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Published in final edited form as: Mol Cell. 2012 Feb 23;45(6):836–843. doi: 10.1016/j.molcel.2012.01.018

TOR Signaling Regulates Ribosome and tRNA Synthesis via LAMMER/Clk and GSK-3 Family Kinases

Jaehoon Lee 1, Robyn D Moir 1, Kerri B McIntosh 2, Ian M Willis 1,3,*
PMCID: PMC3319249  NIHMSID: NIHMS353780  PMID: 22364741

Summary

Target of Rapamycin (TOR)-dependent signaling and the control of cell growth is deregulated in many cancers. However, the signaling molecules downstream of TOR that co-ordinately regulate the synthesis of ribosomes and tRNAs are not well defined. Here we show in yeast that conserved kinases of the LAMMER/Cdc-like and GSK-3 families function downstream of TOR complex 1 to repress ribosome and tRNA synthesis in response to nutrient limitation and other types of cellular stress. As a part of this response, we found that the LAMMER kinase Kns1 is differentially expressed, hyperphosphorylated and accumulates in the nucleus after rapamycin treatment whereupon it primes the phosphorylation of the RNA polymerase III subunit Rpc53 by a specific GSK-3 family member, Mck1. In cooperation with another polymerase subunit, Rpc11, this phosphorylation of Rpc53 modifies the function of the enzyme and together with dephosphorylation of the Maf1 repressor inhibits the growth-promoting activity of RNA polymerase III transcription.

Introduction

The nutrient and stress-sensitive Target of Rapamycin (TOR) signaling pathway regulates lifespan, cell growth and metabolism and influences the occurrence of age-related diseases such as cancer, diabetes and cardiovascular disease (Wullschleger et al., 2006; Guertin and Sabatini, 2007). These effects are achieved by TOR kinase regulation of fundamental cellular processes including transcription by all three nuclear RNA polymerases for the synthesis of ribosomes and their tRNA substrates (Wullschleger et al., 2006). TOR-dependent signaling and transcription by RNA polymerases I and III is deregulated in many cancers (Guertin and Sabatini, 2007; White, 2008) and changes in the level of RNA polymerase III transcription have been shown to promote or suppress cell transformation and tumor formation (Marshall et al., 2008; Johnson et al., 2008). However, the signaling molecules and transcription components downstream of TOR that control cell growth by co-ordinately regulating the synthesis of ribosomes and tRNAs have not been fully defined.

Maf1 is a conserved repressor of RNA polymerase (pol) III transcription and functions to regulate the synthesis of tRNAs, 5S rRNA and other pol III transcripts under diverse conditions of nutrient limitation and cellular stress (Upadhya et al., 2002). In budding yeast, the repression of tRNA gene transcription by Maf1 is negatively regulated by protein kinase A, by the rapamycin-sensitive TOR kinase (TORC1) and by the TORC1-regulated kinase Sch9. These kinases phosphorylate multiple sites in Maf1, inhibiting its interaction with pol III and promoting its accumulation in the cytoplasm (Moir et al., 2006; Lee et al., 2009; Huber et al., 2009; Wei and Zheng, 2009). However, a Maf1 mutant that is nuclear and cannot be phosphorylated at these negative regulatory sites still requires exposure of cells to nutritional or environmental stress to repress transcription (Moir et al., 2006; Huber et al., 2009). This suggests that additional signaling steps are required to enable Maf1-dependent repression. Our examination of this hypothesis has led to the identification of two kinases acting downstream of TORC1 whose function is required to repress ribosome and tRNA synthesis. We define a role for RNA polymerase III as a TORC1-regulated target of these kinases and propose a mechanism that integrates the effect of TORC1 on both RNA polymerase III and Maf1 to achieve transcriptional repression.

