  
Akio Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Cdk Inhibitor ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cdk Phosphorylation ◽  
Cdc28 Kinase

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 andCLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable inpho85Δ cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.

scholarly journals Mutation of Cyclin/cdk Phosphorylation Sites in HsCdc6 Disrupts a Late Step in Initiation of DNA Replication in Human Cells

2000 ◽  
Vol 11 (12) ◽  
pp. 4117-4130 ◽  
Author(s):  
Utz Herbig ◽  
Jason W. Griffith ◽  
Ellen Fanning
Keyword(s):  
Dna Replication ◽  
Inhibitory Effect ◽  
Human Cells ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinases ◽  
Late Step ◽  
Initiation Of Dna Replication ◽  
Cdk Phosphorylation

Cyclin-dependent kinases (Cdk) are essential for promoting the initiation of DNA replication, presumably by phosphorylating key regulatory proteins that are involved in triggering the G1/S transition. Human Cdc6 (HsCdc6), a protein required for initiation of DNA replication, is phosphorylated by Cdk in vitro and in vivo. Here we report that HsCdc6 with mutations at potential Cdk phosphorylation sites was poorly phosphorylated in vitro by Cdk, but retained all other biochemical activities of the wild-type protein tested. Microinjection of mutant HsCdc6 proteins into human cells blocked initiation of DNA replication or slowed S phase progression. The inhibitory effect of mutant HsCdc6 was lost at the G1/S transition, indicating that phosphorylation of HsCdc6 by Cdk is critical for a late step in initiation of DNA replication in human cells.

scholarly journals A CDK-regulated chromatin segregase promoting chromosome replication

2020 ◽  
Author(s):  
Erika Chacin ◽  
Priyanka Bansal ◽  
Karl-Uwe Reusswig ◽  
Luis M. Diaz-Santin ◽  
Pedro Ortega ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Genome Instability ◽  
Chromosome Replication ◽  
S Phase ◽  
Chromatin Dynamics ◽  
Replicative Stress ◽  
Cdk Phosphorylation ◽  
Nucleosome Disassembly

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood.Here, we report the identification of a novel class of chromatin remodeling enzyme, entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+-ATPase that assembles into ~ 1 MDa hexameric complexes capable of segregating histones from DNA. Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that Yta7’s enzymatic activity is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication.Our results present a novel mechanism of how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.

scholarly journals Functional Interactions between Herpesvirus Oncoprotein MEQ and Cell Cycle Regulator CDK2

1999 ◽  
Vol 73 (5) ◽  
pp. 4208-4219 ◽  
Author(s):  
Juinn-Lin Liu ◽  
Ying Ye ◽  
Zheng Qian ◽  
Yongyi Qian ◽  
Dennis J. Templeton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Phosphorylation Site ◽  
Fibroblast Cell ◽  
S Phase ◽  
Morphological Transformation ◽  
Necrosis Factor Alpha ◽  
Coiled Bodies ◽  
Cdk Phosphorylation

ABSTRACT Marek’s disease virus, an avian alphaherpesvirus, has been used as an excellent model to study herpesvirus oncogenesis. One of its potential oncogenes, MEQ, has been demonstrated to transform a rodent fibroblast cell line, Rat-2, in vitro by inducing morphological transformation and anchorage- and serum-independent growth and by protecting cells from apoptosis induced by tumor necrosis factor alpha, C2-ceramide, UV irradiation, or serum deprivation. In this report, we show that there is a cell cycle-dependent colocalization of MEQ protein and cyclin-dependent kinase 2 (CDK2) in coiled bodies and the nucleolar periphery during the G1/S boundary and early S phase. To our knowledge, this is the first demonstration that CDK2 is found to localize to coiled bodies. Such an in vivo association and possibly subsequent phosphorylation may result in the cytoplasmic translocation of MEQ protein. Indeed, MEQ is expressed in both the nucleus and the cytoplasm during the G1/S boundary and early S phase. In addition, we were able to show in vitro phosphorylation of MEQ by CDKs. We have mapped the CDK phosphorylation site of MEQ to be serine 42, a residue in the proximity of the bZIP domain. An indirect-immunofluorescence study of the MEQ S42D mutant, in which the CDK phosphorylation site was mutated to a charged residue, reveals more prominent cytoplasmic localization. This lends further support to the notion that the translocation of MEQ is regulated by phosphorylation. Furthermore, phosphorylation of MEQ by CDKs drastically reduces the DNA binding activity of MEQ, which may in part account for the lack of retention of MEQ oncoprotein in the nucleus. Interestingly, the localization of CDK2 in coiled bodies and the nucleolar periphery is observed only in MEQ-transformed Rat-2 cells, implicating MEQ in modifying the subcellular localization of CDK2. Taken together, our data suggest that there is a novel reciprocal modulation between the herpesvirus oncoprotein MEQ and CDK2.

