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. 2013 Apr;33(7):1273-84.
doi: 10.1128/MCB.01556-12. Epub 2013 Jan 22.

Phosphate-activated Cyclin-Dependent Kinase Stabilizes G1 Cyclin to Trigger Cell Cycle Entry

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Phosphate-activated Cyclin-Dependent Kinase Stabilizes G1 Cyclin to Trigger Cell Cycle Entry

S Menoyo et al. Mol Cell Biol. .
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Abstract

G1 cyclins, in association with a cyclin-dependent kinase (CDK), are universal activators of the transcriptional G1-S machinery during entry into the cell cycle. Regulation of cyclin degradation is crucial for coordinating progression through the cell cycle, but the mechanisms that modulate cyclin stability to control cell cycle entry are still unknown. Here, we show that a lack of phosphate downregulates Cln3 cyclin and leads to G1 arrest in Saccharomyces cerevisiae. The stability of Cln3 protein is diminished in strains with low activity of Pho85, a phosphate-sensing CDK. Cln3 is an in vitro substrate of Pho85, and both proteins interact in vivo. More interestingly, cells that carry a CLN3 allele encoding aspartic acid substitutions at the sites of Pho85 phosphorylation maintain high levels of Cln3 independently of Pho85 activity. Moreover, these cells do not properly arrest in G1 in the absence of phosphate and they die prematurely. Finally, the activity of Pho85 is essential for accumulating Cln3 and for reentering the cell cycle after phosphate refeeding. Taken together, our data indicate that Cln3 is a molecular target of the Pho85 kinase that is required to modulate cell cycle entry in response to environmental changes in nutrient availability.

