Mitotic Role for the Cdc28 Protein Kinase of Saccharomyces cerevisiae (original) (raw)
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The Journal of cell biology, 1987
The product of the Saccharomyces cerevisiae gene CDC28, a protein kinase required for initiation of the cell division cycle, was localized within yeast cells. By using immunofluorescence methods, the CDC28 product was shown to be primarily cytoplasmic in distribution. The gene product was localized largely to the particulate fraction by differential centrifugation after mechanical disruption in aqueous buffers. The particulate association was not affected by the presence of nonionic detergent. To refine this localization further, a procedure was developed for the preparation of yeast cytoplasmic matrices which resemble the cytoskeletons of vertebrate cells on the basis of methodology, immunochemistry, and gross ultrastructure. A portion of the CDC28 product was found Dr.
Molecular and Cellular Biology, 1989
Whereas the Cdc28 protein kinase of the budding yeast Saccharomyces cerevisiae plays an essential role in cell cycle progression during the G1 interval, a function in the progression from the G2 interval into M phase has been inferred for its homologs, including the Cdc2Hs protein kinase of humans. To better understand these apparently disparate roles, we constructed a yeast strain in which the resident CDC28 gene was replaced by its human homolog, CDC2Hs. This transgenic yeast strain was able to perform the G1 functions attributed to the Cdc28 protein kinase, including the ability to grow and divide normally, to respond to environmental signals that induce G1 arrest, and to regulate the Cdc2Hs protein kinase appropriately in response to these signals.
Molecular and Cellular Biology, 1989
The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for cell cycle initiation. In an attempt to identify genes encoding proteins that interact with the Cdc28 protein kinase, high-copy plasmid suppressors of a temperature-sensitive cdc28 mutation were isolated. One such suppressor, CKS1, was found to encode an 18-kilodalton protein that shared a high degree of homology with the suc1+ protein (p13) of Schizosaccharomyces pombe (67% amino acid sequence identity). Disruption of the chromosomal CKS1 gene conferred a G1 arrest phenotype similar to that of cdc28 mutants. The presence of the 18-kilodalton Cks1 protein in yeast lysates was demonstrated by using Cks-1 specific antiserum. Furthermore, the Cks1 protein was shown to be physically associated with active forms of the Cdc28 protein kinase. These data suggest that Cks1 is an essential component of the Cdc28 protein kinase complex.
Cdc28 activates exit from mitosis in budding yeast
The Journal of cell biology, 2000
The activity of the cyclin-dependent kinase 1 (Cdk1), Cdc28, inhibits the transition from anaphase to G1 in budding yeast. CDC28-T18V, Y19F (CDC28-VF), a mutant that lacks inhibitory phosphorylation sites, delays the exit from mitosis and is hypersensitive to perturbations that arrest cells in mitosis. Surprisingly, this behavior is not due to a lack of inhibitory phosphorylation or increased kinase activity, but reflects reduced activity of the anaphase-promoting complex (APC), a defect shared with other mutants that lower Cdc28/Clb activity in mitosis. CDC28-VF has reduced Cdc20- dependent APC activity in mitosis, but normal Hct1- dependent APC activity in the G1 phase of the cell cycle. The defect in Cdc20-dependent APC activity in CDC28-VF correlates with reduced association of Cdc20 with the APC. The defects of CDC28-VF suggest that Cdc28 activity is required to induce the metaphase to anaphase transition and initiate the transition from anaphase to G1 in budding yeast.
Journal of Cell Biology, 1987
The product of the Saccharomyces cerevisiae gene CDC28, a protein kinase required for initiation of the cell division cycle, was localized within yeast cells. By using immunofluorescence methods, the CDC28 product was shown to be primarily cytoplasmic in distribution. The gene product was localized largely to the particulate fraction by differential centrifugation after mechanical disruption in aqueous buffers. The particulate association was not affected by the presence of nonionic detergent. To refine this localization further, a procedure was developed for the preparation of yeast cytoplasmic matrices which resemble the cytoskeletons of vertebrate cells on the basis of methodology, immunochemistry, and gross ultrastructure. A portion of the CDC28 product was found to be tightly associated with these detergent-insoluble cytoplasmic matrices by both immunoflourescence and immunoblotting procedures. Although, for technical reasons, precise quantitation was not possible, it is estimated that a minimum of 2-15 % of the total CDC28 product pool is involved in the association with the insoluble matrix. Alcohol dehydrogenase, a soluble cytoplasmic protein, was found not to be associated with the cytoplasmic matrices at any detectable level, whereas, in contrast, ,o10--40% of the total cellular actin, a bonafide cytoskeletal protein, was present in these structures. The proportion of CDC28 gene product associated with the particulate fraction, and perhaps the insoluble matrix, appears to be substantially decreased during the preparation of spheroplasts.