Results and Discussion

Since several pol III subunits are known to be phosphorylated, we considered that the polymerase might be subject to phosphoregulation by the TOR pathway. To test this hypothesis, we performed in vivo 32P labeling before and after treatment of cells with rapamycin to inhibit TORC1 signaling. Immunoprecipitation of pol III from labeled cell extracts revealed numerous bands whose sizes correspond to subunits identified in phosphoproteomic studies (Figure S1A). Notably, the band corresponding to Rpc53 was qualitatively different following rapamycin treatment. Western blotting confirmed that rapamycin induced the conversion of fast migrating forms of Rpc53 into a slower migrating species (Figures 1A and S1B). This change in Rpc53 mobility was also seen under other repressing conditions such as alkylating DNA damage, ER stress, plasma membrane stretching and growth to stationary phase and could be reversed by phosphatase treatment (Figure 1A). Thus, the data show that Rpc53 hyperphosphorylation correlates with the repression of pol III transcription under diverse conditions in a similar but reciprocal manner to Maf1 which is dephosphorylated under the same conditions (Upadhya et al., 2002; Moir et al., 2006).

Figure 1. Hyperphosphorylation of Rpc53 under conditions that repress ribosome and tRNA synthesis requires the kinases Kns1 and Mck1.

Figure 1

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A Rpc53-HA immunoblotting of extracts prepared from a wild-type yeast strain (W303, OD600~0.5) before or after treatment with rapamycin (Rap), methyl methane sulfonate (MMS), chlorpromazine (CPZ) or tunicamycin (Tu) or after growth to stationary phase (OD600~10). The stationary phase extract was further treated with alkaline phosphatase in the presence or absence of a phosphatase inhibitor cocktail. B Scheme to identify regulators of Rpc53 phosphorylation. An RPC53-HA allele was introduced into 272 mutant strains containing gene-deletions of kinases, phosphatases and their regulators (Table S2) using SGA methods. Haploid double drug resistant strains were grown as indicated for extract preparation and immunoblotting. Deletions of only two genes (KNS1 and MCK1) were found to compromise the phosphorylation of Rpc53. A representative western blot is shown for wild-type, kns1Δ and mck1Δ strains. C Extracts of strains deleted for different GSK3 family members were prepared before or after treatment with rapamycin. D Extracts of wild-type and kns1Δ strains were prepared before or after treatment with different repressing conditions as in panel A. E Extracts of an mck1Δ strain were prepared before or after treatment with different repressing conditions as in panel A. See Supplemental Experimental Procedures for details.

To identify activities associated with rapamycin-induced hyperphosphorylation of Rpc53, we used synthetic genetic array (SGA) methodology to introduce an Rpc53-HA allele into a set of 272 deletion strains representing kinases, phosphatases and regulatory molecules involved in cellular signaling. The resulting haploid double mutants were grown to early log phase, treated with rapamycin and extracts were analyzed by western blotting. This screen uncovered two conserved CMGC family kinases, Kns1 and Mck1, deletions of which abolished the appearance of hyperphosphorylated Rpc53 in response to rapamycin (Figure 1B). Kns1 is the sole member of the LAMMER/Cdc-like (Clk) kinase family in yeast; little is known about its substrates, localization and functions. In metazoans, LAMMER kinases are best known for their regulation of alternative mRNA splicing via SR protein phosphorylation (Prasad and Manley, 2003). However, knowledge about their regulation of other processes and their association with signaling pathways is limited. Recent work has found that Clk2, one of four mammalian LAMMER kinases, is activated by Akt phosphorylation and mediates insulin signaling by repressing PGC1-α, a critical transcriptional coactivator of energy metabolism (Rodgers et al., 2010). Mck1 is one of four members of the glycogen synthase kinase 3 (GSK3) family in yeast. GSK3 kinases are key regulators of many cellular processes including metabolism, cell growth and apoptosis (Jope and Johnson, 2004). Like Clk2, mammalian GSK3 is a known target of Akt and is additionally phosphorylated by ribosomal S6 kinase, a downstream target of mTORC1 (Jope and Johnson, 2004; Zhang et al., 2006). The association of LAMMER and GSK3 kinases with the PI3 kinase-Akt-mTOR pathway in mammals is consistent with our finding that Kns1 and Mck1 are downstream components of the rapamycin-sensitive TORC1 signaling pathway in yeast. Notably, despite the presence of four GSK3 kinases in yeast, the ability to block Rpc53 hyperphosphorylation in response to rapamycin was highly specific to the mck1Δ strain (Figure 1C). In addition to nutritional regulation, our results indicate a role for both Kns1 and Mck1 in signaling to Rpc53 in response to other types of cellular stress: Deletion of either kinase also prevented hyperphosphorylation of Rpc53 in response to MMS, tunicamycin and chlorpromazine treatments (Figures 1D and 1E). The same effect was also seen for growth to stationary phase in the kns1Δ but not the mck1Δ strain (Figures 1D, 1E and S1C) indicating the increased complexity of signaling and the involvement of other kinases under this condition. Importantly, rapamycin-induced hyperphosphorylation of Rpc53 by Kns1 and Mck1 is not dependent on the Maf1 repressor and Maf1 dephosphorylation following rapamycin treatment is not dependent on these kinases (Figure S1D and S1E).

