TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae

Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously.     

Regulation of TORC2 function and localization by Rab5 GTPases in Saccharomyces cerevisiae

The evolutionarily conserved Target of Rapamycin (TOR) complex-2 (TORC2) is an essential regulator of plasma membrane homeostasis in budding yeast (Saccharomyces cerevisiae). In this yeast, TORC2 phosphorylates and activates the effector protein kinase Ypk1 and its paralog Ypk2. These protein kinases, in turn, carry out all the crucial functions of TORC2 by phosphorylating and thereby controlling the activity of at least a dozen downstream substrates. A previously uncharacterized interplay between the Rab5 GTPases and TORC2 signaling was uncovered through analysis of a newly suspected Ypk1 target. Muk1, one of two guanine nucleotide exchange factors for the Rab5 GTPases, was found to be a physiologically relevant Ypk1 substrate; and, genetic analysis indicates that Ypk1-mediated phosphorylation activates the guanine nucleotide exchange activity of Muk1. Second, it was demonstrated both in vivo and in vitro that the GTP-bound state of the Rab5 GTPase Vps21/Ypt51 physically associates with TORC2 and acts as a direct positive effector required for full TORC2 activity. These interrelationships provide a self-reinforcing control circuit for sustained up-regulation of TORC2-Ypk1 signaling. In this overview, we summarize the experimental basis of these findings, their implications, and speculate as to the molecular basis for Rab5-mediated TORC2 activation.     

The requirement for the hydrophobic motif phosphorylation of Ypk1 in yeast differs depending on the downstream events, including endocytosis, cell growth, and resistance to a sphingolipid biosynthesis inhibitor, ISP-1

ISP-1 inhibits de novo sphingolipid biosynthesis and induces growth defects in both mammals and yeast (Saccharomyces cerevisiae). In our previous study, YPK1/SLI2 was identified as one of multicopy suppressor genes for ISP-1 in yeast. Ypk1 is proposed to be a downstream serine/threonine kinase of the sphingolipid signaling pathway in yeast. Other than resistance against ISP-1, Ypk1 is involved in at least two downstream events, namely cell growth and endocytosis. In this study, the effect of mutants of Ypk1 on these three downstream events was investigated. Among Ypk1 mutants, no 'kinase-dead' mutants complemented the defects in any of these three downstream events in the ypk1 null strain. One of the hydrophobic motif phosphorylation-deficient mutants of Ypk1, Ypk1(T662A) had the moderate kinase activity compared with the wild-type Ypk1. Ypk1(T662A) and the wild-type Ypk1 completely restored the slow-growth phenotype and fluid-phase endocytosis defect of the ypk1 null strain. However, unlike the wild-type Ypk1, Ypk1(T662A) lost the ability for the recovery of the ISP-1 resistance in the ypk1 null strain. Furthermore, the expression of Ypk1(T662A) in the wild-type strain showed a dominant-negative effect on the ISP-1-resistance activity. On the other hand, the cell growth revertant of the ypk1 null strain still showed the hypersensitive phenotype to ISP-1. These data suggest that the ISP-1-resistance pathway is under the regulation of the hydrophobic motif phosphorylation and is separated from the other pathways downstream of Ypk1.     

Target of rapamycin complex 2 signals to downstream effector yeast protein kinase 2 (Ypk2) through adheres-voraciously-to-target-of-rapamycin-2 protein 1 (Avo1) in Saccharomyces cerevisiae

