title: Role of the N-terminal region in covalent modification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: comparison of phosphorylation and ADP-ribosylation.
abstract: The effect of cyclic AMP (cAMP)-dependent phosphorylation and ADP-ribosylation on the activities of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), was investigated in order to determine the role of the N-terminus in covalent modification of the enzyme. The bifunctional enzyme was demonstrated to be a substrate in vitro for arginine-specific ADP-ribosyltransferase: 2 mol of ADP-ribose was incorporated per mol of subunit. The Km values for NAD+ and PFK-2/FBPase-2 were 14 microM and 0.4 microM respectively. A synthetic peptide (Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln) corresponding to the site phosphorylated by cAMP-dependent protein kinase was ADP-ribosylated on all three arginine residues. Analysis of ADP-ribosylation of analogue peptides containing only two arginine residues, with the third replaced by alanine, revealed that ADP-ribosylation occurred predominantly on the two most C-terminal arginine residues. Sequencing of the ADP-ribosylated native enzyme also demonstrated that the preferred sites were at Arg-29 and Arg-30, which are just N-terminal to Ser-32, whose phosphorylation is catalysed by cAMP-dependent protein kinase (PKA). ADP-ribosylation was independent of the phosphorylation state of the enzyme. Furthermore, ADP-ribosylation of the enzyme decreased its recognition by liver-specific anti-bifunctional-enzyme antibodies directed to its unique N-terminal region. ADP-ribosylation of PFK-2/FBPase-2 blocked its phosphorylation by PKA, and decreased its PFK-2 activity, but did not alter FBPase-2 activity. In contrast, cAMP-dependent phosphorylation inhibited the kinase and activated the bisphosphatase. These results demonstrate that ADP-ribosylation of arginine residues just N-terminal to the site phosphorylated by PKA modulate PFK-2 activity by an electrostatic and/or steric mechanism which does not involved uncoupling of N- and C-terminal interactions as seen with cAMP-dependent phosphorylation.
authors.affiliation: Departament de Ciències Fisiològiques Humanes i de la Nutrició, Universitat de Barcelona, Hospitalet de Llobregat, Spain.
authors.forename: J L
authors.initials: JL
authors.lastname: Rosa
authors.forename: J X
authors.initials: JX
authors.lastname: Pérez
authors.forename: F
authors.initials: F
authors.lastname: Ventura
authors.forename: A
authors.initials: A
authors.lastname: Tauler
authors.forename: J
authors.initials: J
authors.lastname: Gil
authors.forename: M
authors.initials: M
authors.lastname: Shimoyama
authors.forename: S J
authors.initials: SJ
authors.lastname: Pilkis
authors.forename: R
authors.initials: R
authors.lastname: Bartrons
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010710:Phosphates; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010455:Peptides; D010710:Phosphates; D000246:Adenosine Diphosphate Ribose; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases