title: Heart 6-phosphofructo-2-kinase activation by insulin results from Ser-466 and Ser-483 phosphorylation and requires 3-phosphoinositide-dependent kinase-1, but not protein kinase B.
abstract: Previous studies have shown that (i) the insulin-induced activation of heart 6-phosphofructo-2-kinase (PFK-2) is wortmannin-sensitive, but is insensitive to rapamycin, suggesting the involvement of phosphatidylinositol 3-kinase; and (ii) protein kinase B (PKB) activates PFK-2 in vitro by phosphorylating Ser-466 and Ser-483. In this work, we have studied the effects of phosphorylation of these residues on PFK-2 activity by replacing each or both residues with glutamate. Mutation of Ser-466 increased the V(max) of PFK-2, whereas mutation of Ser-483 decreased citrate inhibition. Mutation of both residues was required to decrease the K(m) for fructose 6-phosphate. We also studied the insulin-induced activation of heart PFK-2 in transfection experiments performed in human embryonic kidney 293 cells. Insulin activated transfected PFK-2 by phosphorylating Ser-466 and Ser-483. Kinase-dead (KD) PKB and KD 3-phosphoinositide-dependent kinase-1 (PDK-1) cotransfectants acted as dominant negatives because both prevented the insulin-induced activation of PKB as well as the inactivation of glycogen-synthase kinase-3, an established substrate of PKB. However, the insulin-induced activation of PFK-2 was prevented only by KD PDK-1, but not by KD PKB. These results indicate that the insulin-induced activation of heart PFK-2 is mediated by a PDK-1-activated protein kinase other than PKB.
authors.affiliation: Hormone and Metabolic Research Unit, Université catholique de Louvain, Institute of Cellular Pathology, Avenue Hippocrate, 75, B-1200 Brussels, Belgium.
authors.forename: L
authors.initials: L
authors.lastname: Bertrand
authors.forename: D R
authors.initials: DR
authors.lastname: Alessi
authors.forename: J
authors.initials: J
authors.lastname: Deprez
authors.forename: M
authors.initials: M
authors.lastname: Deak
authors.forename: E
authors.initials: E
authors.lastname: Viaene
authors.forename: M H
authors.initials: MH
authors.lastname: Rider
authors.forename: L
authors.initials: L
authors.lastname: Hue
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D025481:Phosphofructokinase-2; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
chemical_list: D007328:Insulin; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D025481:Phosphofructokinase-2; D010768:Phosphoserine; D017853:Phosphotransferases (Alcohol Group Acceptor); D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins; D012694:Serine