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

Abstract

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.

Keywords: Cyclic AMP (cAMP); GAD8; Glucose; Mitogen-activated Protein Kinase (MAPK); Protein Kinase A (PKA); S. pombe; Stress Response; TORC2.

FIGURE 1.

Gad8 Ser-546 phosphorylation and Gad8 activity are dependent on glucose availability and are diminished in response to stresses. A, Gad8 kinase activity is dependent on TORC2 activity. A wild type strain expressing no tagged gad8⁺ or wild type (WT), Δtor1, Δsat1, or gad8-KD (gad8^K259D, a kinase-dead allele) strains carrying the gad8-HA allele were grown to mid-log phase in YE medium. Gad8-HA was immunoprecipitated and assayed for its activity using a peptide of Fkh2 as a substrate (Fkh2-GST). Phosphorylation of Fkh2 or phosphorylation of Gad8 at Ser-546 was detected with anti-phospho-AKT substrate or anti-Gad8 phosphospecific antibodies, respectively. B, Gad8 kinase activity and Gad8 Ser-546 phosphorylation are diminished in the absence of glucose or in response to stresses. Wild type cells with no tag or cells expressing gad8-HA were grown to mid-log phase and left untreated in rich (YE) or minimal (EMM) media or treated for 1 h with 1 m KCl, 1 m NaCl, 0.2 m CaCl₂, 1 m sorbitol, 200 nm rapamycin or transferred for 1 h to EMM containing proline as the only nitrogen source (EMM-proline), EMM with no nitrogen source (EMM-N), or no carbon source (EMM-G). C, Gad8 kinase activity and Gad8 Ser-546 phosphorylation are rapidly reduced in response to KCl. Wild type cells with no tag or cells expressing gad8-HA were grown to mid-log phase in YE and treated for the indicated times with 1 m KCl. D, Gad8 activity and phosphorylation at Ser-546 are not affected by DNA stress, oxidative stress, reducing conditions, or cell wall stress. Wild type cells with no tag or cells expressing the gad8-HA were grown to mid-log phase and left untreated in rich (YE) media or treated for 1 h with 12 mm hydroxyurea, 0.03% methyl-methanesulfonate (MMS), 40 μm CPT, 1 μm H₂O₂, 15 mm β-mercaptoethanol (βME), or 0.01% SDS. E, calcineurin inhibitor FK506 activates Gad8. Cells were grown as described above and left untreated (YE) or treated for 1 h with FK506 (2 μg/ml). F, metabolic suppressor 2-deoxyglucose has no effect on Gad8 activity. Cells were grown as described above and left untreated (YE) or treated for 1 h with 100 μg/ml 2-DG.

FIGURE 2.

Gad8 Ser-546 phosphorylation and Gad8 activity rapidly respond to changes in glucose availability. A, Gad8 activity and phosphorylation at Ser-546 are rapidly reduced in the absence of glucose. Wild type cells with no tag or cells expressing gad8-HA were grown to mid-log phase in YE and then shifted for 1 h to EMM with 2% glucose or to EMM without glucose for the indicated time (minutes). B, re-feeding of glucose to starved cells re-activates Gad8. Wild type cells with no tag or cells expressing gad8-HA were grown to mid-log phase and shifted to EMM with or without glucose. After 1 h of starvation, 2% glucose was added for the indicated times (minutes).

FIGURE 3.

