Adenovirus-mediated overexpression of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in gluconeogenic rat hepatoma cells. Paradoxical effect on Fru-2,6-P2 levels.
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been postulated to be a metabolic signaling enzyme, which acts as a switch between glycolysis and gluconeogenesis in mammalian liver by regulating the level of fructose 2,6-bisphosphate. The effect of overexpressing the bifunctional enzyme was studied in FAO cells transduced with recombinant adenoviral constructs of either the wild-type enzyme or a double mutant that has no bisphosphatase activity or protein kinase phosphorylation site. With both constructs, the mRNA and protein were overexpressed by 150- and 40-fold, respectively. Addition of cAMP to cells overexpressing the wild-type enzyme increased the S0.5 for fructose 6-phosphate of the kinase by 1.5-fold but had no effect on the overexpressed double mutant. When the wild-type enzyme was overexpressed, there was a decrease in fructose 2,6-bisphosphate levels, even though 6-phosphofructo-2-kinase maximal activity increased more than 22-fold and was in excess of fructose-2,6-bisphosphatase maximal activity. The kinase:bisphosphatase maximal activity ratio was decreased, indicating that the overexpressed enzyme was phosphorylated by cAMP-dependent protein kinase. Overexpression of the double mutant resulted in a 28-fold increase in kinase maximal activity and a 3-4-fold increase in fructose 2,6-bisphosphate levels. Overexpression of this form inhibited the rate of glucose production from dihydroxyacetone by 90% and stimulated the rate of lactate plus pyruvate production by 200%. In contrast, overexpression of the wild-type enzyme enhanced glucose production and inhibited lactate plus pyruvate production. These results provide direct support for fructose 2,6-bisphosphate as a regulator of gluconeogenic/glycolytic pathway flux and suggest that regulation of bifunctional enzyme activities by covalent modification is more important than the amount of the protein.
Argaud
D
D
Department of Physiology and Biophysics, SUNY at Stony Brook 11794, USA.
Lange
A J
AJ
Becker
T C
TC
Okar
D A
DA
el-Maghrabi
M R
MR
Newgard
C B
CB
Pilkis
S J
SJ
D000256:Adenoviridae; D000818:Animals; D002460:Cell Line; D005635:Fructosediphosphates / Q000378:metabolism*; D005822:Genetic Vectors; D005943:Gluconeogenesis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000378:metabolism; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis* / Q000378:metabolism; D010770:Phosphotransferases / Q000096:biosynthesis* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D005635:Fructosediphosphates; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D011994:Recombinant Proteins; C027652:fructose 2,6-diphosphate; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases