scholarly journals Activating Phosphorylation of the Kin28p Subunit of Yeast TFIIH by Cak1p

1999 ◽  
Vol 19 (7) ◽  
pp. 4774-4787 ◽  
Author(s):  
Jonathan Kimmelman ◽  
Philipp Kaldis ◽  
Christoph J. Hengartner ◽  
Geoffrey M. Laff ◽  
Sang Seok Koh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Large Subunit ◽  
Conditional Allele ◽  
Assembly Factor ◽  
Biochemical Observation

ABSTRACT Cyclin-dependent kinase (CDK)-activating kinases (CAKs) carry out essential activating phosphorylations of CDKs such as Cdc2 and Cdk2. The catalytic subunit of mammalian CAK, MO15/Cdk7, also functions as a subunit of the general transcription factor TFIIH. However, these functions are split in budding yeast, where Kin28p functions as the kinase subunit of TFIIH and Cak1p functions as a CAK. We show that Kin28p, which is itself a CDK, also contains a site of activating phosphorylation on Thr-162. The kinase activity of a T162A mutant of Kin28p is reduced by ∼75 to 80% compared to that of wild-type Kin28p. Moreover, cells containing kin28T162A and a conditional allele of TFB3 (the ortholog of the mammalian MAT1 protein, an assembly factor for MO15 and cyclin H) are severely compromised and display a significant further reduction in Kin28p activity. This finding provides in vivo support for the previous biochemical observation that MO15-cyclin H complexes can be activated either by activating phosphorylation of MO15 or by binding to MAT1. Finally, we show that Kin28p is no longer phosphorylated on Thr-162 following inactivation of Cak1p in vivo, that Cak1p can phosphorylate Kin28p on Thr-162 in vitro, and that this phosphorylation stimulates the CTD kinase activity of Kin28p. Thus, Kin28p joins Cdc28p, the major cell cycle Cdk in budding yeast, as a physiological Cak1p substrate. These findings indicate that although MO15 and Cak1p constitute different forms of CAK, both control the cell cycle and the phosphorylation of the C-terminal domain of the large subunit of RNA polymerase II by TFIIH.

scholarly journals Drosophila Cdk8, a kinase partner of cyclin C that interacts with the large subunit of RNA polymerase II.

1996 ◽  
Vol 7 (4) ◽  
pp. 505-513 ◽  
Author(s):  
V Leclerc ◽  
J P Tassan ◽  
P H O'Farrell ◽  
E A Nigg ◽  
P Léopold
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Eukaryotic Cell ◽  
Knock Out ◽  
Cyclin C

A number of cyclins have been described, most of which act together with their catalytic partners, the cyclin-dependent kinases (Cdks), to regulate events in the eukaryotic cell cycle. Cyclin C was originally identified by a genetic screen for human and Drosophila cDNAs that complement a triple knock-out of the CLN genes in Saccharomyces cerevisiae. Unlike other cyclins identified in this complementation screen, there has been no evidence that cyclin C has a cell-cycle role in the cognate organism. Here we report that cyclin C is a nuclear protein present in a multiprotein complex. It interacts both in vitro and in vivo with Cdk8, a novel protein-kinase of the Cdk family, structurally related to the yeast Srb10 kinase. We also show that Cdk8 can interact in vivo with the large subunit of RNA polymerase II and that a kinase activity that phosphorylates the RNA polymerase II large subunit is present in Cdk8 immunoprecipitates. Based on these observations and sequence similarity to the kinase/cyclin pair Srb10/Srb11 in S. cerevisiae, we suggest that cyclin C and Cdk8 control RNA polymerase II function.

scholarly journals KIN28 encodes a C-terminal domain kinase that controls mRNA transcription in Saccharomyces cerevisiae but lacks cyclin-dependent kinase-activating kinase (CAK) activity.

1995 ◽  
Vol 15 (6) ◽  
pp. 2983-2992 ◽  
Author(s):  
M J Cismowski ◽  
G M Laff ◽  
M J Solomon ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Kinase Activity ◽  
Cyclin Dependent Kinase ◽  
Mrna Transcription ◽  
Terminal Domain ◽  
Cdk Activating Kinase

