Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases.
Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases.
复制标题
DOI：
--
复制
发表时间：
1986-02
期刊：
影响因子：
--
通讯作者：
D. Glass;M. El‐Maghrabi;S. Pilkis
中科院分区：
文献类型：
--
作者：
D. Glass;M. El‐Maghrabi;S. Pilkis
利用与6-磷酸果糖-2-激酶/果糖-2，6-P_2酶(Murray，K.J.，El-Maghrabi，M.R.，Kountz，P.D.，Lukas，T.J.，Soderling，T.R.和Pilkis，S.J.(1984)J.Biol)中的磷酸化位点相对应的合成肽，研究了cAMP依赖和cGMP依赖的蛋白激酶的特异性。化学。259、7673-7681)作为底物。Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln是由cAMP依赖的蛋白激酶催化亚基在其2个丝氨酸残基中的第一个残基上磷酸化的。Km(4微米)和Vmax(14摩尔/分钟/毫克)与天然6-磷酸果糖-2-激酶/果糖-2，6-P酶中该位点的磷酸化水平相当。只含有两个精氨酸的模拟多肽被磷酸化，其动力学常数比亲本多肽差。这些结果表明，其磷酸化位点的氨基酸序列是使6-磷酸果糖-2-激酶/果糖-2，6-P_2酶成为cAMP依赖的蛋白激酶的良好底物的主要决定因素。虽然6-磷酸果糖-2-激酶/果糖-2，6-P_2酶不被cGMP依赖的蛋白激酶磷酸化，但与cAMP依赖的磷酸化位点对应的合成肽是一个相对较好的底物(Km=33微米，Vmax=1mumol/min/mg)。因此，依赖cGMP的蛋白激酶不能磷酸化天然的6-磷酸果糖-2-激酶/果糖-2，6-P_2酶，而不是磷酸化位点上的初级序列。含有α-Ser-Ser或-Thr-Ser部分的多肽均可被cGMP依赖的激酶磷酸化为1.0mol磷酸/摩尔多肽，但磷酸分布在两个羟基氨基酸之间。在三种精氨酸和这些可磷酸化的氨基酸之间用Pro取代甘氨酸，会导致蛋白激酶选择性地磷酸化苏氨酰基或第一丝氨基残基，并将Vmax提高4-6倍。这些结果与Pro允许邻近的苏氨酰残基容易地被cGMP依赖的蛋白激酶磷酸化的作用是一致的。
The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6-phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo-2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP-dependent protein kinase, the synthetic peptide corresponding to the cAMP-dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP-dependent protein kinase.