Autophosphorylation Affects Substrate-Binding Affinity of Tobacco Ca2+-Dependent Protein Kinase1
Autophosphorylation Affects Substrate-Binding Affinity of Tobacco Ca2+-Dependent Protein Kinase1
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DOI：
10.1104/pp.17.00515
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发表时间：
2017-08-01
期刊：
影响因子：
7.4
通讯作者：
Takahashi, Yohsuke
中科院分区：
文献类型：
--
作者：
Ito, Takeshi;Ishida, Sarahmi;Takahashi, Yohsuke
蛋白激酶调节多种生理过程。由于许多激酶保留了固有的自磷酸化能力，自磷酸化似乎是细胞信号传导的最重要机制之一。然而，自磷酸化的生理功能在很大程度上仍然是未知的，除了通过磷酸化催化结构域中的激活环的自激活。芽生长抑制因子（RSG）是参与赤霉素（GA）反馈调节的转录因子。烟草（Nicotiana tabacum）Ca 2+依赖性蛋白激酶NtCDPK 1磷酸化RSG，导致RSG的负调节。NtCDPK 1以前被证明是以Ca 2+依赖的方式自磷酸化。在这里，我们研究了NtCDPK 1中自磷酸化的功能重要性。Ser-6和Thr-21被鉴定为NtCDPK 1的自磷酸化位点。自磷酸化不仅降低了NtCDPK 1与RSG的结合亲和力，而且抑制了NtCDPK 1的同源二聚化。此外，自磷酸化降低了RSG的磷酸化效率，但增加了髓鞘碱性蛋白的磷酸化效率。NtCDPK 1的Ser-6和Thr-21在植物中响应于GAs而被磷酸化。用Ala取代这些自磷酸化位点增强了NtCDPK 1过表达诱导的种子在萌发过程中对GA生物合成抑制剂的敏感性。这些结果提示了CDPKs自身磷酸化的新功能，即自身磷酸化可以阻止底物的过度磷酸化，改变CDPKs的底物偏好性。
Protein kinases regulate diverse physiological processes. Because many kinases preserve inherent autophosphorylation capability, autophosphorylation appears to be one of the most important mechanisms for cellular signaling. However, physiological functions of autophosphorylation are still largely unknown, other than the self-activation by phosphorylation of activation loop in the catalytic domain. REPRESSION OF SHOOT GROWTH (RSG) is the transcription factor involved in gibberellin (GA) feedback regulation. The tobacco (Nicotiana tabacum) Ca2+-dependent protein kinase, NtCDPK1, phosphorylates RSG, resulting in the negative regulation of RSG. NtCDPK1 was previously shown to be autophosphorylated in a Ca2+-dependent manner. Here, we investigated the functional importance of autophosphorylation in NtCDPK1. Ser-6 and Thr-21 were identified as autophosphorylation sites of NtCDPK1. Autophosphorylation not only reduced the binding affinity of NtCDPK1 for RSG, but also inhibited the homodimerization of NtCDPK1. Furthermore, autophosphorylation decreased the phosphorylation efficiency of RSG yet increased that of myelin basic protein. Ser-6 and Thr-21 of NtCDPK1 were phosphorylated in response to GAs in plants. The substitution of these autophosphorylation sites with Ala enhanced the NtCDPK1 overexpression-induced sensitization of seeds to a GA biosynthetic inhibitor during germination. These results suggest new functions of autophosphorylation in CDPKs, namely, autophosphorylation can prevent the excessive phosphorylation of substrates and alter the substrate preference of CDPKs.