scholarly journals Cdk Phosphorylation of a Nucleoporin Controls Localization of Active Genes through the Cell Cycle

2010 ◽  
Vol 21 (19) ◽  
pp. 3421-3432 ◽  
Author(s):  
Donna Garvey Brickner ◽  
Jason H. Brickner
Keyword(s):  
Cell Cycle ◽  
Nuclear Periphery ◽  
S Phase ◽  
Nuclear Pore ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Initiation Of Dna Replication ◽  
Cdk Phosphorylation ◽  
Inducible Genes ◽  
Active Genes

Many inducible genes in yeast are targeted to the nuclear pore complex when active. We find that the peripheral localization of the INO1 and GAL1 genes is regulated through the cell cycle. Active INO1 and GAL1 localized at the nuclear periphery during G1, became nucleoplasmic during S-phase, and then returned to the nuclear periphery during G2/M. Loss of peripheral targeting followed the initiation of DNA replication and was lost in cells lacking a cyclin-dependent kinase (Cdk) inhibitor. Furthermore, the Cdk1 kinase and two Cdk phosphorylation sites in the nucleoporin Nup1 were required for peripheral targeting of INO1 and GAL1. Introduction of aspartic acid residues in place of either of these two sites in Nup1 bypassed the requirement for Cdk1 and resulted in targeting of INO1 and GAL1 to the nuclear periphery during S-phase. Thus, phosphorylation of a nuclear pore component by cyclin dependent kinase controls the localization of active genes to the nuclear periphery through the cell cycle.

scholarly journals A CDK-regulated chromatin segregase promoting chromosome replication

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Erika Chacin ◽  
Priyanka Bansal ◽  
Karl-Uwe Reusswig ◽  
Luis M. Diaz-Santin ◽  
Pedro Ortega ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Genome Instability ◽  
Chromosome Replication ◽  
S Phase ◽  
Chromatin Dynamics ◽  
Replicative Stress ◽  
Cdk Phosphorylation ◽  
Nucleosome Disassembly

AbstractThe replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme—Yta7—entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into ~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.

scholarly journals Rad53 Checkpoint Kinase Phosphorylation Site Preference Identified in the Swi6 Protein of Saccharomyces cerevisiae

2003 ◽  
Vol 23 (10) ◽  
pp. 3405-3416 ◽  
Author(s):  
Julia M. Sidorova ◽  
Linda L. Breeden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Phosphorylation Site ◽  
Site Preference ◽  
Phosphorylation Sites ◽  
Checkpoint Kinase ◽  
Checkpoint Signaling ◽  
And Function

ABSTRACT Rad53 of Saccharomyces cerevisiae is a checkpoint kinase whose structure and function are conserved among eukaryotes. When a cell detects damaged DNA, Rad53 activity is dramatically increased, which ultimately leads to changes in DNA replication, repair, and cell division. Despite its central role in checkpoint signaling, little is known about Rad53 substrates or substrate specificity. A number of proteins are implicated as Rad53 substrates; however, the evidence remains indirect. Previously, we have provided evidence that Swi6, a subunit of the Swi4/Swi6 late-G1-specific transcriptional activator, is a substrate of Rad53 in the G1/S DNA damage checkpoint. In the present study we identify Rad53 phosphorylation sites in Swi6 in vitro and demonstrate that at least one of them is targeted by Rad53 in vivo. Mutations in these phosphorylation sites in Swi6 shorten but do not eliminate the Rad53-dependent delay of the G1-to-S transition after DNA damage. We derive a consensus for Rad53 site preference at positions −2 and +2 (−2/+2) and identify its potential substrates in the yeast proteome. Finally, we present evidence that one of these candidates, the cohesin complex subunit Scc1 undergoes DNA damage-dependent phosphorylation, which is in part dependent on Rad53.

Functionally homologous DNA replication genes in fission and budding yeast

1999 ◽  
Vol 112 (14) ◽  
pp. 2381-2390
Author(s):  
M. Sanchez ◽  
A. Calzada ◽  
A. Bueno
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Kinase Activity ◽  
Budding Yeast ◽  
S Phase ◽  
Yeast Gene ◽  
Cdc2 Kinase ◽  
Initiation Of Dna Replication

The cdc18(+) gene of the fission yeast Schizosaccharomyces pombe is involved in the initiation of DNA replication as well as in coupling the S phase to mitosis. In this work, we show that the Saccharomyces cerevisiae CDC6 gene complements cdc18-K46 ts and cdc18 deletion mutant S. pombe strains. The budding yeast gene suppresses both the initiation and the checkpoint defects associated with the lack of cdc18(+). The Cdc6 protein interacts in vivo with Cdc2 kinase complexes. Interestingly, Cdc6 is an in vitro substrate for Cdc13/Cdc2 and Cig1/Cdc2, but not for Cig2/Cdc2-associated kinases. Overexpression of Cdc6 in fission yeast induces multiple rounds of S-phase in the absence of mitosis and cell division. This CDC6-dependent continuous DNA synthesis phenotype is independent of the presence of a functional cdc18(+) gene product and, significantly, requires only Cig2/Cdc2-associated kinase activity. Finally, these S. pombe over-replicating cells do not require any protein synthesis other than that of Cdc6. Our data strongly suggest that CDC6 and cdc18(+) are functional homologues and also support the idea that controls restricting genome duplication diverge in fission and budding yeast.