The Saccharomyces cerevisiae gene KIN28 is a member of the cyclin-dependent kinase (CDK) family. The Kin28 protein shares extensive sequence identity with the vertebrate CDK-activating kinase MO15 (Cdk7), which phosphorylates CDKs in vitro on a critical threonine residue. Kin28 and MO15 have recently been found to copurify with the transcription factor IIH (TFIIH) holoenzyme of yeast and human cells, respectively. Although TFIIH is capable of phosphorylating the C-terminal domain (CTD) of RNA polymerase II, it has been unclear whether Kin28 is the physiologically relevant CTD kinase or what role CTD phosphorylation plays in transcription. In this study, we used a thermosensitive allele of KIN28 and a hemagglutinin epitope-tagged Kin28 protein to investigate Kin28 function in transcription and in the cell cycle. We show that Kin28 acts as a positive regulator of mRNA transcription in vivo and possesses CTD kinase activity in vitro. However, Kin28 neither regulates the phosphorylation state of the yeast cell cycle CDK, Cdc28, nor possesses CDK-activating kinase activity in vitro. We conclude that Kin28 is a strong candidate for the physiological CTD kinase of S. cerevisiae and that Kin28 function is required for mRNA transcription.

scholarly journals Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.

1993 ◽  
Vol 13 (5) ◽  
pp. 2899-2908 ◽  
Author(s):  
A L Jackson ◽  
P M Pahl ◽  
K Harrison ◽  
J Rosamond ◽  
R A Sclafani
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Protein Interactions ◽  
Kinase Activity ◽  
Mitotic Cell ◽  
Common Point ◽  
Temperature Sensitive ◽  
Cdc7 Kinase

Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the G1/S boundary by using either cultures synchronized with alpha factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob1-1, which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob1-1 mutation cannot bypass all events in G1 phase because it fails to suppress temperature-sensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.

scholarly journals Specific Inhibition of Elm1 Kinase Activity Reveals Functions Required for Early G1 Events

2003 ◽  
Vol 23 (17) ◽  
pp. 6327-6337 ◽  
Author(s):  
Aparna Sreenivasan ◽  
Anthony C. Bishop ◽  
Kevan M. Shokat ◽  
Douglas R. Kellogg
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Growth ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Specific Inhibition ◽  
Bud Emergence ◽  
Wee1 Kinase ◽  
Yeast Homolog

ABSTRACT In budding yeast, the Elm1 kinase is required for coordination of cell growth and cell division at G2/M. Elm1 is also required for efficient cytokinesis and for regulation of Swe1, the budding yeast homolog of the Wee1 kinase. To further characterize Elm1 function, we engineered an ELM1 allele that can be rapidly and selectively inhibited in vivo. We found that inhibition of Elm1 kinase activity during G2 results in a phenotype similar to the phenotype caused by deletion of the ELM1 gene, as expected. However, inhibition of Elm1 kinase activity earlier in the cell cycle results in a prolonged G1 delay. The G1 requirement for Elm1 kinase activity occurs before bud emergence, polarization of the septins, and synthesis of G1 cyclins. Inhibition of Elm1 kinase activity during early G1 also causes defects in the organization of septins, and inhibition of Elm1 kinase activity in a strain lacking the redundant G1 cyclins CLN1 and CLN2 is lethal. These results demonstrate that the Elm1 kinase plays an important role in G1 events required for bud emergence and septin organization.

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

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

2000 ◽  
Vol 20 (16) ◽  
pp. 5858-5864 ◽  
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.

scholarly journals TOR signaling modulates Cdk8-dependent GAL gene expression in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Nicole Hawe ◽  
Konstantin Mestnikov ◽  
Riley Horvath ◽  
Mariam Eji-Lasisi ◽  
Cindy Lam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Mediator Complex ◽  
Tor Signaling ◽  
A Subunit ◽  
Insight Into

AbstractCdk8 of the RNA Polymerase II mediator complex regulates genes by phosphorylating sequence specific transcription factors. Despite conserved importance for eukaryotic transcriptional regulation, the signals regulating Cdk8 are unknown. Full induction of the yeast GAL genes requires phosphorylation of Gal4 by Cdk8, and we exploited this requirement for growth of gal3 yeast on galactose to identify mutants affecting Cdk8 activity. Several mutants from the screen produced defects in TOR signaling. A mutant designated gal four throttle (gft) 1, gft1, was identified as an allele of hom3, encoding aspartokinase. Defects in gft1/ hom3 caused hypersensitivity to rapamycin, and constitutive nuclear localization of Gat1. Furthermore, mutations of tor1 or tco89, encoding TORC1 components, also prevented GAL expression in gal3 yeast, and tco89 was determined to be allelic to gft7. Disruption of cdc55, encoding a subunit of PP2A regulated by TOR signaling, suppressed the effect of gft1/ hom3, gft7/ tco89, and tor1 mutations on GAL expression in gal3 yeast, but not of cdk8/ srb10 disruptions or Gal4 S699A mutation. Mutations of gft1/ hom3 and tor1 did not affect kinase activity of Cdk8 in vitro, but caused loss of Gal4 phosphorylation in vivo. These observations demonstrate that TOR signaling regulates GAL induction through the activity of PP2A/ Cdc55, and are consistent with the contention that Cdk8-dependent phosphorylation of Gal4 S699 is opposed by PP2A/ Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.

