Autophosphorylation Affects Substrate-Binding Affinity of Tobacco Ca²⁺-Dependent Protein Kinase1

Area of Science:

• Plant molecular biology
• Enzymology
• Cellular signaling

Background:

• Protein kinases are crucial regulators of physiological processes.
• Autophosphorylation is a common kinase mechanism, but its physiological roles beyond self-activation are largely unknown.
• REPRESSION OF SHOOT GROWTH (RSG) is a transcription factor involved in gibberellin (GA) feedback regulation, negatively regulated by tobacco NtCDPK1.

Purpose of the Study:

• To investigate the functional importance of autophosphorylation in NtCDPK1.
• To identify autophosphorylation sites and their impact on NtCDPK1 activity and substrate interaction.
• To elucidate the role of NtCDPK1 autophosphorylation in plant GA responses.

Main Methods:

• Site-directed mutagenesis to identify autophosphorylation sites (Ser-6 and Thr-21).
• In vitro assays to assess NtCDPK1 binding affinity to RSG, homodimerization, and substrate phosphorylation.
• In planta analysis of NtCDPK1 overexpression and GA response.

Main Results:

• Autophosphorylation at Ser-6 and Thr-21 reduced NtCDPK1 binding affinity for RSG and inhibited homodimerization.
• Autophosphorylation decreased RSG phosphorylation efficiency but increased myelin basic protein phosphorylation.
• NtCDPK1 autophosphorylation sites were phosphorylated in response to GAs, and their mutation affected seed germination sensitivity.

Conclusions:

• Autophosphorylation of NtCDPK1 plays a regulatory role in cellular signaling beyond self-activation.
• Autophosphorylation prevents excessive substrate phosphorylation and alters substrate preference in calcium-dependent protein kinases (CDPKs).
• These findings reveal novel functions of autophosphorylation in modulating plant hormone responses and gene regulation.