Glc7/Protein Phosphatase 1 Regulatory Subunits Can Oppose the Ipl1/Aurora Protein Kinase by Redistributing Glc7

ABSTRACT Faithful chromosome segregation depends on the opposing activities of the budding yeast Glc7/PP1 protein phosphatase and Ipl1/Aurora protein kinase. We explored the relationship between Glc7 and Ipl1 and found that the phosphorylation of the Ipl1 substrate, Dam1, was altered by decreased Glc7 activity, whereas Ipl1 levels, localization, and kinase activity were not. These data strongly suggest that Glc7 ensures accurate chromosome segregation by dephosphorylating Ipl1 targets rather than regulating the Ipl1 kinase. To identify potential Glc7 and Ipl1 substrates, we isolated ipl1-321 dosage suppressors. Seven genes (SDS22, BUD14, GIP3, GIP4, SOL1, SOL2, and PEX31) encode newly identified ipl1 dosage suppressors, and all 10 suppressors encode proteins that physically interact with Glc7. The overexpression of the Gip3 and Gip4 suppressors altered Glc7 localization, indicating they are previously unidentified Glc7 regulatory subunits. In addition, the overexpression of Gip3 and Gip4 from the galactose promoter restored Dam1 phosphorylation in ipl1-321 mutant cells and caused wild-type cells to arrest in metaphase with unsegregated chromosomes, suggesting that Gip3 and Gip4 overexpression impairs Glc7's mitotic functions. We therefore propose that the overexpression of Glc7 regulatory subunits can titrate Glc7 away from relevant Ipl1 targets and thereby suppress ipl1-321 cells by restoring the balance of phosphatase/kinase activity.

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Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation

The Journal of Cell Biology ◽

10.1083/jcb.201909136 ◽

2020 ◽

Vol 219 (4) ◽

Cited By ~ 2

Author(s):

Gisela Cairo ◽

Anne M. MacKenzie ◽

Soni Lacefield

Keyword(s):

Chromosome Segregation ◽

Protein Phosphatase ◽

Mitotic Chromosome ◽

Budding Yeast ◽

Meiotic Chromosome ◽

Protein Phosphatase 1 ◽

Aurora B ◽

Chromosome Missegregation ◽

Anaphase Onset ◽

Spindle Microtubules

Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore–microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1–Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome–spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1–Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

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Functional Analysis of the Yeast Glc7-Binding Protein Reg1 Identifies a Protein Phosphatase Type 1-Binding Motif as Essential for Repression of ADH2 Expression

Molecular and Cellular Biology ◽

10.1128/mcb.19.9.6029 ◽

1999 ◽

Vol 19 (9) ◽

pp. 6029-6040 ◽

Cited By ~ 38

Author(s):

Kenneth M. Dombek ◽

Valentina Voronkova ◽

Alexa Raney ◽

Elton T. Young

Keyword(s):

Protein Kinase ◽

Central Region ◽

Protein Phosphatase ◽

Kinase Activity ◽

Protein Kinase Activity ◽

Snf1 Kinase ◽

Protein Phosphatase Type 1 ◽

Mutant Cells ◽

Protein Phosphatase Type

ABSTRACT In Saccharomyces cerevisiae, the protein phosphatase type 1 (PP1)-binding protein Reg1 is required to maintain complete repression of ADH2 expression during growth on glucose. Surprisingly, however, mutant forms of the yeast PP1 homologue Glc7, which are unable to repress expression of another glucose-regulated gene, SUC2, fully repressed ADH2. ConstitutiveADH2 expression in reg1 mutant cells did require Snf1 protein kinase activity like constitutive SUC2expression and was inhibited by unregulated cyclic AMP-dependent protein kinase activity like ADH2 expression in derepressed cells. To further elucidate the functional role of Reg1 in repressingADH2 expression, deletions scanning the entire length of the protein were analyzed. Only the central region of the protein containing the putative PP1-binding sequence RHIHF was found to be indispensable for repression. Introduction of the I466M F468A substitutions into this sequence rendered Reg1 almost nonfunctional. Deletion of the central region or the double substitution prevented Reg1 from significantly interacting with Glc7 in two-hybrid analyses. Previous experimental evidence had indicated that Reg1 might target Glc7 to nuclear substrates such as the Snf1 kinase complex. Subcellular localization of a fully functional Reg1-green fluorescent protein fusion, however, indicated that Reg1 is cytoplasmic and excluded from the nucleus independently of the carbon source. When the level of Adr1 was modestly elevated, ADH2 expression was no longer fully repressed in glc7 mutant cells, providing the first direct evidence that Glc7 can repress ADH2 expression. These results suggest that the Reg1-Glc7 phosphatase is a cytoplasmic component of the machinery responsible for returning Snf1 kinase activity to its basal level and reestablishing glucose repression. This implies that the activated form of the Snf1 kinase complex must cycle between the nucleus and the cytoplasm.

