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).