Figure 1. GP activity is negatively regulated by acetylation.

(A) GP acetylation is increased by deacetylase inhibitor. Flag-tagged GP was expressed in Chang’s cells with or without nicotinamide and trichostatin A (NAM+TSA) treatment. Acetylation levels of Flag-beads purified GP were blotted with pan-anti-acetyllysine antibody (α-AcK). IB and IP denote immunoblotting and immunoprecipitation, respectively.

(B) K470 and K796 are the major acetylation sites in GP. Ectopically expressed Flag-GP and GP^K2R in Chang’s cells were immunopurified and immuno-blotted with pan-anti-acetyllysine antibody.

(C) GP catalytic activity is negatively regulated by acetylation. Both wild type GP and GP^K2Q were expressed in Chang’s liver cells with or without NAM+TSA treatment. Catalytic activity of affinity purified GP proteins were determined and normalized to protein levels. Activity of wild type GP under no treatment condition was set as 100%.

(D) The acetylation target K470 and K796 are important for GP inhibition by the deacetylase inhibitor treatment. GP, GP^K470Q, GP^K796Q and GP^K2Q were each expressed in Chang’s liver cells with (hatched bars) or without (solid bars) NAM+TSA treatment as indicated. Specific activity of each purified enzyme was determined.

(E) Wild-type GP degrades glycogen faster than K2Q mutant. Wild type GP and mutant GP^K2Q were expressed at similar levels in Chang’s liver cells maintained in regular DMEM medium. The medium was replaced by glucose-free DMEM at time 0 and the glycogen degradation rate was measured. All error bars represent standard deviation (SD). n = 3 for each experimental group.