Coordinación MolecuLar Del Metabolismo Hepático De Glucosa Por La 6 - Fosfofructo -2 - Quinasa / Fructosa -2,6-Bisfosfatasa: Complejo glucoQuinasa

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Molecular Endocrinology 21(6):1478–1487 Copyright © 2007 by The Endocrine Society doi: 10.1210/me.2006-0356

Molecular Coordination of Hepatic Glucose Metabolism by the 6-Phosphofructo-2-Kinase/Fructose-2,6Bisphosphatase:Glucokinase Complex
W. Ed Smith, Sara Langer, Chaodong Wu, Simone Baltrusch, and David A. Okar Department of Veterans AffairsMedical Center (W.E.S., D.A.O.), Minneapolis, Minnesota 55417; Institute of Clinical Biochemistry (S.L., S.B.), Hannover Medical School, 30623 Hannover, Germany; and Department of Biochemistry (C.W.), Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455
Glucokinase (GK) and 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBP-2) are each powerfulregulators of hepatic carbohydrate metabolism that have been reported to influence each other’s expression, activities, and cellular location. Here we present the first physical evidence for saturable and reversible binding of GK to the FBP-2 domain of PFK-2/FBP-2 in a 1:1 stoichiometric complex. We confirmed complex formation and stoichiometry by independent methods including affinity resin pull-downassays and fluorescent resonance energy transfer. All suggest that the binding of GK to PFK-2/FBP-2 is weak. Enzymatic assays of the GK:PFK-2/FBP-2 complex suggest a concomitant increase of the kinase-to-bisphosphatase ratio of bifunctional enzyme and activation of GK upon binding. The kinase-to-bisphosphatase ratio is increased by activation of the PFK-2 activity whereas FBP-2 activity is unchanged.This means that the GK-bound PFK-2/FBP-2 produces more of the biofactor fructose-2,6-bisphosphate, a potent activator of 6-phosphofructo-1-kinase, the committing step to glycolysis. Therefore, we conclude that the binding of GK to PFK-2/FBP-2 promotes a coordinated up-regulation of glucose phosphorylation and glycolysis in the liver, i.e. hepatic glucose disposal. The GK:PFK-2/FBP-2 interactionmay also serve as a metabolic signal transduction pathway for the glucose sensor, GK, in the liver. Demonstration of molecular coordination of hepatic carbohydrate metabolism has fundamental relevance to understanding the function of the liver in maintaining fuel homeostasis, particularly in managing excursions in glycemia produced by meal consumption. (Molecular Endocrinology 21: 1478–1487, 2007)HE BIFUNCTIONAL ENZYME 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase (PFK-2/ FBP-2; Bif) is an important regulator of cellular fuel metabolism, especially in the liver (1, 2). Its primary function is to control the intracellular content of the biofactor fructose-2,6-bisphosphate (F-2,6-P2), a potent activator of 6-phosphofructo-1-kinase (PFK-1) (3). Activation of PFK-1 in vivo byincreased hepatic F-2,6-P2 has been conclusively demonstrated (4, 5). In addition to confirming that increased hepatic F-2,6-P2 activates glycolysis and reduces glycemia, some reports also suggested that this metabolic regulator coordinates hepatic glycolysis with lipid metabolism (5).
First Published Online March 20, 2007 Abbreviations: AF, Alexa Fluor; FBP-2, fructose-2.6bisphosphatase; FI,fluorescence intensity; F-2,6-P2, fructose-2,6-bisphosphate; FRET, fluorescent resonance energy transfer; GK, glucokinase; HK1, yeast hexokinase 1; NADP, nicotinamide adenine dinucleotide phosphate; Ni:NTA, nickel-nitrilotriacetic acid; PEG, polyethylene glycol; PFK-1, phosphofructo-1-kinase; PFK-2, 6-phosphofructo-2-kinase; PKI, protein kinase A inhibitor; TPI, triose phosphate isomerase; XSFI, excessfluorescence intensity. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.

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Predicated on yeast 2 hybrid and phage display results suggesting that liver PFK-2/FBP-2 bound to glucokinase (GK; hexokinase 4) (6) and using a graded overexpression of Ser-32-dephosphorylated rat liver...