To examine the biological effect of deleting KNS1 and/or MCK1 on the repression of pol III transcription, pre-tRNA northern analysis was performed before and after rapamycin treatment. Repression of pre-tRNALeu synthesis was reproducibly attenuated in strains deleted for the individual kinases and this defect was further enhanced in the double mutant (Figure 2A). Similar results were obtained when measuring bulk tRNA synthesis in a [3H]-uracil pulse-chase experiment (Figure S2A). Notably, in both experiments, transcription was still reduced albeit to only 50-60% of the non-repressed level after one hour of treatment. Substantial attenuation of repression was also observed in the kns1Δ mck1Δ strain in response to DNA damage or stress on the plasma membrane or the ER, consistent with the loss of Rpc53 hyperphosphorylation under these conditions (Figures S2C, S2D, 1D and 1E). These results demonstrate the importance of Kns1 and Mck1 in mediating nutrient and stress signals to the pol III transcription machinery and at the same time reveal the existence other signaling mechanisms which are able to affect a partial transcriptional response. To examine the relationship between Kns1/Mck1 and Maf1 in repressing transcription, we compared the response to rapamycin in wild type, maf1Δ, kns1Δ mck1Δ and maf1Δ kns1Δ mck1Δ strains. No significant difference was observed between the triple mutant and the maf1Δ strain (Figure. S2B). This indicates that Kns1 and Mck1 cannot repress pol III transcription independently of Maf1 and suggests that the function of these kinases is necessary to enable Maf1-dependent repression.

Figure 2. Deletion of KNS1 and MCK1 compromises rapamycin-mediated repression of ribosome and tRNA synthesis.

Figure 2

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A Northern analysis of a short-lived tRNALeu precursor and the stable U3 snRNA was performed with RNA from early log phase cultures of wild-type, single deletion and double deletion strains before and after rapamycin treatment (left panel). Normalized pre-tRNALeu band intensities were used to determine the level of transcriptional repression relative to the untreated wild-type strain. Results from three biological replicates are plotted (right panel) ± standard deviation. B Northern analysis of the short-lived 20S precursor rRNA and mature 18S rRNA was performed with RNA from the log phase cultures in panel A (left panel). Normalized ratios of 20S:18S rRNA were used to determine the level of transcriptional repression relative to the untreated wild-type strain. Results from biological replicates are plotted (right panel) ± standard deviation. C Quantitation of ribosomal protein (RP) mRNA abundance. RNA preparations were analyzed by RT-PCR for various RP mRNAs using the 2-ΔΔCT method and U1 snRNA as a control across all experiments. Results are expressed relative to the untreated wild-type strain (set to 1.0). For each strain and condition, triplicate technical replicates were analysed from three biological replicates. The data are plotted ± standard deviation. Wild-type and mutant strains are color-coded following the scheme used in panels A and B. Control mRNAs for ACT1 and TUB1 showed no significant change in expression with deletion of Kns1 and/or Mck1 in the presence or absence of rapamycin.

