Alteration of Substrate Specificity: The Variable N-Terminal Domain of Tobacco Ca2+-Dependent Protein Kinase Is Important for Substrate Recognition
Alteration of Substrate Specificity: The Variable N-Terminal Domain of Tobacco Ca2+-Dependent Protein Kinase Is Important for Substrate Recognition
复制标题
DOI：
10.1105/tpc.109.073577
复制
发表时间：
2010-05-01
期刊：
影响因子：
11.6
通讯作者：
Takahashi, Yohsuke
中科院分区：
文献类型：
--
作者：
Ito, Takeshi;Nakata, Masaru;Takahashi, Yohsuke
蛋白激酶是参与多种细胞过程的主要信号分子。然而，蛋白激酶区分特异性底物的分子机制在很大程度上仍然未知。Ca 2+依赖性蛋白激酶（CDPKs）在植物Ca 2+信号转导中起着重要作用。以前，我们发现烟草（Nicotiana tabacum）CDPK 1负调控转录因子抑制芽生长（RSG），这是参与赤霉素反馈调节。在这里，我们发现，CDPK 1的可变N端结构域是必要的识别RSG。CDPK 1可变N-末端结构域中的突变（R10 A）降低了RSG结合和RSG磷酸化，同时保持激酶活性不变。此外，R10 A突变抑制了CDPK 1的体内功能。用Nt CDPK 1的可变N-末端结构域取代拟南芥CDPK的可变N-末端结构域At CPK 9赋予RSG激酶活性。这种嵌合的CDPK在转基因植物中的行为根据可变N-末端结构域的身份。我们的研究结果开启了通过操纵可变N-末端结构域来工程化CDPK的底物特异性的可能性，从而使细胞信号传导途径的合理重新布线成为可能。
Protein kinases are major signaling molecules that are involved in a variety of cellular processes. However, the molecular mechanisms whereby protein kinases discriminate specific substrates are still largely unknown. Ca2+-dependent protein kinases (CDPKs) play central roles in Ca2+ signaling in plants. Previously, we found that a tobacco (Nicotiana tabacum) CDPK1 negatively regulated the transcription factor REPRESSION OF SHOOT GROWTH (RSG), which is involved in gibberellin feedback regulation. Here, we found that the variable N-terminal domain of CDPK1 is necessary for the recognition of RSG. A mutation (R10A) in the variable N-terminal domain of CDPK1 reduced both RSG binding and RSG phosphorylation while leaving kinase activity intact. Furthermore, the R10A mutation suppressed the in vivo function of CDPK1. The substitution of the variable N-terminal domain of an Arabidopsis thaliana CDPK, At CPK9, with that of Nt CDPK1 conferred RSG kinase activities. This chimeric CDPK behaved according to the identity of the variable N-terminal domain in transgenic plants. Our results open the possibility of engineering the substrate specificity of CDPK by manipulation of the variable N-terminal domain, enabling a rational rewiring of cellular signaling pathways.