scholarly journals Swm1/Apc13 Is an Evolutionarily Conserved Subunit of the Anaphase-Promoting Complex Stabilizing the Association of Cdc16 and Cdc27

2004 ◽  
Vol 24 (8) ◽  
pp. 3562-3576 ◽  
Author(s):  
Martin Schwickart ◽  
Jan Havlis ◽  
Bianca Habermann ◽  
Aliona Bogdanova ◽  
Alain Camasses ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Small Subunit ◽  
Cell Cycle Regulators ◽  
Anaphase Promoting Complex ◽  
Ubiquitin Ligase Activity ◽  
Evolutionarily Conserved ◽  
Stable Association

ABSTRACT The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.

scholarly journals Control of Mitotic Events by Nap1 and the Gin4 Kinase

1997 ◽  
Vol 138 (1) ◽  
pp. 119-130 ◽  
Author(s):  
Roger Altman ◽  
Douglas Kellogg
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Affinity Chromatography ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Molecular Pathways ◽  
Cyclin Dependent Kinases ◽  
Mitotic Cyclin ◽  
Bud Growth

Little is known about the pathways used by cyclins and cyclin-dependent kinases to induce the events of the cell cycle. In budding yeast, a protein called Nap1 binds to the mitotic cyclin Clb2, and Nap1 is required for the ability of Clb2 to induce specific mitotic events, but the role played by Nap1 is unclear. We have used genetic and biochemical approaches to identify additional proteins that function with Nap1 in the control of mitotic events. These approaches have both identified a protein kinase called Gin4 that is required for the ability of Clb2 and Nap1 to promote the switch from polar to isotropic bud growth that normally occurs during mitosis. Gin4 is also required for the ability of Clb2 and Nap1 to promote normal progression through mitosis. The Gin4 protein becomes phosphorylated as cells enter mitosis, resulting in the activation of Gin4 kinase activity, and the phosphorylation of Gin4 is dependent upon Nap1 and Clb2 in vivo. Affinity chromatography experiments demonstrate that Gin4 binds tightly to Nap1, indicating that the functions of these two proteins are closely tied within the cell. These results demonstrate that the activation of Gin4 is under the control of Clb2 and Nap1, and they provide an important step towards elucidating the molecular pathways that link cyclin-dependent kinases to the events they control.

scholarly journals Pseudosubstrate Inhibition of the Anaphase-Promoting Complex by Acm1: Regulation by Proteolysis and Cdc28 Phosphorylation

2008 ◽  
Vol 28 (15) ◽  
pp. 4653-4664 ◽  
Author(s):  
Denis Ostapenko ◽  
Janet L. Burton ◽  
Ruiwen Wang ◽  
Mark J. Solomon
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Anaphase Promoting Complex ◽  
Inhibitory Protein ◽  
Ubiquitin Ligase Activity ◽  
Dependent Protein ◽  
Apc Mutation

ABSTRACT The ubiquitin ligase activity of the anaphase-promoting complex (APC)/cyclosome needs to be tightly regulated for proper cell cycle progression. Substrates are recruited to the APC by the Cdc20 and Cdh1 accessory proteins. The Cdh1-APC interaction is inhibited through phosphorylation of Cdh1 by Cdc28, the major cyclin-dependent protein kinase in budding yeast. More recently, Acm1 was reported to be a Cdh1-binding and -inhibitory protein in budding yeast. We found that although Acm1 is an unstable protein and contains the KEN-box and D-box motifs typically found in APC substrates, Acm1 itself is not an APC substrate. Rather, it uses these motifs to compete with substrates for Cdh1 binding, thereby inhibiting their recruitment to the APC. Mutation of these motifs prevented Acm1-Cdh1 binding in vivo and rendered Acm1 inactive both in vitro and in vivo. Acm1 stability was critically dependent on phosphorylation by Cdc28, as Acm1 was destabilized following inhibition of Cdc28, mutation of consensus Cdc28 phosphorylation sites in Acm1, or deletion of the Bmh1 and Bmh2 phosphoprotein-binding proteins. Thus, Cdc28 serves dual roles in inhibiting Cdh1-dependent APC activity during the cell cycle: stabilization of the Cdh1 inhibitor Acm1 and direct phosphorylation of Cdh1 to prevent its association with the APC.

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