The conserved Ser/Thr kinase target of rapamycin (TOR) serves as a central regulator in controlling cell growth-related functions. There exist two distinct TOR complexes, TORC1 and TORC2, each coupling to specific downstream effectors and signaling pathways. In Saccharomyces cerevisiae, TORC2 is involved in regulating actin organization and maintaining cell wall integrity. Ypk2 (yeast protein kinase 2), a member of the cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase family, is a TORC2 substrate known to participate in actin and cell wall regulation. Employing avo3(ts) mutants with defects in TORC2 functions that are suppressible by active Ypk2, we investigated the molecular interactions involved in mediating TORC2 signaling to Ypk2. GST pulldown assays in yeast lysates demonstrated physical interactions between Ypk2 and components of TORC2. In vitro binding assays revealed that Avo1 directly binds to Ypk2. In avo3(ts) mutants, the TORC2-Ypk2 interaction was reduced and could be restored by AVO1 overexpression, highlighting the important role of Avo1 in coupling TORC2 to Ypk2. The interaction was mapped to an internal region (amino acids 600-840) of Avo1 and a C-terminal region of Ypk2. Ypk2(334-677), a truncated form of Ypk2 containing the Avo1-interacting region, was able to interfere with Avo1-Ypk2 interaction in vitro. Overexpressing Ypk2(334-677) in yeast cells resulted in a perturbation of TORC2 functions, causing defective cell wall integrity, aberrant actin organization, and diminished TORC2-dependent Ypk2 phosphorylation evidenced by the loss of an electrophoretic mobility shift. Together, our data support the conclusion that the direct Avo1-Ypk2 interaction is crucial for TORC2 signaling to the downstream Ypk2 pathway.     

Plasma membrane proteins Slm1 and Slm2 mediate activation of the AGC kinase Ypk1 by TORC2 and sphingolipids in S. cerevisiae

The PH domain-containing proteins Slm1 and Slm2 were originally identified as substrates of the rapamycin-insensitive TOR complex 2 (TORC2) and as mediators of signaling by the lipid second messenger phosphatidyl-inositol-4,5-bisphosphate (PI4,5P2) in budding yeast S. cerevisiae. More recently, these proteins have been identified as critical effectors that facilitate phosphorylation and activation of the AGC kinases Ypk1 and Ypk2 by TORC2.¹ Here, we review the molecular basis for this regulation as well as place it within the context of recent findings that have revealed Slm1/2 and TORC2-dependent phosphorylation of Ypk1 is coupled to the biosynthesis of complex sphingolipids and to their levels within the plasma membrane (PM) as well as other forms of PM stress. Together, these studies reveal the existence of an intricate homeostatic feedback mechanism, whereby the activity of these signaling components is linked to the biosynthesis of PM lipids according to cellular need.     

Functional counterparts of mammalian protein kinases PDK1 and SGK in budding yeast

BACKGROUND: In animal cells, recruitment of phosphatidylinositol 3-kinase by growth factor receptors generates 3-phosphoinositides, which stimulate 3-phosphoinositide-dependent protein kinase-1 (PDK1). Activated PDK1 then phosphorylates and activates downstream protein kinases, including protein kinase B (PKB)/c-Akt, p70 S6 kinase, PKC isoforms, and serum- and glucocorticoid-inducible kinase (SGK), thereby eliciting physiological responses. RESULTS: We found that two previously uncharacterised genes of Saccharomyces cerevisiae, which we term PKH1 and PKH2, encode protein kinases with catalytic domains closely resembling those of human and Drosophila PDK1. Both Pkh1 and Pkh2 were essential for cell viability. Expression of human PDK1 in otherwise inviable pkh1Delta pkh2Delta cells permitted growth. In addition, the yeast YPK1 and YKR2 genes were found to encode protein kinases each with a catalytic domain closely resembling that of SGK; both Ypk1 and Ykr2 were also essential for viability. Otherwise inviable ypk1Delta ykr2Delta cells were fully rescued by expression of rat SGK, but not mouse PKB or rat p70 S6 kinase. Purified Pkh1 activated mammalian SGK and PKBalpha in vitro by phosphorylating the same residue as PDK1. Pkh1 activated purified Ypk1 by phosphorylating the equivalent residue (Thr504) and was required for maximal Ypk1 phosphorylation in vivo. Unlike PKB, activation of Ypk1 and SGK by Pkh1 did not require phosphatidylinositol 3,4,5-trisphosphate, consistent with the absence of pleckstrin homology domains in these proteins. The phosphorylation consensus sequence for Ypk1 was similar to that for PKBalpha and SGK. CONCLUSIONS: Pkh1 and Pkh2 function similarly to PDK1, and Ypk1 and Ykr2 to SGK. As in animal cells, these two groups of yeast kinases constitute two tiers of a signalling cascade required for yeast cell growth.     

