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.