Ribosome and tRNA synthesis is co-ordinately regulated under a broad range of conditions (Willis et al., 2004). We therefore asked whether deletion of KNS1 and/or MCK1 exerted similar effects on repression of rDNA transcription by pol I and ribosomal protein (RP) gene transcription by pol II. Northern analysis of the short-lived 20S rRNA precursor revealed substantial accumulation of this species upon rapamycin treatment of the kns1Δ mck1Δ strain consistent with reduced repression of pol I transcription (Figure 2B). Diminished repression of pol I transcription by rapamycin was also seen by pulse-chase labeling of rRNA with [C3H3] methionine (Figure S2E). Similarly, northern blotting demonstrated that rapamycin-mediated repression of two RP genes (RPL3 and RPL28) was attenuated in the kns1Δ mck1Δ strain (Figures S2F and S2G). RT-PCR analysis of seven different RP mRNAs confirmed that repression of these genes was significantly compromised in the kns1Δ mck1Δ strain (Figure 2C). Together these experiments reveal a common role for Kns1 and Mck1 in signaling transcriptional repression of ribosome and tRNA synthesis under conditions where TORC1 kinase activity is inhibited.

TOR signaling is known to control the localization of numerous nutrient-regulated transcription factors and signaling proteins and these changes are often accompanied by changes in their phosphorylation (Beck and Hall, 1999). Given that Kns1 contains 12 known phosphosites (Breitkreutz et al., 2010), we examined its phosphorylation state on denaturing phos-tag acrylamide gels and found that it increases in a rapamycin-dependent manner (Figures 3A and S3C). Kns1 hyperphosphorylation under repressing conditions is largely autocatalytic as these changes are not seen in a kinase-dead KNS1 mutant (Figure 3A). The abundance of Kns1 in log phase cells is relatively low at ~160 molecules per cell (Figures S3A and S3B) and required increased levels of expression for detection by indirect immunofluorescence. Under these conditions, Kns1 was found in the cytoplasm and in the nucleus in a large fraction (~50%) of log phase cells (Figures 3B and 3C). A similar fraction of cells contained detectable fluorescence in the cytoplasm but not in the nucleus while few cells contained nuclear-only fluorescence. These proportions were altered substantially after treatment of the cells with rapamycin. In particular, the fraction of cells exhibiting nuclear-only fluorescence increased about 10-fold while the fraction of cells with fluorescence only in the cytoplasm was reduced (Figures 3B and 3C). Catalytically-inactive Kns1 is not differentially phosphorylated in response to rapamycin (Figure 3A). Thus, TORC1 regulation of Kns1 must be achieved via a different mechanism. We found that the amount of Kns1 protein increases ~2-4 fold in response to either rapamycin or MMS (Figure 3D and 3E) and that elevating the expression of Kns1 is functionally significant since it induces Rpc53 hyperphosphorylation under otherwise optimal growth conditions (Figure 3F). Thus, rapamycin treatment leads to increased expression and autophosphorylation of Kns1 and is accompanied by its accumulation in the nucleus, consistent with a direct role in Rpc53 phosphorylation.

Figure 3. TORC1 activity determines the expression, phosphorylation state and localization of Kns1.

Figure 3

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A Phos-tag™ acrylamide gel analysis of a W303 strain containing wild-type Kns1-myc or a kinase dead (KD) mutant on pRS425. Cells grown to mid-log phase were treated with rapamycin or drug vehicle before extract preparation and myc immunoblotting. Sample loading was normalized for total Kns1 protein (see Figure S3C). B. Kns1-myc localization was determined by indirect immunofluoresence (Alexa 488) in a W303-derived strain where KNS1 expression was driven by the TDH3 promoter. Kns1 nuclear localization is confirmed by the overlap with DAPI nuclear staining (merge). Cells were mock-treated (left panels) or rapamycin-treated (right panels) for 60 min. Images are representative of multiple independent experiments. C Quantitation of Kns1-myc localization. Localization was assigned by manual inspection of >120 untreated cells (black bars) and >170 rapamycin-treated cells (green bars) over several independent fields to the nucleus (colocalization with DAPI), the cytoplasm (outside the DAPI signal) or both compartments (localization throughout cell). Lower and upper limits of fluorescence intensity were set to the background level (no primary antibody) and to the maximum signal per field, respectively. D Time course of Kns1 induction by rapamycin. Samples were resolved by SDS-PAGE (no Phos-tag) and Kns1-myc was quantified and normalized to Tfc1 (as in Figure S3A). The fold change in Kns1 is indicated. E Induction of Kns1 by MMS was determined as in panel D. F Overexpression of Kns1 causes hyperphosphorylation of Rpc53. Wild-type and TDH3pr-Kns1 strains (derived from RPC53del) were treated with rapamycin for 1 hr and extracts were blotted for RPC53-HA. Data in panels D, E and F are representative of multiple experiments.

