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, 24 (24), 4271-8

Regulation of G0 Entry by the Pho80-Pho85 cyclin-CDK Complex

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Regulation of G0 Entry by the Pho80-Pho85 cyclin-CDK Complex

Valeria Wanke et al. EMBO J.

Abstract

Eukaryotic cell proliferation is controlled by growth factors and essential nutrients. In their absence, cells may enter into a quiescent state (G0). In Saccharomyces cerevisiae, the conserved protein kinase A (PKA) and rapamycin-sensitive TOR (TORC1) pathways antagonize G0 entry in response to carbon and/or nitrogen availability primarily by inhibiting the PAS kinase Rim15 function. Here, we show that the phosphate-sensing Pho80-Pho85 cyclin-cyclin-dependent kinase (CDK) complex also participates in Rim15 inhibition through direct phosphorylation, thereby effectively sequestering Rim15 in the cytoplasm via its association with 14-3-3 proteins. Inactivation of either Pho80-Pho85 or TORC1 causes dephosphorylation of the 14-3-3-binding site in Rim15, thus enabling nuclear import of Rim15 and induction of the Rim15-controlled G0 program. Importantly, we also show that Pho80-Pho85 and TORC1 converge on a single amino acid in Rim15. Thus, Rim15 plays a key role in G0 entry through its ability to integrate signaling from the PKA, TORC1, and Pho80-Pho85 pathways.

