Critical Determinants of Substrate Recognition by Cyclin-Dependent Kinase-like 5 (CDKL5)
Critical Determinants of Substrate Recognition by Cyclin-Dependent Kinase-like 5 (CDKL5)
复制标题
DOI：
10.1021/bi501308k
复制
发表时间：
2015-05-19
期刊：
影响因子：
2.9
通讯作者：
Kameshita, Isamu
中科院分区：
文献类型：
--
作者：
Katayama, Syouichi;Sueyoshi, Noriyuki;Kameshita, Isamu
细胞周期蛋白依赖性激酶（CDKL 5）是一种与X连锁神经发育障碍相关的丝氨酸/苏氨酸蛋白激酶。在之前的一项研究中，我们确定了两性物理素1（Amphl）作为CDKL 5的潜在底物，并在Ser-293上确定了一个磷酸化位点。在这项研究中，我们使用Amphl作为模型底物，研究了CDKL 5识别底物的分子机制。Amphl充当有效的CDKL 5底物），而Amph 2（Amphl的结构相关同源物）不被CDKL 5磷酸化。Amphl磷酸化位点周围的序列是RPR（293）SPSQ，而Amph 2中的相应序列是IPK（332）SPSQ。为了确定底物的氨基、Amph 2酸序列特异性，制备Amphl和Amph 2的各种点突变体并通过CDKL 5磷酸化。Amph 2（I329 R）和Amph 1都是有效的CDKL 5底物，但Amph 1（R290 I）不是，这表明β-3位的N-乙酰基残基对于底物识别是关键的：在Amphl磷酸化位点周围的脯氨酰残基中，Pro-291位于P2位置，而Pro-294不位于P +1位置，是CDKL 5磷酸化所必需的。使用Amphl的各种缺失突变体的磷酸化实验揭示，富含脯氨酸的结构域（PRD）（氨基酸247-315）单独不被CDKL 5磷酸化。相比之下，包含PR和CLAP结构域的Amph 1（247-385）充当有效的CDKL 5底物。这些结果一起表明，Amphl中的磷酸化位点序列（RPXSX）和CLAP结构域结构在CDKL 5的识别和磷酸化中起关键作用。
Cyclin-dependent (CDKL5) is a Ser/Thr protein kinase known to be associated-With X-linked neurodevelopmental disorders. In a previous study, we identified amphiphysin 1 (Amphl) as a potential substrate for CDKL5 and identified a single phosphorylation site at Ser-293. In this study, we investigated the molecular mechanisms of substrate recognition by CDKL5 using Amphl as a model substrate. Amphl served as an efficient CDKL5 substrate) whereas Amph2, a structurally related homologue of Amphl, was not phosphorylated by CDKL5. The sequence around the Amphl phosphorylation site is RPR(293)SPSQ, while the corresponding sequence in Amph2 is IPK(332)SPSQ, To define the amino,Amph2 acid sequence specificity of the substrate, various point mutants of Amphl and Amph2 were prepared and phosphorylated by CDKL5. Both 1 283 413 588 Amph2(I329R) and' Amphl served as efficient CDKL5 substrates, but Amphl (R290I) Did not, indicating that the arginyl residue at the P -3 position is critical for substrate recognition: With regard to prolyl residues around the phosphorylation site of Amphl, Pro-291 at the P 2 position, but not Pro-294 at the P +1 position, is indispensable for phosphorylation by CDKL5. Phosphorylation experiments using various deletion mutants of Amphl revealed that the proline-rich domain (PRD) (amino acids 247-315) alone was not phosphorylated by CDKL5. In contrast, Amph1(247-385), which comprised the PR) and CLAP domains, served as an efficient CDKL5 substrate. These results) taken together, suggest that both the phosphorylation site sequence (RPXSX) and the CLAP domain structure in Amphl play crucial roles in recognition and phosphorylation by CDKL5.