Molecular and Cellular Biology, 1996
Entry into mitosis requires activation of cdc2 kinase brought on by its association with cyclin B, phosphorylation of the conserved threonine (Thr-167 in Schizosaccharomyces pombe) in the T loop, and dephosphorylation of the tyrosine residue at position 15. Exit from mitosis, on the other hand, is induced by inactivation of cdc2 activity via cyclin destruction. It has been suggested that in addition to cyclin degradation, dephosphorylation of Thr-167 may also be required for exit from the M phase. Here we show that Saccharomyces cerevisiae cells expressing cdc28-E169 (a CDC28 allele in which the equivalent threonine, Thr-169, has been replaced by glutamic acid) are able to degrade mitotic cyclin Clb2, inactivate the Cdc28/Clb2 kinase, and disassemble the anaphase spindles, suggesting that they exit mitosis normally. The cdc28-E169 allele is active with respect to its mitotic functions, since it complements the mitosis-defective cdc28-1N allele. Whereas replacement of Thr-169 with se...
The EMBO Journal, 1993
It is widely assumed that degradation of mitotic cyclins causes a decrease in mitotic cdc2/CDC28 kinase activity and thereby triggers the metaphase to anaphase transition. Two observations made on the budding yeast Saccharomcyces cerevisiae are inconsistent with this scenario: (i) anaphase occurs in the presence of high levels of kinase in cdc15 mutants and (ii) overproduction of a B-type mitotic cyclin causes arrest not in metaphase as previously reported but in telophase. Kinase destruction is therefore implicated in the exit from mitosis rather than the entry into anaphase. The behaviour of espi mutants shows in addition that kinase destruction can occur in the absence of anaphase completion. The execution of anaphase and the destruction of CDC28 kinase activity therefore appear to take place independently of one another.
Molecular and Cellular Biology, 1999
Saccharomyces cerevisiaeproteins Cdc4 and Cdc20 contain WD40 repeats and participate in proteolytic processes. However, they are thought to act at two different stages of the cell cycle: Cdc4 is involved in the proteolysis of the Cdk inhibitor, Sic1, necessary for G1/S transition, while Cdc20 mediates anaphase-promoting complex-dependent degradation of anaphase inhibitor Pds1, a process necessary for the onset of chromosome segregation. We have isolated three mutant alleles ofCDC4(cdc4-10,cdc4-11, andcdc4-16) which suppress the nuclear division defect ofcdc20-1cells. However, the previously characterized mutationcdc4-1and a new allele,cdc4-12, do not alleviate the defect ofcdc20-1cells. This genetic interaction suggests an additional role for Cdc4 in G2/M. Reexamination of thecdc4-1mutant revealed that, in addition to being defective in the onset of S phase, it is also defective in G2/M transition when released from hydroxyurea-induced S-phase arrest. A second function forCDC4in lat...
Current Biology, 2000
Cdc15p is an essential protein kinase and functions with a group of late mitotic proteins that includes Lte1p, Tem1p, Cdc14p and Dbf2p/Dbf20p to inactivate Cdc28p-Clb2p at the end of mitosis in budding yeast [1,2]. Cdc14p is activated and released from the nucleolus at late anaphase/telophase to dephosphorylate important regulators of Cdc28p-Clb2p such as Hct1p/Cdh1p, Sic1p and Swi5p in a CDC15-dependent manner [3-7]. How Cdc15p itself is regulated is not known. Here, we report that both the phosphorylation and localization of Cdc15p are cell cycle regulated. The extent of phosphorylation of Cdc15p gradually increases during cell-cycle progression until some point during late anaphase/telophase when it is rapidly dephosphorylated. We provide evidence suggesting that Cdc14p is the phosphatase responsible for the dephosphorylation of Cdc15p. Using a Cdc15p fusion protein coupled at its carboxyl terminus to green fluorescent protein (GFP), we found that Cdc15p, like its homologue Cdc7p [8] in fission yeast, localizes to the spindle pole bodies (SPBs) during mitosis. At the end of telophase, a portion of Cdc15p is located at the mother-bud neck, suggesting a possible role for Cdc15p in cytokinesis.