Q936Phosphorylation Regulation of Overexpressed Protein Kinase Ypk1 in Budding Yeast

The CDK (cyclin-dependent kinase) family of enzymes is required for the G1-to-S-phase and G2-to-M-phase transitions during the cell-division cycle of eukaryotes. We have shown previously that the protein kinase CKII catalyses the phosphorylation of Ser-39 in Cdc2 during the G1 phase of the HeLa cell-division cycle [Russo, Vandenberg, Yu, Bae, Franza and Marshak (1992) J. Biol. Chem. 267, 20317–20325]. To identify a functional role for this phosphorylation, we have studied the homologous enzymes in the budding yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of Cdc2, Cdc28, contains a consensus CKII site (Ser-46), which is homologous with that of human Cdc2. Using in vitro kinase assays, metabolic labelling, peptide mapping and phosphoamino acid analysis, we demonstrate that this site is phosphorylated in Cdc28 in vivo as well in vitro. In addition, S. cerevisiae cells in which Ser-46 has been mutated to alanine show a decrease in both cell volume and protein content of 33%, and this effect is most pronounced in the stationary phase. Because cell size in S. cerevisiae is regulated primarily at the G1 stage, we suggest that CKII contributes to the regulation of the cell cycle in budding yeast by phosphorylation of Cdc28 as a checkpoint for G1 progression.

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MAD3 Encodes a Novel Component of the Spindle Checkpoint Which Interacts with Bub3p, Cdc20p, and Mad2p

The Journal of Cell Biology ◽

10.1083/jcb.148.5.871 ◽

2000 ◽

Vol 148 (5) ◽

pp. 871-882 ◽

Cited By ~ 175

Author(s):

Kevin G. Hardwick ◽

Raymond C. Johnston ◽

Dana L. Smith ◽

Andrew W. Murray

Keyword(s):

Cell Cycle ◽

Sequence Analysis ◽

Protein Kinase ◽

Nuclear Protein ◽

Budding Yeast ◽

Spindle Checkpoint ◽

Terminal Region ◽

Loss Of Function ◽

Checkpoint Proteins ◽

Two Hybrid

We show that MAD3 encodes a novel 58-kD nuclear protein which is not essential for viability, but is an integral component of the spindle checkpoint in budding yeast. Sequence analysis reveals two regions of Mad3p that are 46 and 47% identical to sequences in the NH2-terminal region of the budding yeast Bub1 protein kinase. Bub1p is known to bind Bub3p (Roberts et al. 1994) and we use two-hybrid assays and coimmunoprecipitation experiments to show that Mad3p can also bind to Bub3p. In addition, we find that Mad3p interacts with Mad2p and the cell cycle regulator Cdc20p. We show that the two regions of homology between Mad3p and Bub1p are crucial for these interactions and identify loss of function mutations within each domain of Mad3p. We discuss roles for Mad3p and its interactions with other spindle checkpoint proteins and with Cdc20p, the target of the checkpoint.

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Cks1 Is Required for G1Cyclin–Cyclin-Dependent Kinase Activity in Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.20.16.5858-5864.2000 ◽

2000 ◽

Vol 20 (16) ◽

pp. 5858-5864 ◽

Cited By ~ 39

Author(s):

Gregory J. Reynard ◽

William Reynolds ◽

Rati Verma ◽

Raymond J. Deshaies

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Budding Yeast ◽

Protein Kinase Activity ◽

Cyclin Dependent Kinase ◽

Multiple Substrates ◽

Cell Extracts

ABSTRACT p13suc1 (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity. However, the mechanism by which Cks influences the function of cyclin-CDK complexes has remained elusive. We show here that Cks1 is required for the protein kinase activity of budding yeast G1 cyclin-CDK complexes. Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G1cyclin-CDK complexes. In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G1 cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G1 cyclin-CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G1 phase in budding yeast.

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Phosphorylation and spindle pole body localization of the Cdc15p mitotic regulatory protein kinase in budding yeast

Current Biology ◽

10.1016/s0960-9822(00)00382-1 ◽

2000 ◽

Vol 10 (6) ◽

pp. 329-332 ◽

Cited By ~ 62

Author(s):

Shuichan Xu ◽

Han-Kuei Huang ◽

Peter Kaiser ◽

Martin Latterich ◽

Tony Hunter

Keyword(s):

Protein Kinase ◽

Budding Yeast ◽

Regulatory Protein ◽

Spindle Pole Body ◽

Spindle Pole ◽

Pole Body

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WITHDRAWN: The MCM2p-binding is crucial for the function of CDC7-DBF4 protein kinase during the initiation of chromosomal DNA replication in budding yeast

Journal of Biological Chemistry ◽

10.1074/jbc.m603586200 ◽

2006 ◽

Author(s):

W. Nakai

Keyword(s):

Dna Replication ◽

Protein Kinase ◽

Budding Yeast ◽

Chromosomal Dna

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Identification of cut8+ and cek1+, a novel protein kinase gene, which complement a fission yeast mutation that blocks anaphase.

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6361 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6361-6371 ◽

Cited By ~ 25

Author(s):

I Samejima ◽

M Yanagida

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Fission Yeast ◽

Gene Product ◽

Budding Yeast ◽

Essential Gene ◽

Restrictive Temperature ◽

Kinase Gene ◽

Yeast Saccharomyces Cerevisiae ◽

Novel Protein

The fission yeast Schizosaccharomyces pombe [corrected] temperature sensitivity cut8-563 mutation causes chromosome overcondensation and short spindle formation in the absence of sister chromatid separation. The cut8-563 mutation allows cytokinesis before the completion of anaphase, thus producing cells with a cut phenotype. The cut8+ gene product may be required for normal progression of anaphase. Diploidization occurs at the restrictive temperature, and 60 to 70% of the cells surviving after two generations are diploid. These phenotypes are reminiscent of those of budding yeast (Saccharomyces cerevisiae) ctf13 and ctf14 (ndc10) mutations. The cut8+ gene, isolated by complementation of the mutant, predicts a 262-amino-acid protein; the amino and carboxy domains are hydrophilic, while the central domain contains several hydrophobic stretches. It has a weak overall similarity to the budding yeast DBF8 gene product. DBF8 is an essential gene whose mutations result in delay in mitotic progression and chromosome instability. Anti-cut8 antibodies detect a 33-kDa polypeptide. Two multicopy suppressor genes for cut8-563 are identified. They are the cut1+ gene essential for nuclear division, and a new gene (designated cek1+) which encodes a novel protein kinase. The cek1+ gene product is unusually large (1,309 amino acids) and has a 112-amino-acid additional sequence in the kinase domain. The cek1+ gene is not an essential gene. Protein phosphorylation by cek1 may facilitate the progression of anaphase through direct or indirect interaction with the cut8 protein.

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