To investigate the biochemical relationship between Kns1 and Mck1 in the phosphorylation of Rpc53, we purified both GST-tagged kinases from yeast and assayed their activity against recombinant Rpc53. Wild-type GST-Kns1 but not a kinase dead mutant was active for autophosphorylation and strongly phosphorylated Rpc53 (Figure 4A, upper panel). In contrast, wild-type GST-Mck1 which was active in autophosphorylation showed only background levels of Rpc53 phosphorylation, comparable to the kinase-dead GST-Mck1 mutant (Figure 4B, upper panel). Phosphoproteomic studies have identified 11 phosphosites in Rpc53 (see Figure S1A legend). To map the site(s) phosphorylated by Kns1 in vitro, we initially generated two mutants (M1A and M2A), containing clustered alanine substitutions at multiple phosphosites (Figure 4C). The wild-type and M2A mutant Rpc53 proteins were comparable substrates for recombinant Kns1, whereas phosphorylation of the M1A mutant was undetectable (Figure 4D). Further mutagenesis mapped a single phosphorylation site within the M1A cluster at T232 which occurs in a proposed RXXS/TP consensus motif for Kns1 identified in native proteins (Figures 4C and 4D; (Nikolakaki et al., 2002). A hallmark of GSK3 kinases is that their efficient phosphorylation of many substrates requires a priming phosphate at the +4 position of their recognition sequence (Cohen and Frame, 2001). It was potentially significant therefore that the phosphosite at T232 was located at the +4 position in one of two overlapping consensus GSK3 motifs (Figure 4C). Thus, Kns1 phosphorylation of Rpc53 at T232 could prime GSK3 phosphorylation at T228 and subsequently at S224. To test this possibility, we assayed the effect of recombinant Kns1 activity on Rpc53 phosphorylation by GST-Mck1. The presence of Kns1 enabled wild-type GST-Mck1 but not its kinase-dead mutant to phosphorylate Rpc53 and quantitatively converted the fast migrating species containing phosphothreonine at position 232 into a slower migrating species (Figure 4E). This in vitro reaction recapitulates the change in Rpc53 mobility seen in vivo under repressing conditions (Figure 1). The requirement for priming of Rpc53 by Kns1 and the subsequent change in mobility upon phosphorylation by Mck1 explains how deletion of either kinase abolished the appearance of hyperphosphorylated Rpc53 (Figure 1). Consistent with this, yeast cells containing the non-primable RPC53 mutations T232A or M1A or mutations at the adjacent Mck1 sites (S224A and T228A) do not exhibit the slower migrating form of Rpc53 upon treatment with rapamycin (Figure 4F). Moreover, Mck1 exhibited negligible activity against wild-type Rpc53 in the absence of Kns1 or against the T232A mutant protein in the presence of Kns1 (Figure S4B). Finally, Mck1 was able to phosphorylate, albeit weakly, the Rpc53 phosphomimic T232E to produce the slower migrating species (Figure S4B).

Figure 4. Kns1 is a priming kinase for Mck1 phosphorylation of Rpc53.

