Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase
Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase
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DOI：
10.1128/mcb.18.3.1416
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发表时间：
1998-03-01
影响因子：
5.3
通讯作者：
Steinberg, RA
中科院分区：
文献类型：
--
作者：
Cauthron, RD;Carter, KB;Steinberg, RA
环AMP依赖性蛋白激酶或蛋白激酶A的催化亚基在Thr-197上的磷酸化是最佳酶活性所需的，并且发现从动物来源或细菌表达菌株分离的酶在该位点被磷酸化。Thr-197的自磷酸化发生在大肠杆菌中和体外，但是主要由活性，相反，在完整的S49小鼠淋巴瘤细胞中，新合成的蛋白激酶A的Thr-197磷酸化对细胞内蛋白激酶A的激活剂或抑制剂既有效又不敏感。使用[S-35]甲硫氨酸标记的非磷酸化重组催化亚基作为凝胶迁移率变动分析中的底物，我们已经鉴定了蛋白激酶A缺陷的S49细胞提取物中的活性，其磷酸化Thr-197上的催化亚基，用阴离子交换法部分纯化了蛋白激酶A激酶活性，交换和羟基磷灰石层析在ATP的低K-m和快速时程方面是蛋白激酶A磷酸化的有效催化剂，激酶激酶对野生型催化亚基的磷酸化激活了该亚基与蛋白激酶A的假底物肽抑制剂的结合。通过凝胶迁移试验和[γ-P-32]ATP掺入试验，该酶对野生型催化亚基和Met取代Lys-72的失活突变体有活性，但对Ala取代Thr-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，该酶是特异性磷酸化的Thr-197。
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 [S-35]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 K-m for ATP and a rapid time course, Phosphorylation of wild-ty pe 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-P-32]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.