Influence of Ser/Pro-rich domain and kinase domain of double cortin-like protein kinase on microtubule-binding activity
Influence of Ser/Pro-rich domain and kinase domain of double cortin-like protein kinase on microtubule-binding activity
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DOI：
10.1093/jb/mvr013
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发表时间：
2011-05-01
影响因子：
2.7
通讯作者：
Kameshita, Isamu
中科院分区：
文献类型：
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作者：
Nagamine, Tadashi;Shimomura, Sachiko;Kameshita, Isamu
双重皮质醇样蛋白激酶(DCLK)是一种主要在脑内表达的丝氨酸/苏氨酸蛋白激酶。DCLK由三个功能结构域组成：N端双皮质样(DC)结构域、C端激酶区和位于DC和激酶区之间的Ser/Pro-rich(SP)结构域。虽然已知DC结构域介导微管结合，但SP结构域和激酶结构域在微管结合中的功能作用尚不清楚。在本研究中，我们利用不同的缺失突变体和嵌合蛋白研究了斑马鱼DCLK(ZDCLK)的微管结合活性。用免疫细胞化学方法和洗涤剂提取法生化分析方法对不同突变体的微管结合活性进行了评价。当从zDCLK中去掉激活域时，微管结合活性显著增强。虽然zDCLK(DC+SP)突变体显示出很强的微管结合活性，但仅DC结构域的微管结合活性要低得多，这表明zDCLK的SP结构域在增强DC结构域的微管结合活性方面发挥了作用。这些结果表明，DCLK的微管结合活性与激活域和SP结构域有关。
Doublecortin-like protein kinase (DCLK) is a Ser/Thr protein kinase predominantly expressed in brain. DCLK is composed of three functional domains; the N-terminal doublecortin-like (DC) domain, the C-terminal kinase domain and Ser/Pro-rich (SP) domain in between DC and kinase domains. Although the DC domain is known to mediate microtubule association, functional roles of the SP domain and the kinase domain on microtubule association is not known. In this study, we investigated the microtubule-binding activity of zebrafish DCLK (zDCLK) using various deletion mutants and chimeric proteins. The microtubule-binding activity of various mutants of zDCLK was assessed both by immunocytochemical analysis and by biochemical analysis using detergent extraction method. When the kinase domain was removed from zDCLK, the microtubule-binding activity was significantly enhanced. Although the zDCLK(DC + SP) mutant showed a strong microtubule-binding activity, the DC domain alone showed much lower microtubule-binding activity, indicating that the SP domain of zDCLK plays a role in enhancing microtubule-binding activity of the DC domain. These results suggest that both the kinase domain and the SP domain are involved in regulating the microtubule-binding activity of DCLK.