Phosphorylation Of Liver Pyruvate Kinase Research Articles

Characterizing the regulation of pyruvate kinase in response to hibernation in ground squirrel liver (Urocitellus richardsonii).

The Richardson's ground squirrel (Urocitellus richardsonii) undergoes numerous changes to its core physiological and metabolic processes over the months it spends hibernating during the winter. Winter torpor is characterized by an overall reduction in metabolic rate, a lowering of core body temperature, and a switch to preferential consumption of lipids instead of carbohydrates. The alterations in central metabolic pathways are often accomplished by the regulation of key enzymes within the glycolytic pathway. The regulation of one such enzyme, pyruvate kinase (PK), was characterized in the present study in the liver of torpid ground squirrels. PK was purified from liver tissue of euthermic and hibernating U. richardsonii and subsequently assayed to determine the kinetic parameters of the enzyme at 22° and 5°C. Additional studies assessed the relative degree of post-translational modifications in PK from control and hibernating ground squirrels. The results from this study demonstrated significantly lowered maximal activity in the hibernating form of the enzyme and decreased sensitivity to the activator FBP when compared to the control. Immunoblotting demonstrated increased relative serine and threonine phosphorylation (~3 fold) in the hibernating PK. Taken together these results suggest that phosphorylation of liver PK is an important step in inhibiting glycolytic activity in the liver of the Richardson's ground squirrel during torpor.

Phosphorylation of liver pyruvate kinase by Ca ++ /calmodulin-dependent protein kinase: Characterization of two phosphorylation sites

Rat liver pyruvate kinase is phosphorylated by calcium/calmodulin-dependent protein kinase II at serine and threonine residues in a 3-4 kDa CNBr fragment located near the amino terminus. The two sites of phosphorylation were separated by reverse-phase HPLC of a thermolysin digest. Sequence analysis established the sites of phosphorylation as follows: Leu-Arg-Arg-Ala-Ser(PO4)-Val-Ala-Gln-Leu-Thr(PO4)-Gln-Glu.

Cyclic AMP stimulated phosphorylation of liver pyruvate kinase in hepatocytes

The addition of glucagon (10 −6 M) to an incubation mixture containing 32Pi and hepatocytes isolated from livers of rats fed ad libitum results in both a 3-fold increased incorporation of 32P into L-type pyruvate kinase and a decreased catalytic activity. The 32P incorporated into pyruvate kinase was covalently bound to the enzyme as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In addition, exogenous cyclic AMP (10 −3 M) stimulated the phosphorylation and the suppression of catalytic activity to a similar extent. On the other hand, insulin (10 −7 M) had essentially no effect on the incorporation of 32P into pyruvate kinase or on its catalytic activity under the conditions used in this study. These results suggest that phosphorylation of pyruvate kinase invivo is stimulated by glucagon via cyclic AMP and cyclic AMP-dependent protein kinase and that the activity of the enzyme is, at least in part, regulated by a phosphorylation-dephosphorylation mechanism.