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The Sec1p/Munc18 (SM) protein, Vps45p, cycles on and off membranes during vesicle transport

The Journal of Cell Biology ◽

10.1083/jcb.200212078 ◽

2003 ◽

Vol 161 (4) ◽

pp. 691-696 ◽

Cited By ~ 30

Author(s):

Nia J. Bryant ◽

David E. James

Keyword(s):

Protein Phosphatase ◽

Final Stage ◽

Vesicle Transport ◽

Protein Phosphatase 1 ◽

Wild Type ◽

Sm Proteins ◽

Transport Reaction ◽

Specific Manner ◽

Sm Protein ◽

Mutant Cells

Protein phosphatase 1 (PP1, Glc7p) functions in the final stage of SNARE-mediated vesicle transport between docking and fusion. During this process, trans-SNARE complexes, formed between molecules in opposing membranes, convert to cis-complexes, with all participants in the same lipid bilayer. Here, we show that glc7 mutant cells accumulate SNARE complexes. These complexes are clearly different from those found in either wild-type or sec18–1 cells as the Sec1p/Munc18 (SM) protein Vps45p does not bind to them. Given that PP1 controls fusion, the SNARE complexes that accumulate in glc7 mutants likely represent trans-SNARE complexes. Vps45p dissociates from the membrane in the absence of PP1 activity, but rapidly reassociates after its reactivation. These data reveal that SM proteins cycle on and off membranes in a stage-specific manner during the vesicle transport reaction, and suggest that protein phosphorylation plays a key role in the regulation of this cycle.

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Regulation of chromosome segregation by Glc8p, a structural homolog of mammalian inhibitor 2 that functions as both an activator and an inhibitor of yeast protein phosphatase 1.

Molecular and Cellular Biology ◽

10.1128/mcb.15.11.6064 ◽

1995 ◽

Vol 15 (11) ◽

pp. 6064-6074 ◽

Cited By ~ 66

Author(s):

H Y Tung ◽

W Wang ◽

C S Chan

Keyword(s):

Protein Kinase ◽

Chromosome Segregation ◽

Protein Phosphatase ◽

Site Directed Mutagenesis ◽

Protein Phosphatase 1 ◽

Temperature Sensitive ◽

Loss Of Function ◽

Yeast Saccharomyces Cerevisiae ◽

Growth Phenotype

The Ipl1 protein kinase is essential for proper chromosome segregation and cell viability in the budding yeast Saccharomyces cerevisiae. We have previously shown that the temperature-sensitive growth phenotype of conditional ipl1-1ts mutants can be suppressed by a partial loss-of-function mutation in the GLC7 gene, which encodes the catalytic subunit (PP1C) of protein phosphatase 1, thus suggesting that this enzyme acts in opposition to the Ipl1 protein kinase in regulating yeast chromosome segregation. We report here that the Glc8 protein, which is related in primary sequence to mammalian inhibitor 2, also participates in this regulation. Like inhibitor 2, the Glc8 protein is heat stable, exhibits anomalous electrophoretic mobility, and functions in vitro as an inhibitor of yeast as well as rabbit skeletal muscle PP1C. Interestingly, overexpression as well as deletion of the GLC8 gene results in a partial suppression of the temperature-sensitive growth phenotype of ipl1ts mutants and also moderately reduces the amount of protein phosphatase 1 activity which is assayable in crude yeast lysates. In addition, the chromosome missegregation phenotype caused by an increase in the dosage of GLC7 is totally suppressed by the glc8-delta 101::LEU2 deletion mutation. These findings together suggest that the Glc8 protein is involved in vivo in the activation of PP1C and that when the Glc8 protein is overproduced, it may also inhibit PP1C function. Furthermore, site-directed mutagenesis studies of GLC8 suggest that Thr-118 of the Glc8 protein, which is equivalent to Thr-72 of inhibitor 2, may play a central role in the ability of this protein to activate and/or inhibit PP1C in vivo.

