Rosa J L, Pérez J X, Ventura F, Tauler A, Gil J, Shimoyama M, Pilkis S J, Bartrons R
Departament de Ciències Fisiològiques Humanes i de la Nutrició, Universitat de Barcelona, Hospitalet de Llobregat, Spain.
Biochem J. 1995 Jul 1;309 ( Pt 1)(Pt 1):119-25. doi: 10.1042/bj3090119.
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.
为了确定N端在该酶共价修饰中的作用，研究了环磷酸腺苷（cAMP）依赖性磷酸化和ADP核糖基化对大鼠肝脏双功能酶6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶（PFK-2/FBPase-2）活性的影响。双功能酶在体外被证明是精氨酸特异性ADP核糖基转移酶的底物：每摩尔亚基掺入2摩尔ADP核糖。NAD⁺和PFK-2/FBPase-2的Km值分别为14μM和0.4μM。与被cAMP依赖性蛋白激酶磷酸化的位点相对应的合成肽（Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln）在所有三个精氨酸残基上都发生了ADP核糖基化。对仅含两个精氨酸残基且第三个被丙氨酸取代的类似肽的ADP核糖基化分析表明，ADP核糖基化主要发生在两个最靠近C端的精氨酸残基上。对ADP核糖基化的天然酶进行测序也表明，优先位点是在Arg-29和Arg-30，它们正好位于Ser-32的N端，Ser-32的磷酸化由cAMP依赖性蛋白激酶（PKA）催化。ADP核糖基化与酶的磷酸化状态无关。此外，该酶的ADP核糖基化降低了针对其独特N端区域的肝脏特异性抗双功能酶抗体对它的识别。PFK-2/FBPase-2的ADP核糖基化阻止了其被PKA磷酸化，并降低了其PFK-2活性，但没有改变FBPase-2活性。相反，cAMP依赖性磷酸化抑制了激酶并激活了双磷酸酶。这些结果表明，在PKA磷酸化位点的N端的精氨酸残基的ADP核糖基化通过静电和/或空间机制调节PFK-2活性，这与cAMP依赖性磷酸化中所见的N端和C端相互作用的解偶联不同。
