ROLE OF THE N-TERMINAL REGION IN COVALENT MODIFICATION OF 6-PHOSPHOFRUCTO-2-KINASE FRUCTOSE-2,6-BISPHOSPHATASE - COMPARISON OF PHOSPHORYLATION AND ADP-RIBOSYLATION/

The effect-of cyclic AMP (cAMP)-dependent phosphorylation and ADP-ribo sylation on the activities of the rat liver bifunctional enzyme, 6-pho sphofructo-2-kinase/fructose-2,6-bis- phosphatase (PFK-2/FBPase-2), wa s investigated in order to determine the role of the N-terminus in cov alent modification of the enzyme. The bifunctional enzyme was demonstr ated to be a substrate in vitro for arginine-specific ADP-ribosyltrans ferase : 2 mol of ADP-ribose was incorporated per mol of subunit. The K-m values for NAD(+) and PFK-2/FBPase-2 were 14 mu M and 0.4/LM respe ctively. A synthetic peptide (Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile- Pro-Gln) corresponding to the site phosphorylated by cAMP-dependent pr otein kinase was ADP-ribosylated on all three arginine residues. Analy sis of ADP-ribosylation of analogue peptides containing only two argin ine residues, with the third replaced by alanine, revealed that ADP-ri bosylation occurred predominantly on the two most C-terminal arginine residues. Sequencing of the ADP-ribosylated native enzyme also demonst rated that the preferred sites were at Arg-29 and Arg-30, which are ju st N-terminal to Ser-32, whose phosphorylation is catalysed by cAMP-de pendent 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-bifunction al-enzyme antibodies directed to its unique N-terminal region. ADP-rib osylation of PFK-2/FBPase-2 blocked its phosphorylation by PKA, and de creased its PFK-2 activity, but did not alter FBPase-2 activity, In co ntrast, cAMP-dependent phosphorylation inhibited the kinase and activa ted the bisphosphatase. These results demonstrate that ADP-ribosylatio n of arginine residues just N-terminal to the site phosphorylated by P KA modulate PFK-2 activity by an electrostatic and/or steric mechanism which does not involve uncoupling of N- and C-terminal interactions a s seen with cAMP-dependent phosphorylation.