Cauthron R D, Carter K B, Liauw S, Steinberg R A
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Mol Cell Biol. 1998 Mar;18(3):1416-23. doi: 10.1128/MCB.18.3.1416.
Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [35S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low Km for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [gamma-32P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.
环磷酸腺苷依赖性蛋白激酶（或蛋白激酶A）催化亚基在苏氨酸-197位点的磷酸化是实现最佳酶活性所必需的，并且从动物来源或细菌表达菌株中分离出的酶在该位点均被磷酸化。苏氨酸-197的自磷酸化在大肠杆菌中及体外发生，但这是一种效率不高的分子间反应，主要由活性的、先前已磷酸化的分子催化。相比之下，完整的S49小鼠淋巴瘤细胞中新合成的蛋白激酶A在苏氨酸-197位点的磷酸化既高效又不受细胞内蛋白激酶A激活剂或抑制剂的影响。在凝胶迁移率变动分析中，使用[35S]甲硫氨酸标记的、未磷酸化的重组催化亚基作为底物，我们在蛋白激酶A缺陷的S49细胞提取物中鉴定出一种能使催化亚基在苏氨酸-197位点磷酸化的活性。通过阴离子交换和羟基磷灰石层析部分纯化的蛋白激酶A激酶活性，就对ATP的低Km值和快速的反应进程而言，是蛋白激酶A磷酸化的高效催化剂。激酶激酶对野生型催化亚基的磷酸化激活了该亚基，使其能够与蛋白激酶A的假底物肽抑制剂结合。通过凝胶迁移分析和[γ-32P]ATP掺入分析，该酶对野生型催化亚基以及用甲硫氨酸取代赖氨酸-72的无活性突变体有活性，但对用丙氨酸取代苏氨酸-197的突变体无活性。结合突变亚基的结果，磷酸氨基酸分析表明该酶对苏氨酸-197的磷酸化具有特异性。