Figures

Figure 1
Figure 1
Protein kinase-inactivating mutations permit the study of cytoplasmic retention and/or nuclear import of GFP-Rim15. (A) Localization of GFP-Rim15K823Y, GFP-Rim15C1176Y, and GFP-Rim15. Exponentially growing (EXP), rapamycin-treated (RAP), and diauxic (DS; OD600=3.5) rim15Δ (IP31) cells expressing GFP-Rim15K823Y (pFD1008), GFP-Rim15C1176Y (pFD633), or GFP-Rim15 (pFD846) were visualized by fluorescence microscopy. (B) Localization of GFP-Rim15K823Y (pFD1008), GFP-Rim15C1176Y (pFD633), or GFP-Rim15 (pFD846) in exponentially growing wild-type (W303-1A) or msn5Δ (YBL029) cells. Notably, the msn5Δ mutant overexpressing GFP-Rim15 was very sick, which is consistent with the expectation that overproduction (from the ADH1 promoter) of nuclear GFP-Rim15 wild-type protein is detrimental to cell growth. Nevertheless, the overall very weak GFP-Rim15 signal observed in these cells was for the most part localized in the nuclei (WN, weak nuclear staining). N and C denote mainly nuclear and cytoplasmic localization of the GFP-fusion proteins, respectively.
Figure 2
Figure 2
Cytoplasmic retention of GFP-Rim15 depends on its association with 14-3-3 proteins. (A) Domain architecture of Rim15. The various domains of Rim15 include the N-terminal PAS, the C2HC-type zinc-finger (ZnF), the central kinase catalytic domain (gray rectangles) with an insert of 188 amino acids between subdomains VII and VIII, and the C-terminal receiver (REC) domain. As predicted by the Scansite program (http://scansite.mit.edu) the single high-stringency, putative 14-3-3 protein-binding site in Rim15 (scoring in the top 0.165%) flanks amino-acid T1075. (B) Interaction between Rim15KI and Bmh2. Bmh2-HA3 (pTB419) was immunoprecipitated from extracts prepared from wild-type cells (W303-1A) expressing either GST-Rim15KI (pVW900) or GST-Rim15KI-T1075A (pVW902). The input extracts (upper two panels) and the immunoprecipitates (lower panel) were analyzed via immunoblot analysis using anti-GST (upper and lower panels) or anti-HA antibodies (panel in the middle). (C) Localization of GFP-Rim15C1176Y (pFD633), GFP-Rim15C1176Y/T1075A (pVW1017), and GFP-Rim15C1176Y/ΔKI (pVW1068) in exponentially growing rim15Δ (IP31) cells. The numbers indicate the percentage of cells with clear nuclear staining of the GFP-fusion protein; >200 cells were counted. (D, E) Depletion of 14-3-3 proteins results in the nuclear accumulation of GFP-Rim15C1176Y. GFP-Rim15C1176Y (pFD633) expressing bmh1Δ bmh2Δ rim15Δ [pGAL1-BMH2] (CDV235) cells carrying either the control plasmid pLC921 (strain 1) or plasmid pCDV994 (expressing BMH2-HA3 from its own promoter; strain 2) were pregrown on SGal/Raf, transferred to SD medium, and grown exponentially (through repeated dilutions) for the times indicated. Bmh2-HA3 and Bmh2 levels (in strains 1 and 2) were analyzed prior (time zero) and following (24 or 48 h) the transfer of the cells from SGal/Raf to SD medium via immunoblot analysis using anti-HA (top panel) and anti-Bmh2 (lower panel) antibodies (D). GFP-Rim15C1176Y was visualized 24 h following the transfer of the cells from SGal/Raf to SD medium (E). (F) Depletion of 14-3-3 proteins results in transcriptional activation of Rim15-dependent genes. GRE1 and HSP26 transcript levels were determined (via Northern blot analysis as described in Pedruzzi et al, 2000) at the times indicated following transfer of bmh1Δ bmh2Δ [pGAL1-BMH2] (SL1470) and bmh1Δ bmh2Δ rim15Δ [pGAL1-BMH2] (CDV235) cells from SGal/Raf to SD medium. Depletion of Bmh2 was monitored via immunoblot analysis using anti-Bmh2 antibodies (top panel).
Figure 3
Figure 3
The Pho80–Pho85 cyclin–CDK phosphorylates the T1075-containing 14-3-3-binding site in Rim15 and stimulates its cytoplasmic retention. (A) Interaction between Rim15 and Pho85. GST-Rim15 (pNB566; lanes 1–4) and GST (YCpIF2-GST; lane 5) were precipitated from extracts prepared from wild-type (KT1961) cells coexpressing HA2-Pho85 (pIP774; lanes 1 and 5), HA2-Tpk1 (pCDV503; lane 2), HA2-Tps1 (pAR502; lane 3), or Bud14-HA3 (pFD662; lane 4). Cell lysates (Input) and GST pull-down fractions were subjected to PAGE and immunoblots were probed using anti-GST or anti-HA antibodies as indicated. Tpk1 and Tps1 (both previously identified as Rim15-interacting proteins; Reinders et al, 1998) served as positive controls, while Bud14 served as a negative control. (B) In vitro phosphorylation of T1075 in Rim15. GST-Rim15KI (pVW995), GST-Rim15KI-T1075A (pVW997), and GST (pGEX3X), expressed and purified from bacteria, were used as in vitro substrates (see top panel for input blot) in protein kinase assays using equal amounts of GST-Pho80 and either HA2-Pho85 or protein kinase inactive HA2-Pho85E53A. The protein input bands (top panel) and the phosphorylation levels of the Rim15KI variants (lower panel) were quantified (see Materials and methods). The average ratio of [32P]GST-Rim15KI-T1075/GST-Rim15KI-T1075 (from four independent experiments) is expressed in percent of the corresponding ratio for wild-type GST-Rim15KI (set at 100% following the deduction of the background signal in the E53A lane) and is indicated below the relevant lane. (C) In vivo phosphorylation of T1075 in Rim15. GST-Rim15 (WT; pNB566) and GST-Rim15T1075A (T1075A; pLC824) were purified from exponentially growing rim15Δ (IP31) cells and analyzed by immunoblotting (using either anti-GST antibodies or phospho-specific antibodies against Rim15-pT1075) following treatment with phosphatase inhibitors (PPI) alone or λ-phosphatase (λ-PPase)±PPI. (D) Rim15-myc13 (pVW904) from exponentially growing rim15Δ (IP31) and pho85Δ rim15Δ (CDV201-3B) mutants were analyzed for phosphorylation of T1075 using Rim15-pT1075 phospho-specific antibodies. Equal amounts of the fusion proteins were verified by immunoblotting using anti-myc antibodies. (E) Localization of GFP-Rim15C1176Y (pFD633) in exponentially growing rim15Δ (IP31), pcl1Δ pcl2Δ rim15Δ (CDV237-9D), pho85Δ rim15Δ (CDV201-3B), and pho80Δ rim15Δ (CDV237-7A) mutant cells. The numbers indicate the percentage of cells with clear nuclear staining of the GFP-fusion protein; >200 cells were counted.
Figure 4
Figure 4
The Pho80–Pho85 cyclin–CDK complex antagonizes induction of Rim15-dependent G0 traits. (A, B) Induction of GRE1-lacZ expression (from integrative plasmid pIP490; A) and trehalose levels (B; for method see Pedruzzi et al, 2000) as wild-type (KT1961), rim15Δ (IP31), pho85Δ (IP48-3C), pho85Δ rim15Δ (CDV201-3B), pho80Δ (CDV237-10C), and pho80Δ rim15Δ (CDV237-7A) mutant cells were grown to stationary phase on YPD medium. (C) Stationary phase survival of wild-type (•), rim15Δ (○), pho85Δ (▪), and pho85Δ rim15Δ (□) mutant cells (for strains see [A, B]). (D) Glycogen levels in 4-day old batch cultures (visualized after exposure for 1 min to iodine vapor; for strains see [A, B]). (E) Both phosphate starvation and rapamycin treatment reduce the extent of phosphorylation of the T1075 residue in Rim15. Exponentially growing rim15Δ (IP31) cells expressing Rim15-myc13 (pVW904) were starved for phosphate (−P) or treated with rapamycin (+RAP) for the indicated times. Detection of fusion protein levels and pT1075 were as in Figure 3D. (F) Exponentially growing rim15Δ (IP31) cells expressing GFP-Rim15C1176Y (pFD633; EXP) were treated for 45 min with rapamycin (+RAP), or submitted for 45 min to phosphate starvation in the absence (−P) or in the presence (−P/+RAP) of rapamycin and subsequently visualized by fluorescence microscopy. The numbers indicate the percentage of cells with clear nuclear staining of the GFP-fusion protein; >200 cells were counted.
Figure 5
Figure 5
Model for the regulation of Rim15 by nutrient-sensory kinases. Cytoplasmic Rim15, anchored through its binding to 14-3-3 proteins, is kept inactivate through PKA-mediated phosphorylation (1). TORC1 inactivation results (either due to the activation of a phosphatase[s] and/or inactivation of a protein kinase) in dephosphorylation of pT1075 in Rim15 with concomitant abrogation of cytoplasmic retention of Rim15 by 14-3-3 proteins (2). Following its nuclear import (3), Rim15 presumably escapes from further PKA-mediated inhibition (4). Active Rim15 (drawn in black) induces the G0 program, initiates an autophosphorylation process (illustrated with a circle around Rim15), and accelerates its Msn5-mediated nuclear export. The Pho80–Pho85 cyclin–CDK complex phosphorylates T1075 of Rim15 (5), thereby promoting (following export of Rim15; 6) the re-association between Rim15 and 14-3-3 proteins in the cytoplasm (7). The nutrient sensing protein kinases PKA, TOR (in TORC1), and Pho80-Pho85 sense glucose, nutrients (nitrogen), and phosphate (Pi), respectively (Wilson and Roach, 2002). See text for further details.

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