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.