Skip to main content
. 2014 Jun 13;289(31):21727–21737. doi: 10.1074/jbc.M114.573824

FIGURE 1.

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 (gad8K259D, 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.