Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan.
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan.
PLoS One. 2018 Apr 23;13(4):e0196357. doi: 10.1371/journal.pone.0196357. eCollection 2018.
Tobacco (Nicotiana tabacum) Ca2+-dependent protein kinase 1 (NtCDPK1) is involved in feedback regulation of the plant hormone gibberellin through the phosphorylation of the transcription factor, REPRESSION OF SHOOT GROWTH (RSG). Previously, Ser-6 and Thr-21 were identified as autophosphorylation sites in NtCDPK1. Autophosphorylation of Ser-6 and Thr-21 not only decreases the binding affinity of NtCDPK1 for RSG, but also inhibits the homodimerization of NtCDPK1. Furthermore, autophosphorylation decreases the phosphorylation efficiency of RSG. We demonstrated that Ser-6 and Thr-21 of NtCDPK1 are phosphorylated in response to GAs in plants. The substitution of these autophosphorylation sites with Ala enhances the NtCDPK1 overexpression-induced sensitization of seeds to a GA biosynthetic inhibitor during germination. These findings suggested that autophosphorylation of Ser-6 and Thr-21 prevents excessive phosphorylation of RSG. In this study, we attempted to determine which autophosphorylation site is responsible for the functional regulation of NtCDPK1. Ser-6 was autophosphorylated within 1 min, whereas Thr-21 required over 5 min to be completely autophosphorylated. Furthermore, we found that Ser-6 and Thr-21 were autophosphorylated by inter- and intramolecular mechanisms, respectively, which may be reflected in the faster autophosphorylation of Ser-6. Although both autophosphorylation sites were involved in the reduction of the binding affinity of NtCDPK1 for RSG and the inhibition of NtCDPK1 homodimerization, autophosphorylation of Ser-6 alone was sufficient to decrease the kinase activity of NtCDPK1 for RSG. These results suggest that autophosphorylation of Ser-6 is important for the rapid reduction of NtCDPK1 kinase activity for RSG, whereas that of Thr-21 may play an auxiliary role.
烟草（Nicotiana tabacum）钙依赖蛋白激酶 1（NtCDPK1）通过磷酸化转录因子 REPRESSION OF SHOOT GROWTH（RSG）参与植物激素赤霉素的反馈调节。先前，鉴定出 Ser-6 和 Thr-21 是 NtCDPK1 的自身磷酸化位点。Ser-6 和 Thr-21 的自身磷酸化不仅降低了 NtCDPK1 与 RSG 的结合亲和力，而且抑制了 NtCDPK1 的同源二聚化。此外，自身磷酸化降低了 RSG 的磷酸化效率。我们证明了植物中 NtCDPK1 的 Ser-6 和 Thr-21 响应 GA 被磷酸化。用 Ala 取代这些自身磷酸化位点可增强 NtCDPK1 过表达对种子萌发过程中 GA 生物合成抑制剂的敏感性。这些发现表明，Ser-6 和 Thr-21 的自身磷酸化可防止 RSG 的过度磷酸化。在这项研究中，我们试图确定哪个自身磷酸化位点负责 NtCDPK1 的功能调节。Ser-6 在 1 分钟内自身磷酸化，而 Thr-21 则需要超过 5 分钟才能完全自身磷酸化。此外，我们发现 Ser-6 和 Thr-21 分别通过分子内和分子间机制进行自身磷酸化，这可能反映在 Ser-6 的更快自身磷酸化上。尽管这两个自身磷酸化位点都参与了降低 NtCDPK1 与 RSG 的结合亲和力和抑制 NtCDPK1 同源二聚化，但 Ser-6 的自身磷酸化足以降低 NtCDPK1 对 RSG 的激酶活性。这些结果表明，Ser-6 的自身磷酸化对于快速降低 NtCDPK1 对 RSG 的激酶活性很重要，而 Thr-21 的自身磷酸化可能起辅助作用。