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.     

Plasma membrane proteins Slm1 and Slm2 mediate activation of the AGC kinase Ypk1 by TORC2 and sphingolipids in S. cerevisiae

The PH domain-containing proteins Slm1 and Slm2 were originally identified as substrates of the rapamycin-insensitive TOR complex 2 (TORC2) and as mediators of signaling by the lipid second messenger phosphatidyl-inositol-4,5-bisphosphate (PI4,5P2) in budding yeast S. cerevisiae. More recently, these proteins have been identified as critical effectors that facilitate phosphorylation and activation of the AGC kinases Ypk1 and Ypk2 by TORC2.¹ Here, we review the molecular basis for this regulation as well as place it within the context of recent findings that have revealed Slm1/2 and TORC2-dependent phosphorylation of Ypk1 is coupled to the biosynthesis of complex sphingolipids and to their levels within the plasma membrane (PM) as well as other forms of PM stress. Together, these studies reveal the existence of an intricate homeostatic feedback mechanism, whereby the activity of these signaling components is linked to the biosynthesis of PM lipids according to cellular need.     

Vacuolar Localization via the N-terminal Domain of Sch9 is Required for TORC1-dependent Phosphorylation and Downstream Signal Transduction

The budding yeast Sch9 kinase (functional orthologue of the mammalian S6 kinase) is a major effector of the Target of Rapamycin Complex 1 (TORC1) complex in the regulation of cell growth in response to nutrient availability and stress. Sch9 is partially localized at the vacuolar surface, where it is phosphorylated by TORC1. The recruitment of Sch9 on the vacuole is mediated by direct interaction between phospholipids of the vacuolar membrane and the region of Sch9 encompassing amino acid residues 1-390, which contains a C2 domain. Since many C2 domains mediate phospholipid binding, it had been suggested that the C2 domain of Sch9 mediates its vacuolar recruitment. However, the in vivo requirement of the C2 domain for Sch9 localization had not been demonstrated, and the phenotypic consequences of Sch9 delocalization remained unknown. Here, by examining cellular localization, phosphorylation state and growth phenotypes of Sch9 truncation mutants, we show that deletion of the N-terminal domain of Sch9 (aa 1-182), but not the C2 domain (aa 183-399), impairs vacuolar localization and TORC1-dependent phosphorylation of Sch9, while causing growth defects similar to those observed in sch9Δ cells. These defects can be reversed either via artificial tethering of the protein to the vacuole, or by introducing phosphomimetic mutations at the TORC1 target sites, suggesting that Sch9 localization on the vacuole is needed for the TORC1-dependent activation of the kinase. Our study uncovers a key role for the N-terminal domain of Sch9 and provides new mechanistic insight into the regulation of a major TORC1 signaling branch.     

Target of rapamycin and LST8 proteins associate with membranes from the endoplasmic reticulum in the unicellular green alga Chlamydomonas reinhardtii

The highly conserved target of rapamycin (TOR) kinase is a central controller of cell growth in all eukaryotes. TOR exists in two functionally and structurally distinct complexes, termed TOR complex 1 (TORC1) and TORC2. LST8 is a TOR-interacting protein that is present in both TORC1 and TORC2. Here we report the identification and characterization of TOR and LST8 in large protein complexes in the model photosynthetic green alga Chlamydomonas reinhardtii. We demonstrate that Chlamydomonas LST8 is part of a rapamycin-sensitive TOR complex in this green alga. Biochemical fractionation and indirect immunofluorescence microscopy studies indicate that TOR and LST8 exist in high-molecular-mass complexes that associate with microsomal membranes and are particularly abundant in the peri-basal body region in Chlamydomonas cells. A Saccharomyces cerevisiae complementation assay demonstrates that Chlamydomonas LST8 is able to functionally and structurally replace endogenous yeast LST8 and allows us to propose that binding of LST8 to TOR is essential for cell growth.     

