Autophosphorylation of Ser-6 via an intermolecular mechanism is important for the rapid reduction of NtCDPK1 kinase activity for substrate RSG.

Autophosphorylation of Ser-6 via an intermolecular mechanism is important for the rapid reduction of NtCDPK1 kinase activity for substrate RSG.

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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 click here 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 vegetable glycerin for sale NtCDPK1 kinase activity for RSG, whereas that of Thr-21 may play an auxiliary role.