Pivotal Role of the C-terminal DW-motif in Mediating Inhibition of Pyruvate Dehydrogenase Kinase 2 by Dichloroacetate
Pivotal Role of the C-terminal DW-motif in Mediating Inhibition of Pyruvate Dehydrogenase Kinase 2 by Dichloroacetate
复制标题
DOI：
10.1074/jbc.m109.065557
复制
发表时间：
2009-12-04
影响因子：
4.8
通讯作者：
Chuang, David T.
中科院分区：
文献类型：
--
作者：
Li, Jun;Kato, Masato;Chuang, David T.
线粒体丙酮酸脱氢酶复合物 (PDC) 通过丙酮酸脱氢酶激酶 (PDK) 亚型 1-4 催化的磷酸化下调。 PDK 同工型的过度表达导致 PDC 活性降低在癌症和糖尿病中普遍存在。在本研究中，我们研究了不变的 C 端 DW 基序在二氯乙酸 (DCA) 抑制人 PDK2 中的作用。在 PDK2 同二聚体的另一个亚基中的 DW 基序（Asp-382 和 Trp-383）及其相互作用残基（Tyr-145 和 Arg-149）中进行了取代。单突变体和双突变体显示出 20-60% 的残余活性，这些活性不受 PDC 核心的刺激。 R149A和Y145F/R149A突变体对DCA的表观IC50值显着增加，而对DCA的结合亲和力与野生型PDK2相当。 R149A 和 Y145F 变体都表现出对 ADP 和 ATP 相似的亲和力增加，模仿 DCA 的作用。 R149A 和 DW 基序突变 (D382A/W383A) 阻止 PDC 的硫辛酰结构域与这些突变体的结合，类似于 DCA 和 ADP 存在下的野生型 PDK2。相比之下，二氢硫辛酰胺模拟物 AZD7545 的结合在这些 PDK2 变体中基本上不受影响。我们的结果阐明了 DW 基序在介导 DCA、核苷酸和硫辛酰结构域结合位点之间的通讯中的关键作用。该信号网络将 PDK2 锁定在非活性闭合构象中，与没有 DCA 和 ADP 的活性开放构象保持平衡。这些结果表明 DW 基序锚定位点是抑制癌症和糖尿病中异常 PDK 活性的药物靶点。
The mitochondrial pyruvate dehydrogenase complex (PDC) is down-regulated by phosphorylation catalyzed by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. Overexpression of PDK isoforms and therefore reduced PDC activity prevails in cancer and diabetes. In the present study, we investigated the role of the invariant C-terminal DW-motif in inhibition of human PDK2 by dichloroacetate (DCA). Substitutions were made in the DW-motif (Asp-382 and Trp-383) and its interacting residues (Tyr-145 and Arg-149) in the other subunit of PDK2 homodimer. Single and double mutants show 20-60% residual activities that are not stimulated by the PDC core. The R149A and Y145F/R149A mutants show drastic increases in apparent IC50 values for DCA, whereas binding affinities for DCA are comparable with wild-type PDK2. Both R149A and Y145F variants exhibit increased similar affinities for ADP and ATP, mimicking the effects of DCA. The R149A and the DW-motif mutations (D382A/W383A) forestall binding of the lipoyl domain of PDC to these mutants, analogous to wild-type PDK2 in the presence of DCA and ADP. In contrast, the binding of a dihydrolipoamide mimetic AZD7545 is largely unaffected in these PDK2 variants. Our results illuminate the pivotal role of the DW-motif in mediating communications between the DCA-, the nucleotide-, and the lipoyl domain-binding sites. This signaling network locks PDK2 in the inactive closed conformation, which is in equilibrium with the active open conformation without DCA and ADP. These results implicate the DW-motif anchoring site as a drug target for the inhibition of aberrant PDK activity in cancer and diabetes.