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Yeast Fin1-PP1 dephosphorylates an Ipl1 substrate, Ndc80, to remove Bub1-Bub3 checkpoint proteins from the kinetochore during anaphase

PLoS Genetics ◽

10.1371/journal.pgen.1009592 ◽

2021 ◽

Vol 17 (5) ◽

pp. e1009592

Author(s):

Michael Bokros ◽

Delaney Sherwin ◽

Marie-Helene Kabbaj ◽

Yanchang Wang

Keyword(s):

Chromosome Segregation ◽

Protein Phosphatase ◽

Spindle Assembly Checkpoint ◽

Kinase Activity ◽

Protein Phosphatase 1 ◽

Regulatory Subunit ◽

Cyclin Dependent Kinase ◽

Checkpoint Proteins ◽

Anaphase Onset ◽

Chromosome Attachment

The spindle assembly checkpoint (SAC) prevents anaphase onset in response to chromosome attachment defects, and SAC silencing is essential for anaphase onset. Following anaphase onset, activated Cdc14 phosphatase dephosphorylates the substrates of cyclin-dependent kinase to facilitate anaphase progression and mitotic exit. In budding yeast, Cdc14 dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. We previously showed that kinetochore-localized Fin1-PP1 promotes the removal of the SAC protein Bub1 from the kinetochore during anaphase. We report here that Fin1-PP1 also promotes kinetochore removal of Bub3, the Bub1 partner, but has no effect on another SAC protein Mad1. Moreover, the kinetochore localization of Bub1-Bub3 during anaphase requires Aurora B/Ipl1 kinase activity. We further showed that Fin1-PP1 facilitates the dephosphorylation of kinetochore protein Ndc80, a known Ipl1 substrate. This dephosphorylation reduces kinetochore association of Bub1-Bub3 during anaphase. In addition, we found that untimely Ndc80 dephosphorylation causes viability loss in response to tensionless chromosome attachments. These results suggest that timely localization of Fin1-PP1 to the kinetochore controls the functional window of SAC and is therefore critical for faithful chromosome segregation.

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Type 1 protein phosphatase acts in opposition to IpL1 protein kinase in regulating yeast chromosome segregation.

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4731 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4731-4740 ◽

Cited By ~ 187

Author(s):

L Francisco ◽

W Wang ◽

C S Chan

Keyword(s):

Protein Kinase ◽

Chromosome Segregation ◽

Protein Phosphatase ◽

High Fidelity ◽

Yeast Saccharomyces Cerevisiae ◽

M Phase ◽

Mutant Cells ◽

Putative Protein Kinase

The IPL1 gene is required for high-fidelity chromosome segregation in the budding yeast Saccharomyces cerevisiae. Conditional ipl1ts mutants missegregate chromosomes severely at 37 degrees C. Here, we report that IPL1 encodes an essential putative protein kinase whose function is required during the later part of each cell cycle. At 26 degrees C, the permissive growth temperature, ipl1 mutant cells are defective in the recovery from a transient G2/M-phase arrest caused by the antimicrotubule drug nocodazole. In an effort to identify additional gene products that participate with the Ipl1 protein kinase in regulating chromosome segregation in yeast, a truncated version of the previously identified DIS2S1/GLC7 gene was isolated as a dosage-dependent suppressor of ipl1ts mutations. DIS2S1/GLC7 is predicted to encode a catalytic subunit (PP1C) of type 1 protein phosphatase. Overexpression of the full-length DIS2S1/GLC7 gene results in chromosome missegregation in wild-type cells and exacerbates the mutant phenotype in ipl1 cells. In addition, the glc7-1 mutation can partially suppress the ipl1-1 mutation. These results suggest that type 1 protein phosphatase acts in opposition to the Ipl1 protein kinase in vivo to ensure the high fidelity of chromosome segregation.

