Role of the PH domain in regulating in vitro autophosphorylation events required for reconstitution of PDK1 catalytic activity
Role of the PH domain in regulating in vitro autophosphorylation events required for reconstitution of PDK1 catalytic activity
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DOI：
10.1016/j.bioorg.2006.05.002
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发表时间：
2006-08-01
影响因子：
5.1
通讯作者：
Harris, Thomas K.
中科院分区：
文献类型：
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作者：
Gao, Xinxin;Harris, Thomas K.
磷酸肌醇依赖性蛋白激酶-1（PDK 1）除了其催化结构域外，还含有一个C-末端普列克底物蛋白同源（PH）结构域，其结合膜结合的磷脂酰肌醇（3，4，5）-三磷酸[PI（3，4，5）P-3]第二信使。在这里，我们报告在体外动力学，磷酸肽映射，和寡聚化的研究，解决的PH结构域在调节特定的自磷酸化事件，这是PDK 1催化活化所需的作用。首先，通过用λ蛋白磷酸酶（λ PP）处理产生N-末端HiS（6）标记的全长（HiS 6-PDK 1）和催化结构域构建体[HiS 6-PDKI（Delta PH）]的“无活性”未磷酸化形式。λ PP处理的His（6）-PDK 1（Delta PH）催化活性的重建需要活化环Ser-241磷酸化，这仅在“活性”PDK 1的反式加成时发生，表观双分子速率常数（app）k1（S241）= 374 +/- 29 M-1 s（-1）。相比之下，全长λ PP处理的HiS（6）-PDK 1催化Ser-241顺式自磷酸化，表观一级速率常数为（app）k1（S241）=（5.0 +/- 11.5）x 10（-4）s-1，但保持“无活性”。λ PP处理的HiS 6-PDKI催化活性的重建仅在含有PI（314，5）P3的囊泡存在下自磷酸化时发生。PI（3，4，5）P3与PH结构域的结合激活了20倍的表观一级Ser-241自磷酸化[（app）k1（S241）=（1.1 +/- 0.1）x 10（-2）s（-1）]，并促进双相Thr-513反式自磷酸化[（app）k2（T513）=（4.9 +/- 1.1）× 10（2）M-1 s（-1）和（app）k3（T513）=（1.5 +/- 0.2）× 10（3）M-1 s（-1）]。诱变研究的结果表明，Thr-513磷酸化可能会导致解离的自抑制接触之间形成的连续调节PH和催化激酶结构域。(c)2006爱思唯尔公司All rights reserved.
In addition to its catalytic domain, phosphoinsositide-dependent protein kinase-1 (PDK1) contains a C-terminal pleckstrin homology (PH) domain, which binds the membrane-bound phosphatidylinositol (3,4,5)-triphosphate [PI(3,4,5)P-3] second messenger. Here, we report in vitro kinetic, phosphopeptide mapping, and oligomerization studies that address the role of the PH domain in regulating specific autophosphorylation events, which are required for PDK1 catalytic activation. First, 'inactive' unphosphorylated forms of N-terminal HiS(6) tagged full length (HiS6-PDK1) and catalytic domain constructs [HiS6-PDKI(Delta PH)] were generated by treatment with Lambda protein phosphatase (lambda PP). Reconstitution of lambda PP-treated His(6)-PDK1(Delta PH) catalytic activity required activation loop Ser-241 phosphorylation, which occurred only upon trans-addition of 'active' PDK1 with an apparent bimolecular rate constant of (app)k1(S241) = 374 +/- 29 M-1 s(-1). In contrast, full length lambda PP-treated HiS(6)-PDK1 catalyzed Ser-241 cis-autophosphorylation with an apparent first-order rate constant of (app)k1(S241) = (5.0 +/- 11.5) x 10(-4) s-1 but remained 'inactive'. Reconstitution of lambda PP-treated HiS6-PDKI catalytic activity occurred only when autophosphorylated in the presence of PI(314,5)P3 containing vesicles. PI(3,4,5)P3 binding to the PH domain activated apparent first-order Ser-241 autophosphorylation by 20-fold [(app)k1(S241) = (1.1 +/- 0.1) x 10(-2) s(-1)] and also promoted biphasic Thr-513 trans-autophosphorylation [(app)k2(T513) = (4.9 +/- 1.1) x 10(2) M-1 s(-1) and (app)k3(T513) = (1.5 +/- 0.2) x 10(3) M-1 s(-1)]. The results of mutagenesis studies suggest that Thr-513 phosphorylation may cause dissociation of autoinhibitory contacts formed between the contiguous regulatory PH and catalytic kinase domains. (c) 2006 Elsevier Inc. All rights reserved.