Roles for PI(3,5)P2 in nutrient sensing through TORC1

TORC1, a conserved protein kinase, regulates cell growth in response to nutrients. Localization of mammalian TORC1 to lysosomes is essential for TORC1 activation. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂), an endosomal signaling lipid, is implicated in insulin-dependent stimulation of TORC1 activity in adipocytes. This raises the question of whether PI(3,5)P₂ is an essential general regulator of TORC1. Moreover, the subcellular location where PI(3,5)P₂ regulates TORC1 was not known. Here we report that PI(3,5)P₂ is required for TORC1 activity in yeast and regulates TORC1 on the vacuole (lysosome). Furthermore, we show that the TORC1 substrate, Sch9 (a homologue of mammalian S6K), is recruited to the vacuole by direct interaction with PI(3,5)P₂, where it is phosphorylated by TORC1. Of importance, we find that PI(3,5)P₂ is required for multiple downstream pathways via TORC1-dependent phosphorylation of additional targets, including Atg13, the modification of which inhibits autophagy, and phosphorylation of Npr1, which releases its inhibitory function and allows nutrient-dependent endocytosis. These findings reveal PI(3,5)P₂ as a general regulator of TORC1 and suggest that PI(3,5)P₂ provides a platform for TORC1 signaling from lysosomes.     

Pkh1 and Pkh2 differentially phosphorylate and activate Ypk1 and Ykr2 and define protein kinase modules required for maintenance of cell wall integrity

Saccharomyces cerevisiae Pkh1 and Pkh2 are functionally redundant homologs of mammalian protein kinase, phosphoinositide-dependent protein kinase-1. They activate two closely related, functionally redundant enzymes, Ypk1 and Ykr2 (homologs of mammalian protein kinase, serum- and glucocorticoid-inducible protein kinase). We found that Ypk1 has a more prominent role than Ykr2 in mediating their shared essential function. Considerable evidence demonstrated that Pkh1 preferentially activates Ypk1, whereas Pkh2 preferentially activates Ykr2. Loss of Pkh1 (but not Pkh2) reduced Ypk1 activity; conversely, Pkh1 overexpression increased Ypk1 activity more than Pkh2 overexpression. Loss of Pkh2 reduced Ykr2 activity; correspondingly, Pkh2 overexpression increased Ykr2 activity more than Pkh1 overexpression. When overexpressed, a catalytically active C-terminal fragment (kinase domain) of Ypk1 was growth inhibitory; loss of Pkh1 (but not Pkh2) alleviated toxicity. Loss of Pkh2 (but not Pkh1) exacerbated the slow growth phenotype of a ypk1Delta strain. This Pkh1-Ypk1 and Pkh2-Ykr2 dichotomy is not absolute because all double mutants (pkh1Delta ypk1Delta, pkh2Delta ypk1Delta, pkh1Delta ykr2Delta, and pkh2Delta ykr2Delta) were viable. Compartmentation contributes to selectivity because Pkh1 and Ypk1 were located exclusively in the cytosol, whereas Pkh2 and Ykr2 entered the nucleus. At restrictive temperature, ypk1-1(ts) ykr2Delta cells lysed rapidly, but not in medium containing osmotic support. Dosage and extragenic suppressors were selected. Overexpression of Exg1 (major exoglucanase), or loss of Kex2 (endoprotease involved in Exg1 processing), rescued growth at high temperature. Viability was also maintained by PKC1 overexpression or an activated allele of the downstream protein kinase (BCK1-20). Conversely, absence of Mpk1 (distal mitogen-activated protein kinase of the PKC1 pathway) was lethal in ypk1-1(ts) ykr2Delta cells. Thus, Pkh1-Ypk1 and Pkh2-Ykr2 function in a novel pathway for cell wall integrity that acts in parallel with the Pkc1-dependent pathway.