Figures

Fig 1
Fig 1
Phosphate deprivation leads to G1 arrest. Wild-type cells were grown exponentially in synthetic complete medium. At time zero, cells were harvested and incubated either in the same medium (+PO42−) or in a medium without phosphate (−PO42−). At the indicated times, samples were collected and then subjected to several analyses: total cell number (A), DNA content (B), percentage of budding (C), and cell volume (D). (B) The DNA contents of wild-type cells incubated in a medium without a nitrogen source (−N) are shown. (C) The percentages of budding of wild-type cells carrying a centromeric plasmid with the CLN2 gene expressed from an ADH promoter are shown. Data ± standard deviations from three independent experiments are shown.
Fig 2
Fig 2
Phosphate deprivation leads to the downregulation of Cln3p. (A) Strain YAN32 (triple tagged: Cln2-HA, Clb5-TAP, and Sic1-Myc) was grown exponentially in synthetic complete medium. At time zero, the cells were harvested and incubated in phosphate (PO42−)-free medium. At the indicated times, samples were recovered and then analyzed for different proteins by immunoblotting using specific antibodies. (B) The wild-type strain was treated and sampled as described in Materials and Methods. Transcripts were analyzed using RT-PCR with specific primers. Data ± standard deviations from three independent experiments are shown. (C) Strains YPC702 (SWI6-TAP), YNR11 (WHI5-TAP), and YNR55 (CLN3-MYC) were treated, sampled, and analyzed as described for panel A.
Fig 3
Fig 3
Pho85 inactivation leads to downregulation of Cln3. (A) Wild-type (wt) and pho85Δ cells were grown exponentially in YPD and then assessed for levels of Cln3-Myc (left), and wild-type cells were grown in synthetic complete medium with (+PO42−) or without (−PO42−) phosphate (right). Samples were taken after 6 h, and levels of Cln3-Myc were monitored. (B) Cells from the YAM67 strain were incubated with either 1-Na PP1 (a specific pho85-as inhibitor) or drug vehicle (dimethyl sulfoxide [DMSO]). Samples were taken at the indicated times, and Cln3-Myc was analyzed by immunoblotting. Data ± standard deviations from three independent experiments are shown. Cln3p, phosphorylated Cln3.
Fig 4
Fig 4
Phosphate controls cellular levels of Cln3 by modulating the PHO pathway. (A) Schematic of the PHO pathway. During phosphate starvation, Vip1 causes an increase in the levels of inositol heptakisphosphate (IP7), which binds to and changes the conformation state of Pho81, leading to the inactivation of Pho85/Pho80 complexes. (B) Relative amounts of Cln3-Myc in different strains. Wild-type (wt), pho81Δ, and vip1Δ strains were grown in synthetic complete medium with (+PO42−) or without (−PO42−) phosphate. After 5 h, the levels of Cln3-Myc were quantified by immunoblotting using monoclonal antibodies. Cells of the Gal1-PHO85 strain (a wild-type strain that carries a centromeric plasmid with PHO85 expressed under the Gal1 promoter) were grown for 5 h in synthetic complete medium with galactose as a carbon source, either with or without phosphate. (C) Pho80 is necessary to maintain high levels of Cln3. As described for panel B, wild-type (GAL-Ø) and GAL1-PHO80 strains were grown in synthetic complete medium in the presence of galactose without phosphate (−PO42−). Levels of Cln3-Myc were quantified by immunoblotting using monoclonal antibodies. (D) Pho80 is necessary to maintain high levels of Cln3. Quantification of data in panel E (data ± standard deviations from four independent experiments) is shown. (E) Pho80 is necessary to maintain high levels of Cln3. A plasmid with a Cln3-Myc epitope tag was introduced in strains with the indicated mutations for deficiency of the different Pho85 cyclins. After 3 h of exponential growth in YPD, the levels of Cln3-Myc were analyzed by immunoblotting.
Fig 5
Fig 5
Pho85 activity increases the half-life of Cln3 protein. (A) Different strains with a genomic tagged version of CLN3 were sampled and then analyzed for CLN3 mRNA levels (by RT-PCR) or Cln3-Myc levels (by immunoblotting). Wild-type cells were grown exponentially in synthetic complete medium with (wt) or without (wt −PO42−) phosphate, pho85Δ strain cells were grown in the same complete medium with phosphate, and GAL-PHO85 cells were grown for 5 h in synthetic complete medium with galactose as a carbon source. (B) YAM67 strain cells were incubated with either 1-Na PP1 (a specific pho85-as inhibitor) or drug vehicle (DMSO). Forty minutes later (time zero), cycloheximide (CHX) was added to the cultures (final concentration, 10 μg/ml). At the indicated times, samples were taken and analyzed for Cln3-Myc levels by immunoblotting. Data ± standard deviations from four independent experiments are shown. t1/2, half-life. (C) Downregulation of autophagy does not restore the diminished Cln3 levels of pho85Δ mutants. Strains of the indicated genotypes were grown exponentially in phosphate-rich medium, and Cln3-Myc was analyzed by immunoblot assay using specific antibodies. (D) Absence of the PEST region restores Cln3 levels in pho85Δ cells. Wild-type or pho85Δ cells carrying a plasmid with a cln3-1 allele without the PEST region (41) were grown exponentially in YPD. Cln3-HA levels were measured by immunoblotting. (E) Absence of UBC4 restores Cln3 levels in a pho85Δ strain. Strains of the indicated genotypes carrying a genomic Myc-tagged version of Cln3 were grown exponentially in phosphate-rich medium. Cells were harvested, and Cln3 levels were evaluated by immunoblotting.
Fig 6
Fig 6
Cln3 is phosphorylated in vitro by Pho85/Pho80. (A) Schematic representation of Cln3 with the different functional domains. The PEST region is indicated by shading, and the putative target residues for Pho85 by arrows. NLS, nuclear localization signal. (B) In vitro kinase assay of Pho85/Pho80 on Cln3. Recombinant Pho85 and GST-Pho80, purified from bacteria, were incubated with the C-terminal half of Cln3 (also from bacteria) containing the PEST region with the indicated mutations or the wild-type sequence (see Materials and Methods and Table 2). Pho4, a well-known substrate of Pho85/Pho80, was included as a control for the Pho85/Pho80 activity. The arrows indicate Cln3 protein. (C) In vitro kinase assay of Cdc28-TAP and the Cln3 mutants with the indicated mutations or the wild-type sequence. IgG-Sepharose beads were used to pull down Cdc28 either from a no-tag strain (−) or the Cdc28-TAP tag strain (+). Sic1 was included as a control for the Cdc28 kinase activity.
Fig 7
Fig 7
In vivo phosphorylation of S449 and T520 is essential to maintain Cln3 levels. (A) Pho80 and Cln3 interact in vivo. Yeast extracts (WCE [whole-cell extracts]) containing untagged Pho80 or Pho80-TAP were pulled down with IgG-Sepharose. Tagged Cln3-Myc (from its chromosomal locus) was detected using specific antibodies (IP [immunoprecipitate]). (B) Alanine replacement of S449 and T520 destabilizes Cln3. Wild-type cells were transformed with a centromeric plasmid bearing a Cln3-Myc or a 2-Ala (S449A T520A) mutant version and grown for 4 h in rich medium. The amount of Cln3 was determined by immunoblotting. (C) Quantification of experiment whose results are shown in panel B. Data ± standard deviations of three independent experiments are shown. (D) Aspartic acid replacement of S449 and T520 stabilizes Cln3. Wild-type (in phosphate-deficient medium) or pho85Δ (in phosphate-rich medium) cells bearing a plasmid with different versions of CLN3 were grown exponentially for 4 h. Cln3 levels were analyzed by immunoblotting. To simplify the nomenclature, S449D means a double substitution of Asp into the Ser 449 and the previous residue to mimic the double-negative charge that represents the phosphate group (see Materials and Methods), and the same is true for T520D. (E) Quantification of experiments whose results are shown in panel D. Data ± standard deviations of three independent experiments are shown.
Fig 8
Fig 8
Pho85 activity is necessary for proper G1 arrest and cell cycle reentry. (A) Proposed model of Pho85 activity. In phosphate-rich medium, Pho85/Pho80 complexes remain highly active. Under such conditions, Pho85 phosphorylates and inactivates Pho4 and Rim15 (25) and, conversely, activates cyclin Cln3. (B) Proper regulation of Pho85 activity is essential for survival under conditions of phosphate deprivation. Cells were incubated in synthetic complete medium for 7 days, at which point cells were collected and assessed for viability by colony counting (left). The percentage of budding was analyzed by counting no less than 200 cells under the microscope (right). Data ± standard deviations from three independent experiments are shown. (C) Pho85 activity is necessary for reentry into the cell cycle after refeeding. Cells transformed with a centromeric plasmid bearing Cln3-Myc were deprived of phosphate for 7 h (time zero) and then refed. At various times, samples were collected and analyzed for DNA content by flow cytometry (top). Times (min) are indicated at left; “+PO42−” indicates the initiation of refeeding. Cln3 levels were monitored by immunoblotting (bottom) at times (min) indicated above the gel. (D) pho85Δ progresses through G1, with a small delay, after α-factor exit. Wild-type and pho85Δ cells were synchronized with α-factor for 3 h and then released into fresh medium at 30°C. At various times, samples were collected. Times (min) are indicated at left; “α-factor release” indicates the time of release following synchronization. Total DNA content was measured as described in Materials and Methods, except that propidium iodide was used instead of SYBR green.

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