scholarly journals A Heterodimer of Thioredoxin and IB2 Cooperates with Sec18p (NSF) to Promote Yeast Vacuole Inheritance

1997 ◽  
Vol 136 (2) ◽  
pp. 299-306 ◽  
Author(s):  
Zuoyu Xu ◽  
Andreas Mayer ◽  
Eric Muller ◽  
William Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Early Stage ◽  
Redox Chemistry ◽  
Soluble Proteins ◽  
S Phase ◽  
Cell Extracts ◽  
Vacuole Inheritance ◽  
Cytosolic Inhibitor

Early in S phase, the vacuole (lysosome) of Saccharomyces cerevisiae projects a stream of vesicles and membranous tubules into the bud where they fuse and establish the daughter vacuole. This inheritance reaction can be studied in vitro with isolated vacuoles. Rapid and efficient homotypic fusion between saltwashed vacuoles requires the addition of only two purified soluble proteins, Sec18p (NSF) and LMA1, a novel heterodimer with a thioredoxin subunit. We now report the identity of the second subunit of LMA1 as IB2, a previously identified cytosolic inhibitor of vacuolar proteinase B. Both subunits are needed for efficient vacuole inheritance in vivo and for the LMA1 activity in cell extracts. Each subunit acts via a novel mechanism, as the thioredoxin subunit is not acting through redox chemistry and LMA1 is still needed for the fusion of vacuoles which do not contain proteinase B. Both Sec18p and LMA1 act at an early stage of the in vitro reaction. Though LMA1 does not stimulate Sec18p-mediated Sec17p release, LMA1 cannot fulfill its function before Sec18p. Upon Sec17p/Sec18p action, vacuoles become labile but are rapidly stabilized by LMA1. The action of LMA1 and Sec18p is thus coupled and ordered. These data establish LMA1 as a novel factor in trafficking of yeast vacuoles.

scholarly journals Cdc34 C-terminal tail phosphorylation regulates Skp1/cullin/F-box (SCF)-mediated ubiquitination and cell cycle progression

2007 ◽  
Vol 405 (3) ◽  
pp. 569-581 ◽  
Author(s):  
Martin Sadowski ◽  
Amanda Mawson ◽  
Rohan Baker ◽  
Boris Sarcevic
Keyword(s):  
Cell Cycle ◽  
Catalytic Activity ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Phosphorylation Sites ◽  
Tail Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cycle Progression

The ubiquitin-conjugating enzyme Cdc34 (cell division cycle 34) plays an essential role in promoting the G1–S-phase transition of the eukaryotic cell cycle and is phosphorylated in vivo. In the present study, we investigated if phosphorylation regulates Cdc34 function. We mapped the in vivo phosphorylation sites on budding yeast Cdc34 (yCdc34; Ser207 and Ser216) and human Cdc34 (hCdc34 Ser203, Ser222 and Ser231) to serine residues in the acidic tail domain, a region that is critical for Cdc34's cell cycle function. CK2 (protein kinase CK2) phosphorylates both yCdc34 and hCdc34 on these sites in vitro. CK2-mediated phosphorylation increased yCdc34 ubiquitination activity towards the yeast Saccharomyces cerevisiae Sic1 in vitro, when assayed in the presence of its cognate SCFCdc4 E3 ligase [where SCF is Skp1 (S-phase kinase-associated protein 1)/cullin/F-box]. Similarly, mutation of the yCdc34 phosphorylation sites to alanine, aspartate or glutamate residues altered Cdc34–SCFCdc4-mediated Sic1 ubiquitination activity. Similar results were obtained when yCdc34's ubiquitination activity was assayed in the absence of SCFCdc4, indicating that phosphorylation regulates the intrinsic catalytic activity of Cdc34. To evaluate the in vivo consequences of altered Cdc34 activity, wild-type yCdc34 and the phosphosite mutants were introduced into an S. cerevisiae cdc34 deletion strain and, following synchronization in G1-phase, progression through the cell cycle was monitored. Consistent with the increased ubiquitination activity in vitro, cells expressing the phosphosite mutants with higher catalytic activity exhibited accelerated cell cycle progression and Sic1 degradation. These studies demonstrate that CK2-mediated phosphorylation of Cdc34 on the acidic tail domain stimulates Cdc34–SCFCdc4 ubiquitination activity and cell cycle progression.

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