Figure 4

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A. Equal amounts of wild-type or kinase-dead (KD) GST-Kns1, detected by immunoblotting (α-GST), were used to phosphorylate recombinant Rpc53 (C53-His) with [γ-32P]ATP. B. Equal amounts of wild-type or kinase-dead GST-Mck1, detected by immunoblotting (α-His5), were used to phosphorylate Rpc53 as in panel A. Note that the low level of GST-Mck1 and Rpc53 phosphorylation seen with kinase-dead GST-Mck1 is due to a copurifying kinase. C. Schematic showing the known phosphosites in Rpc53 and the location of mutants M1A and M2A, which contained triple phosphosite substitutions to alanine. The sequence around the M1 site is shown with a proposed Kns1 motif (RXXS/TP) underlined and the phosphosite boxed in grey. Two overlapping GSK3 motifs (S/TXXXS/T) direct Mck1 phosphorylation of primed substrates (blue arrows) at phosphosites boxed in grey. D. In vitro phosphorylation of wild-type and mutant Rpc53 proteins by recombinant wild-type and kinase-dead Kns1 with [γ-32P]ATP. Equal loading of Rpc53 is shown by western blot (WB) E. Rpc53 phosphorylation was performed as above by titratingequal amounts of wild-type or kinase-dead GST-Mck1 in the presence of a constant amount of recombinant Kns1. F. Plasmid shuffling was used to introduce the indicated RPC53-HA alleles into a BY4741-related strain containing a chromosomal deletion of RPC53. Extracts were analyzed by immunblotting before and after rapamycin treatment. G The wild-type and M1A mutant Rpc53 strains shown in panel F were transformed with a plasmid containing S. pombe Rpc11 under GAL promoter control followed by deletion of the chromosomal S. cerevisiae RPC11 gene (rpc11Δ∷natR). Northern analysis of pre-tRNALeu and U4 snRNA was performed before and after rapamycin treatment. H. Normalized pre-tRNALeu band intensities were used to determine the level of transcription relative to the untreated wild-type Rpc53 strain. Results from three biological replicates are plotted ± s.d. I Model of TOR regulation of pol III transcription. Nutrients and stress regulate Maf1 via Sch9 and PKA and regulate pol III via Kns1 and Mck1. Under nutrient replete conditions Sch9 and PKA phosphorylate Maf1 and inhibit its ability to repress transcription. This inhibition of Maf1 is relieved under stress conditions which also result in the phosphorylation of Rpc53 by Kns1 and Mck1 and changes in polymerase function. Rpc53 and its partner Rpc37 function in transcription elongation and interact with Rpc11 which functions in proofreading, termination, facilitated recycling and cooperates with Rpc53 in enabling repression by Maf1. The inability of Maf1 to inhibit elongating or recycling polymerases suggests that Kns1 and Mck1 phosphorylation of Rpc53 leads to inhibition of recycling or promotes polymerase dissociation after termination to allow interactions with Maf1 that prevent subsequent reinitiation (see text for details). Dotted lines represent interactions that are indirect (e.g. Sch9 to PKA, (Soulard et al., 2010) and TORC1-regulated expression of Kns1, Figure 3D). Arrows and bars indicate positive and negative consequences, respectively, on a protein function or process.

While Kns1 and Mck1 function together in phosphorylating Rpc53, the enhanced capacity of the kns1Δ mck1Δ double mutant to attenuate pol III repression relative to the single mutants (Figures 2A and S2A) indicates that these kinases must also function independently of one another in their response to repressing conditions. Accordingly, other targets of these kinases are predicted to participate, along with Rpc53, in the transcriptional response to TORC1 inhibition. In support of this, we found that mutation of the Kns1 and Mck1 phosphorylation sites in Rpc53 is not sufficient to attenuate repression of pol III transcription by rapamycin (Figure S4A). In considering other proteins that might function with Rpc53 to repress transcription, we focussed on Rpc11 since its stable association with the polymerase has been reported to depend on Rpc53 and its partner Rpc37 (Landrieux et al., 2006). Rpc11 functions in exoribonucleolytic proofreading, termination and facilitated reinitiation, a process that enables high rates of pol III transcription in vitro by retaining the polymerase on the template after termination and allowing it to be recycled for multiple rounds of initiation (Dieci and Sentenac, 1996; Landrieux et al., 2006; Iben et al., 2011). Importantly, pol III molecules engaged in transcription or undergoing facilitated reinitiation are resistant to repression by Maf1 implying that polymerase recycling is interrupted during Maf1-dependent repression (Cabart et al., 2008; Vannini et al., 2010). To examine the contribution of Rpc11 in transcriptional repression we used a sensitized strain where the essential function of Rpc11 is provided by its S. pombe homolog. In this context, the Rpc53 M1A mutant showed elevated transcription during log phase growth and a defect in repression following rapamycin treatment (Figures 4G and 4H). This confirms the negative regulatory role of Rpc53 phosphorylation at the M1A sites and demonstrates a cooperative function of Rpc11 in transcriptional repression. These findings are consistent with a mechanism of repression where changes in Rpc53 and Rpc11 disrupt facilitated recycling or favor polymerase dissociation from the template after termination, thereby allowing an interaction between Maf1 and pol III that inhibits reinitiation (Fig. 4I).