Glucose is the minimal requirement for Gad8. A, regulation of Gad8 phosphorylation and activity in response to glucose or KCl is independent of protein synthesis. Cells were grown to mid-log and shifted to EMM without glucose or to EMM containing 1 m KCl for 1 h. Following glucose starvation, 2% glucose was re-added for 1 h (+*). When indicated, cycloheximide (100 μg/ml) was added for 30 min. Gad8 in vitro kinase activity and Ser-546 phosphorylation were detected as described above. B, glucose is necessary for Gad8 activation. Cells were grown to mid-log and left untreated (YE) or washed and incubated for 1 h in PBS supplemented with proline (10 mm), NH₄Cl (5 mm), glucose (2%), FK506 (2 μg/ml), or glucose (2%) and FK506 (2 μg/ml). Gad8 in vitro kinase activity and phosphorylation at Ser-546 were detected as described above. C, re-feeding of glucose to cells incubated in PBS is enough to re-activate Gad8. Cells were grown to mid-log phase and then incubated for 1 h in PBS. 2% glucose was added for the indicated times. Gad8 in vitro kinase activity and phosphorylation status at Ser-546 were determined as above. D, glucose is the most efficient carbon source for activation of Gad8. Cells were incubated for 1 h in EMM with no carbon source (−) or EMM supplemented with glucose (2%), low glucose (0.2%), glycerol (3%), sucrose (2%), succinate (2%), galactose (2%), raffinose (2%) or leucine (2%). Gad8 in vitro kinase activity and Ser-546 phosphorylation were determined as above.

FIGURE 4.

Gad8 activity depends on the PKA pathway. A, wild type, Δgit3, Δgpa2, Δgpb1, Δpka1, Δssp1, or Δssp2 mutant cells were grown to mid-log phase. Gad8 in vitro kinase activity and phosphorylation status at Ser-546 were determined as above. B, suppression of Gad8 activity in glucose-depleted conditions is reversed by constitutive activation of the PKA pathway. Wild type (WT) cells or cells lacking pde1⁺ (Δpde1), encoding for phosphodiesterase, were grown to mid-log phase and incubated for 1 h in EMM with or without glucose (2%). Gad8 in vitro kinase activity and phosphorylation status at Ser-546 was determined as above. C, overexpression of gad8⁺ suppresses the genotoxic sensitivity of mutant cells in the PKA pathway. Serial dilutions of exponentially growing wild type (WT), Δgad8, Δgit3, Δgpa2, Δgpb1, or Δpka1 strains transformed with empty vector (pREP1) or pREP1-gad8⁺ were spotted on rich medium (YE) with or without CPT (7.5 μm).

FIGURE 5.

Pmk1-MAPK pathway negatively regulates Gad8 activity. A, Pmk1-MAPK pathway negatively regulates Gad8 activity in response to glucose depletion. Wild type (WT) cells or cells lacking rho2⁺ (Δrho2), pck2⁺ (Δpck2), or pmk1⁺ (Δpmk1) were grown as described by mid-log phase, washed, and incubated for 1 h in EMM with or without glucose (2%). Gad8 in vitro kinase activity and Ser-546 phosphorylation were determined as above. B, Pmk1-MAPK pathway negatively regulates Gad8 activity in response to osmotic stress. Wild type cells or cells lacking rho2⁺ (Δrho2), pck2⁺ (Δpck2), or pmk1⁺ (Δpmk1) were grown to mid-log phase, washed, and incubated for 1 h in YE with or without KCl (1 m), C, constitutive activation of the PKA relieves the suppression of Gad8 activity in salt stress. Wild type cells or cells lacking pde1⁺ (Δpde1) were grown to mid-log phase, washed, and incubated for 1 h in YE with or without KCl (1 m) as indicated. D, Rst2, a transcription factor downstream of Pka1, is not involved in the regulation of Gad8 activity. Wild type or Δrst2 cells were grown to mid-log phase. Gad8 in vitro kinase activity and Ser-546 phosphorylation were determined as above.

FIGURE 6.

Working model. The TORC2-Gad8 pathway is positively regulated by cAMP/PKA1 and negatively regulated by the PmK1-MAPK pathway. In the presence of glucose, the PKA pathway is activated in a cAMP-dependent manner, leading to the activation of TORC2-Gad8. The Pmk1-MAPK pathway is activated under glucose starvation conditions, leading to inhibition of TORC2-Gad8, via inhibition of the Pka1 pathway or via an independent mechanism.