TOR Complex 2- independent mutations in the regulatory PIF pocket of Gad8AKT1/SGK1 define separate branches of the stress response mechanisms in fission yeast.
ABSTRACT: The Target of rapamycin (TOR) protein kinase forms part of TOR complex 1 (TORC1) and TOR complex 2 (TORC2), two multi-subunit protein complexes that regulate growth, proliferation, survival and developmental processes by phosphorylation and activation of AGC-family kinases. In the fission yeast, Schizosaccharomyces pombe, TORC2 and its target, the AGC kinase Gad8 (an orthologue of human AKT or SGK1) are required for viability under stress conditions and for developmental processes in response to starvation cues. In this study, we describe the isolation of gad8 mutant alleles that bypass the requirement for TORC2 and reveal a separation of function of TORC2 and Gad8 under stress conditions. In particular, osmotic and nutritional stress responses appear to form a separate branch from genotoxic stress responses downstream of TORC2-Gad8. Interestingly, TORC2-independent mutations map into the regulatory PIF pocket of Gad8, a highly conserved motif in AGC kinases that regulates substrate binding in PDK1 (phosphoinositide dependent kinase-1) and kinase activity in several AGC kinases. Gad8 activation is thought to require a two-step mechanism, in which phosphorylation by TORC2 allows further phosphorylation and activation by Ksg1 (an orthologue of PDK1). We focus on the Gad8-K263C mutation and demonstrate that it renders the Gad8 kinase activity independent of TORC2 in vitro and independent of the phosphorylation sites of TORC2 in vivo. Molecular dynamics simulations of Gad8-K263C revealed abnormal high flexibility at T387, the phosphorylation site for Ksg1, suggesting a mechanism for the TORC2-independent Gad8 activity. Significantly, the K263 residue is highly conserved in the family of AGC-kinases, which may suggest a general way of keeping their activity in check when acting downstream of TOR complexes.
Project description:From yeast to human, TOR (target of rapamycin) kinase plays pivotal roles in coupling extracellular stimuli to cell growth and metabolism. TOR kinase functions in two distinct protein complexes, TOR complex 1 (TORC1) and 2 (TORC2), which phosphorylate and activate different AGC-family protein kinases. TORC1 is controlled by the small GTPase Rheb, but little is known about TORC2 regulators.We have identified the Ryh1 GTPase, a human Rab6 ortholog, as an activator of TORC2 signaling in the fission yeast Schizosaccharomyces pombe. Mutational inactivation of Ryh1 or its guanine nucleotide exchange factor compromises the TORC2-dependent phosphorylation of the AGC-family Gad8 kinase. In addition, the effector domain of Ryh1 is important for its physical interaction with TORC2 and for stimulation of TORC2 signaling. Thus, GTP-bound Ryh1 is likely to be the active form stimulatory to TORC2-Gad8 signaling. Consistently, expression of the GTP-locked mutant Ryh1 is sufficient to promote interaction between TORC2 and Gad8 and to induce Gad8 hyperphosphorylation. The loss of functional Ryh1, TORC2, or Gad8 brings about similar vacuolar fragmentation and stress sensitivity, further corroborating their involvement in a common cellular process. Human Rab6 can substitute Ryh1 in S. pombe, and therefore Rab6 may be a potential activator of TORC2 in mammals.In its GTP-bound form, Ryh1, an evolutionarily conserved Rab GTPase, activates TORC2 signaling to the AGC kinase Gad8. The Ryh1 GTPase and the TORC2-Gad8 pathway are required for vacuolar integrity and cellular stress resistance in S. pombe.
Project description:TOR (target of rapamycin) signaling coordinates cell growth, metabolism, and cell division through tight control of signaling via two complexes, TORC1 and TORC2. Here, we show that fission yeast TOR kinases and mTOR are phosphorylated on an evolutionarily conserved residue of their ATP-binding domain. The Gad8 kinase (AKT homologue) phosphorylates fission yeast Tor1 at this threonine (T1972) to reduce activity. A T1972A mutation that blocked phosphorylation increased Tor1 activity and stress resistance. Nitrogen starvation of fission yeast inhibited TOR signaling to arrest cell cycle progression in G1 phase and promoted sexual differentiation. Starvation and a Gad8/T1972-dependent decrease in Tor1 (TORC2) activity was essential for efficient cell cycle arrest and differentiation. Experiments in human cell lines recapitulated these yeast observations, as mTOR was phosphorylated on T2173 in an AKT-dependent manner. In addition, a T2173A mutation increased mTOR activity. Thus, TOR kinase activity can be reduced through AGC kinase-controlled phosphorylation to generate physiologically significant changes in TOR signaling.