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Type 1 protein phosphatase acts in opposition to IpL1 protein kinase in regulating yeast chromosome segregation

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4731-4740.1994 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4731-4740

Author(s):

L Francisco ◽

W Wang ◽

C S Chan

Keyword(s):

Protein Kinase ◽

Chromosome Segregation ◽

Protein Phosphatase ◽

High Fidelity ◽

Yeast Saccharomyces Cerevisiae ◽

M Phase ◽

Mutant Cells ◽

Putative Protein Kinase

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Involvement of a Protein Kinase Activity Inducer in DNA Double Strand Break Repair and Radioresistance of Deinococcus radiodurans

Journal of Bacteriology ◽

10.1128/jb.00026-08 ◽

2008 ◽

Vol 190 (11) ◽

pp. 3948-3954 ◽

Cited By ~ 32

Author(s):

Yogendra S. Rajpurohit ◽

Roja Gopalakrishnan ◽

Hari S. Misra

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Deinococcus Radiodurans ◽

Strand Break ◽

Pyrroloquinoline Quinone ◽

Protein Kinase Activity ◽

Wild Type ◽

Dna Double Strand Break ◽

Break Repair ◽

Mutant Cells

ABSTRACT Transgenic bacteria producing pyrroloquinoline quinone, a known cofactor for dehydrogenases and an inducer of a periplasmic protein kinase activity, show resistance to both oxidative stress and protection from nonoxidative effects of radiation and DNA-damaging agents. Deinococcus radiodurans R1 encodes an active pyrroloquinoline quinone synthase, and constitutive synthesis of pyrroloquinoline quinone occurred in wild-type bacteria. Disruption of a genomic copy of pqqE resulted in cells that lacked this cofactor. The mutant showed a nearly 3-log decrease in γ radiation resistance and a 2-log decrease in mitomycin C tolerance compared to wild-type cells. The mutant cells did not show sensitivity to UVC radiation. Expression of pyrroloquinoline quinone synthase in trans showed that there was functional complementation of γ resistance and mitomycin C tolerance in the pqqE mutant. The sensitivity to γ radiation was due to impairment or slow kinetics of DNA double strand break repair. Low levels of 32P incorporation were observed in total soluble proteins of mutant cells compared to the wild type. The results suggest that pyrroloquinoline quinone has a regulatory role as a cofactor for dehydrogenases and an inducer of selected protein kinase activity in radiation resistance and DNA strand break repair in a radioresistant bacterium.

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Cdc37 has distinct roles in protein kinase quality control that protect nascent chains from degradation and promote posttranslational maturation

The Journal of Cell Biology ◽

10.1083/jcb.200604106 ◽

2007 ◽

Vol 176 (3) ◽

pp. 319-328 ◽

Cited By ~ 68

Author(s):

Atin K. Mandal ◽

Paul Lee ◽

Jennifer A. Chen ◽

Nadinath Nillegoda ◽

Alana Heller ◽

...

Keyword(s):

Protein Kinase ◽

Mutant Strain ◽

Kinase Activity ◽

Wild Type Strain ◽

Wild Type ◽

General Role ◽

Rapid Degradation ◽

In The Wild ◽

Lipid Kinases ◽

Mutant Cells

Cdc37 is a molecular chaperone that functions with Hsp90 to promote protein kinase folding. Analysis of 65 Saccharomyces cerevisiae protein kinases (∼50% of the kinome) in a cdc37 mutant strain showed that 51 had decreased abundance compared with levels in the wild-type strain. Several lipid kinases also accumulated in reduced amounts in the cdc37 mutant strain. Results from our pulse-labeling studies showed that Cdc37 protects nascent kinase chains from rapid degradation shortly after synthesis. This degradation phenotype was suppressed when cdc37 mutant cells were grown at reduced temperatures, although this did not lead to a full restoration of kinase activity. We propose that Cdc37 functions at distinct steps in kinase biogenesis that involves protecting nascent chains from rapid degradation followed by its folding function in association with Hsp90. Our studies demonstrate that Cdc37 has a general role in kinome biogenesis.

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