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

Adiel Cohen,Martin Kupiec,Ronit Weisman

doi:10.1074/jbc.m114.573824

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.

Highlights

TORC2 is a conserved protein complex that regulates multiple aspects of cell survival and proliferation
TORC2-dependent Gad8 Phosphorylation and Gad8 Kinase Activity Are Decreased in Response to Glucose Depletion, Ionic or Osmotic Stress—To monitor TORC2-Gad8 activity in response to extracellular changes, we developed a simple, nonradioactive in vitro kinase assay for Gad8 [34]
Constitutive activation of the cAMP/protein kinase A (PKA) pathway by disruption of the cAMP phosphodiesterase pde1ϩ resulted in hyperactivation of TORC2-Gad8 under glucose starvation or KCl stress

Summary

Background

TORC2 is a conserved protein complex that regulates multiple aspects of cell survival and proliferation. Pmk, similar to human extracellular signalregulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad phosphorylation and activation. Inhibition of TORC2-Gad occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Glucose starvation leads to a decrease in Pka activity, which allows entrance into sexual development and activation of gluconeogenesis, partially through phosphorylation of the Zn2ϩ finger transcription factor Rst2 [23] Another branch involved in glucose signaling is mediated by AMP kinase, called Ssp in S. pombe, which is activated under glucose-limiting condition. In response to glucose starvation or osmotic stress, the small GTPase Rho positively regulates Pmk1-MAPK through the protein kinase C ortholog Pck2 [27]. Our data locate TORC2-Gad downstream of two major signaling pathways that respond to extracellular signals and provide the first insights into the mechanisms that underlie TORC2 activation

EXPERIMENTAL PROCEDURES

RESULTS

23 Ϯ 2 27 Ϯ 3 23 Ϯ 4

DISCUSSION