Project description:Cell proliferation, metabolism, migration and survival are coordinated through the tight control of two target of rapamycin (TOR) kinase complexes: TORC1 and TORC2. Here, we show that a novel phosphorylation of fission yeast Gad8 (AGC kinase) on the evolutionarily conserved threonine 6 (Thr6) prevents the physical association between Gad8 and TORC2. Accordingly, this block to protein interactions by Gad8 Thr6 phosphorylation decreases TORC2-controlled activation of Gad8. Likewise, phosphorylation of Gad8 Thr6, possibly by PKC, prevents the association of Gad8 with TORC2 thereby increasing TORC2 activity, because it reduces Gad8-mediated feedback inhibition of TORC2. Consistently, the introduction of a Gad8 T6D mutant, that mimics phosphorylation, increased TORC2 activity. Increased PKC(Pck2) expression prevented Gad8-TORC2 binding and so reduced the TORC2-mediated phosphorylation of Gad8 serine 546 that activates Gad8. Interestingly, independent of the Ser546 phosphorylation status, Gad8 Thr6 phosphorylation is important for remodelling the actin cytoskeleton and survival upon potassium ion and heat stresses. In contrast, Ser546 phosphorylation is required for the control of G1 arrest, mating, cell length at division and vascular size. Finally, these findings reveal a novel mode of TORC2 activation that is essential for cell survival following stress.
Project description:TOR (Target Of Rapamycin) signalling coordinates cell growth and division in response to changes in the nutritional environment of the cell. TOR kinases form two distinct complexes: TORC1 and TORC2. In mammals, the TORC1 controlled S6K1 kinase phosphorylates the ribosomal protein S6 thereby co-ordinating cell size and nutritional status. We show that the Schizosaccharomyces pombe AGC kinase Gad8 co-immunoprecipitates with the ribosomal protein S6 (Rps6) and regulates its phosphorylation status. It has previously been shown that Gad8 is phosphorylated by TORC2. Consistent with this, we find that TORC2 as well as TORC1 modulates Rps6 phosphorylation. Therefore, S6 phosphorylation in fission yeast actually represents a read-out of the combined activities of TORC1 and TORC2. In contrast, we find that the in vivo phosphorylation status of Maf1 (a repressor of RNA polymerase III) specifically correlates with TORC1 activity.
Project description:The target of rapamycin (TOR) protein kinase forms multi-subunit TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which exhibit distinct substrate specificities. Sin1 is one of the TORC2-specific subunit essential for phosphorylation and activation of certain AGC-family kinases. Here, we show that Sin1 is dispensable for the catalytic activity of TORC2, but its conserved region in the middle (Sin1CRIM) forms a discrete domain that specifically binds the TORC2 substrate kinases. Sin1CRIM fused to a different TORC2 subunit can recruit the TORC2 substrate Gad8 for phosphorylation even in the sin1 null mutant of fission yeast. The solution structure of Sin1CRIM shows a ubiquitin-like fold with a characteristic acidic loop, which is essential for interaction with the TORC2 substrates. The specific substrate-recognition function is conserved in human Sin1CRIM, which may represent a potential target for novel anticancer drugs that prevent activation of the mTORC2 substrates such as AKT.
Project description:Phosphoinositide-dependent protein kinase 1 (PDK1) plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases. This family includes protein kinase C (PKC), protein kinase B (PKB), p70/p90 ribosomal S6 kinases (RSK and S6K), and the catalytic subunit of cAMP-dependent protein kinase (PKA). Although PDK1 phosphorylates and activates PKC, PKB, and RSK in vivo, PDK1 regulation of PKA remains controversial. We isolated ksg1, the fission yeast ortholog of mammalian PDK1, as a suppressor of growth defects caused by loss of the stress-activated MAP kinase, Spc1. Here, we demonstrate that Ksg1 is required for activation of PKA. Cells containing the ksg1.12 thermolabile allele exhibit pleiotropic phenotypes, including the failure to arrest in G(1) and an inability to conjugate. The ksg1.12 allele strongly suppresses defects associated with unregulated PKA. Pka1, the catalytic subunit of cAMP-dependent protein kinase, is phosphorylated in vivo at Thr-356, which is located in the activation loop of the kinase and corresponds to Thr-197 in mammalian PKA. Phosphorylation of Thr-356 is required for in vivo activation of Pka1 and is dependent upon Ksg1. These data provide experimental evidence that PKA is a physiological substrate for PDK1.
