Consequences of lysine 72 mutation on the phosphorylation and activation state of cAMP-dependent kinase
Consequences of lysine 72 mutation on the phosphorylation and activation state of cAMP-dependent kinase
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DOI：
10.1074/jbc.m407586200
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发表时间：
2005-03-11
影响因子：
4.8
通讯作者：
Taylor, SS
中科院分区：
文献类型：
--
作者：
Iyer, GH;Moore, MJ;Taylor, SS
获得失活激酶的一般策略是利用激酶核心中关键保守残基的突变，并且 cAMP 依赖性激酶中的等效 Lys(72) 通常用于生成“死”激酶。在这里，我们分析了这种突变对激酶结构和功能的影响。 Lys(72) 突变为组氨酸 (K72H) 产生无活性的酶，未磷酸化。用外源激酶 (PDK-1) 处理会产生仅在 Thr(197) 处磷酸化的突变体，该突变体保持非活性，但仍然能够结合 ATP。 K72H 中的 Ser(338) 不能自磷酸化，也不能在分子间过程中被活性野生型 C 亚基磷酸化。 Lys(72) 突变体一旦在 Thr(197) 上磷酸化，就能以高亲和力与 RIalpha 亚基结合。因此，死亡的激酶仍然可以充当结合底物和抑制剂的支架；只有磷酰基转移有缺陷。使用 C 亚基活性的有效抑制剂 H-89，还获得了大肠杆菌表达的 C 亚基的未磷酸化状态。该蛋白能够在 PDK-1 存在的情况下成熟为活性形式，并且能够在 Ser(338) 上进行二次自磷酸化。与 H-89 处理的野生型蛋白不同，突变蛋白 (K72H) 在 Thr(197) 磷酸化后不能进行后续的顺式自磷酸化。使用这两种底物和哺乳动物表达的 PDK-1，我们可以阐明 C 亚基激活的可能的两步过程：PDK-1 或 PDK-1 样酶对激活环 Thr(197) 进行初始磷酸化，然后在 Ser(338) 处进行第二个顺式自磷酸化步骤。
General strategies to obtain inactive kinases have utilized mutation of key conserved residues in the kinase core, and the equivalent Lys(72) in cAMP-dependent kinase has often been used to generate a "dead" kinase. Here, we have analyzed the consequences of this mutation on kinase structure and function. Mutation of Lys(72) to histidine (K72H) generated an inactive enzyme, which was unphosphorylated. Treatment with an exogenous kinase (PDK-1) resulted in a mutant that was phosphorylated only at Thr(197) and remained inactive but nevertheless capable of binding ATP. Ser(338) in K72H cannot be autophosphorylated, nor can it be phosphorylated in an intermolecular process by active wild type C-subunit. The Lys(72) mutant, once phosphorylated on Thr(197), can bind with high affinity to the RIalpha subunits. Thus a dead kinase can still act as a scaffold for binding substrates and inhibitors; it is only phosphoryl transfer that is defective. Using a potent inhibitor of C-subunit activity, H-89, Escherichia coli-expressed C-subunit was also obtained in its unphosphorylated state. This protein is able to mature into its active form in the presence of PDK-1 and is able to undergo secondary autophosphorylation on Ser(338). Unlike the H-89-treated wild type protein, the mutant protein ( K72H) cannot undergo the subsequent cis autophosphorylation following phosphorylation at Thr(197). Using these two substrates and mammalian-expressed PDK-1, we can elucidate a possible two-step process for the activation of the C-subunit: initial phosphorylation on the activation loop at Thr(197) by PDK-1, or a PDK-1-like enzyme, followed by second cis autophosphorylation step at Ser(338).