The posttranslational modification of pol III function by Kns1 and Mck1, two downstream kinases in the rapamycin-sensitive TORC1 pathway, reveals a previously unknown branch to the regulatory system controlling protein synthetic capacity (Fig. 4I). The conservation of the components of this system suggests that similar regulatory mechanisms are likely to operate in higher eukaryotes. For example, the recent discovery that the ribosome is an activating cofactor for TORC2 in both yeast and mammals has revealed that TORC1 regulation of ribosome synthesis coordinates TORC2-regulated functions with cell growth (Zinzalla et al., 2011). Thus, the conservation of LAMMER and GSK3 family kinases and their role in regulating ribosome synthesis in yeast suggests that they contribute to ribosome-mediated control of TORC2. Finally, we note that TORC1 regulates growth-related processes besides ribosome and tRNA synthesis (Wullschleger et al., 2006). Our findings suggest that an examination of LAMMER and GSK3 kinase function in other rapamycin-sensitive processes is likely to be fruitful.

Experimental Procedures

Yeast strains and methods

Yeast strains werederived from S288C or W303 (Table S1) and were cultured in YPD or synthetic complete media as required, with or without drug treatments to repress transcription (see Supplemental Experimental Procedures). SGA methods were used to introduce an RPC53-HA∷hphR allele constructed in strain Y7092 into a mini-array of yeast gene-deletion strains (Table S2) by sequential robotic pinning (RoToR, Singer Instruments) (Tong and Boone, 2007). Methods for fixation of cells, immunofluorescence microscopy and analysis of optical sections have been described (Moir et al., 2006).

Analysis of RNA

Methods for [C3H3] methionine and [5,6-3H]-uracil pulse-chase labeling and for RNA preparation and Northern analysis have been reported elsewhere (Upadhya et al., 2002; Moir et al., 2006; Huber et al., 2009). For real-time PCR analysis of mRNAs, DNase-treated total RNA was reverse transcribed and the cDNA was quantified using Taqman probes on an ABI Prism 7900HT system (Zhao et al., 2006).

Proteins and in vitro kinase assays

The preparation of whole cell extracts for immunoblotting employed cells harvested at room temperature by low speed centrifugation and denaturing lysis as reported elsewhere (Moir et al., 2006; Huber et al., 2009). Phos-tag™ acrylamide was obtained from the NARD Institute and was used at a final concentration of 25 μM. The purification of GST-kinases from yeast and recombinant proteins from E. coli and their use for in vitro kinase assays was performed accordingly to published methods (Moir et al., 2006; Lee et al., 2009). Additional details are provided in the Supplemental Experimental Procedures.

Supplementary Material

01
02

Highlights.

  • Kns1 and Mck1 are conserved protein kinases that function downstream of TORC1

  • Efficient repression of ribosome and tRNA synthesis requires Kns1 and Mck1

  • RNA polymerase III is a target of nutrient and stress signaling pathways

  • Phosphorylation of Rpc53 in cooperation with Rpc11 enables repression by Maf1

Acknowledgments

We thank Jon Warner for comments on the manuscript, Mari Carmen Fernandez Ramirez for strain construction, George Kassavetis for the galactose-inducible S. pombe Rpc11 plasmid and Rich Maraia for S. pombe Rpc11 antibodies. This work was supported by NIH grant RO1 GM085177 (I.M.W.). K.B.M was supported by NIH grant R01 GM025532 to Jon Warner.

Footnotes

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Supplementary Materials

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NIHMS353780-supplement-01.doc (3.7MB, doc)
02
NIHMS353780-supplement-02.xls (93KB, xls)

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