Project description:Protein kinases AKT and PKBR1 of Dictyostelium belong to the AGC protein kinase superfamily. AKT and PKBR1 are phosphorylated at similar sites by phosphoinositide-dependent kinase 1 (PDK1) and TORC2 kinases; however, they have different subcellular localizing domains. AKT has a phosphoinositide 3-kinase (PI3K)/phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)]-regulated PH (pleckstrin homology) domain whereas PKBR1 is myristoylated and persistently membrane localized. Using strains defective for PI3K/PtdIns(3,4,5)P(3)-, PDK1- and TORC2-signaling or strains that express phospho-site mutants of AKT and PKBR1, we dissect the different roles of PI3K/PtdIns(3,4,5)P(3), PDK1 and TORC2. We show that activation of AKT and PKBR1 requires PDK1-site phosphorylation, but that phosphorylation by TORC2 is insufficient for AKT or PKBR1 activation. However, PDK1-site phosphorylation is dependent on phosphorylation by TORC2, which suggests that there is regulatory coordination among PDK1, TORC2 and their phospho-site targets. This defines a separate input for signaling in control of chemotaxis and dependency on PDK1 function. We also demonstrate that PDK1 in Dictyostelium functions independently of PI3K/PtdIns(3,4,5)P(3). Finally, we show that AKT and PKBR1 exhibit substrate selectivity and identify two novel lipid-interacting proteins preferentially phosphorylated by AKT. Despite certain similarities, AKT and PKBR1 have distinct regulatory paths that impact activation and effector targeting, with PDK1 serving a central role.
Project description:TOR proteins reside in two distinct complexes, TOR complexes 1 and 2 (TORC1 and TORC2), that are central for the regulation of cellular growth, proliferation, and survival. TOR is also the target for the immunosuppressive and anticancer drug rapamycin. In Schizosaccharomyces pombe, disruption of the TSC complex, mutations in which can lead to the tuberous sclerosis syndrome in humans, results in a rapamycin-sensitive phenotype under poor nitrogen conditions. We show here that the sensitivity to rapamycin is mediated via inhibition of TORC1 and suppressed by overexpression of isp7(+), a member of the family of 2-oxoglutarate-Fe(II)-dependent oxygenase genes. The transcript level of isp7(+) is negatively regulated by TORC1 but positively regulated by TORC2. Yet we find extensive similarity between the transcriptome of cells disrupted for isp7(+) and cells mutated in the catalytic subunit of TORC1. Moreover, Isp7 regulates amino acid permease expression in a fashion similar to that of TORC1 and opposite that of TORC2. Overexpression of isp7(+) induces TORC1-dependent phosphorylation of ribosomal protein Rps6 while inhibiting TORC2-dependent phosphorylation and activation of the AGC-like kinase Gad8. Taken together, our findings suggest a central role for Isp7 in amino acid homeostasis and the presence of isp7(+)-dependent regulatory loops that affect both TORC1 and TORC2.
Project description:The Target Of Rapamycin (TOR) is an evolutionarily conserved protein kinase that forms 2 distinct protein complexes referred to as TOR complex 1 (TORC1) and 2 (TORC2). Recent extensive studies have demonstrated that TORC1 is under the control of the small GTPases Rheb and Rag that funnel multiple input signals including those derived from nutritional sources; however, information is scarce as to the regulation of TORC2. A previous study using the model system provided by the fission yeast Schizosaccharomyces pombe identified Ryh1, a Rab-family GTPase, as an activator of TORC2. Here, we show that the nucleotide-binding state of Ryh1 is regulated in response to glucose, mediating this major nutrient signal to TORC2. In glucose-rich growth media, the GTP-bound form of Ryh1 induces TORC2-dependent phosphorylation of Gad8, a downstream target of TORC2 in fission yeast. Upon glucose deprivation, Ryh1 becomes inactive, which turns off the TORC2-Gad8 pathway. During glucose starvation, however, Gad8 phosphorylation by TORC2 gradually recovers independently of Ryh1, implying an additional TORC2 activator that is regulated negatively by glucose. The paired positive and negative regulatory mechanisms may allow fine-tuning of the TORC2-Gad8 pathway, which is essential for growth under glucose-limited environment.
Project description:In eukaryotic cells, the highly conserved Target of Rapamycin (TOR) and the Mitogen Activated Protein Kinase (MAPK) signaling pathways elicit adaptive responses to extra- and intracellular conditions by regulating essential cellular functions. However, the nature of the functional relationships between both pathways is not fully understood. In the fission yeast Schizosaccharomyces pombe the cell integrity MAPK pathway (CIP) regulates morphogenesis, cell wall structure and ionic homeostasis. We show that the Rab GTPase Ryh1, a TORC2 complex activator, cross-activates the CIP and its core member, the MAPK Pmk1, by two distinct mechanisms. The first one involves TORC2 and its downstream effector, Akt ortholog Gad8, which together with TORC1 target Psk1 increase protein levels of the PKC ortholog Pck2 during cell wall stress or glucose starvation. Also, Ryh1 activates Pmk1 in a TORC2-independent fashion by prompting plasma membrane trafficking and stabilization of upstream activators of the MAPK cascade, including PDK ortholog Ksg1 or Rho1 GEF Rgf1. Besides, stress-activated Pmk1 cross-inhibits Ryh1 signaling by decreasing the GTPase activation cycle, and this ensures cell growth during alterations in phosphoinositide metabolism. Our results reveal a highly intricate cross-regulatory relationship between both pathways that warrants adequate cell adaptation and survival in response to environmental changes.