Colbran J L, Francis S H, Leach A B, Thomas M K, Jiang H, McAllister L M, Corbin J D
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615.
J Biol Chem. 1992 May 15;267(14):9589-94.
Bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) is a potent and relatively specific substrate for cGMP-dependent protein kinase (cGK) as compared to cAMP-dependent protein kinase (cAK) (Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265, 14971-14978). A synthetic peptide, RKISASEFDRPLR (BPDEtide), was synthesized corresponding to the sequence surrounding the phosphorylation site in cG-BPDE. BPDEtide retained the cGK/cAK kinase specificity demonstrated by native cG-BPDE: the apparent Km of BPDEtide for cGK was 5-fold lower than that for cAK (Km = 68 and 320 microM, respectively). Vmax values were 11 mumol/min/mg for cGK and 3.2 mumol/min/mg for cAK. The peptide was not phosphorylated to a measurable extent by protein kinase C or by calcium/calmodulin-dependent protein kinase II. Thus, the primary amino acid sequence of the peptide substrate was sufficient to confer kinase specificity. Studies in crude tissue extracts indicated that BPDEtide was the most selective peptide substrate documented for measuring cGK activity. Peptide analogs of BPDEtide were synthesized to determine the contribution of specific residues to cGK or cAK substrate specificity. Substitution of a Lys for the amino-terminal Arg did not reduce cGK/cAK specificity; neither did the exchange of an Ala for the non-phosphorylated Ser nor the removal of the 3 carboxyl-terminal residues. A truncated BPDEtide (RKISASE) served equally well as substrate (Km approximately 90 microM) for both kinases. However, restoration of the Phe, to yield RKISASEF, reproduced the original cGK/cAK specificity for BPDEtide (Km = 120 and 480 microM, respectively), primarily by decreasing the affinity of cAK. Addition of a carboxyl-terminal Phe to the peptide RKRSRAE (derived from the sequence of the cGK phosphorylation site in histone H2B) or to the peptide LRRASLG (derived from the sequence of the cAK phosphorylation site in pyruvate kinase) also improved the cGK/cAK specificity by decreasing the affinity of cAK. These data suggested that the Phe in each substrate tested is a negative determinant for cAK.
与环磷酸腺苷依赖性蛋白激酶（cAK）相比，牛肺环磷酸鸟苷结合型环磷酸鸟苷特异性磷酸二酯酶（cG - BPDE）是环磷酸鸟苷依赖性蛋白激酶（cGK）的一种有效且相对特异的底物（托马斯，M.K.，弗朗西斯，S.H.，和科尔宾，J.D.（1990）《生物化学杂志》265，14971 - 14978）。合成了一种与cG - BPDE中磷酸化位点周围序列相对应的合成肽，RKISASEFDRPLR（BPDE肽）。BPDE肽保留了天然cG - BPDE所显示的cGK/cAK激酶特异性：BPDE肽对cGK的表观米氏常数比对cAK低5倍（米氏常数分别为68和320微摩尔）。cGK的最大反应速度值为11微摩尔/分钟/毫克，cAK为3.2微摩尔/分钟/毫克。该肽未被蛋白激酶C或钙/钙调蛋白依赖性蛋白激酶II磷酸化至可测量的程度。因此，肽底物的一级氨基酸序列足以赋予激酶特异性。在粗组织提取物中的研究表明，BPDE肽是记录在案的用于测量cGK活性的最具选择性的肽底物。合成了BPDE肽的类似物以确定特定残基对cGK或cAK底物特异性的贡献。用赖氨酸取代氨基末端的精氨酸不会降低cGK/cAK特异性；用丙氨酸取代非磷酸化的丝氨酸或去除3个羧基末端残基也不会降低其特异性。一种截短的BPDE肽（RKISASE）对两种激酶而言同样是良好的底物（米氏常数约为90微摩尔）。然而，恢复苯丙氨酸以产生RKISASEF，再现了BPDE肽原来的cGK/cAK特异性（米氏常数分别为120和480微摩尔），主要是通过降低cAK的亲和力。在肽RKRSRAE（源自组蛋白H2B中cGK磷酸化位点的序列）或肽LRRASLG（源自丙酮酸激酶中cAK磷酸化位点的序列）的羧基末端添加苯丙氨酸，也通过降低cAK的亲和力提高了cGK/cAK特异性。这些数据表明，所测试的每种底物中的苯丙氨酸是cAK的负向决定因素。
