Abstract
RSG is a transcriptional activator that regulates endogenous amounts of GAs. Function of RSG is regulated by subcellular localization, which is controlled by endogenous amounts of GAs through phosphorylation of Ser-114. We have identified tobacco calcium-dependent protein kinase (NtCDPK) as a protein kinase that specifically phosphorylates Ser-114 of RSG. Although CDPK family consists of many CDPK isoforms and the target sequences of CDPKs are conserved, the mechanisms of isoform-specific substrate specificity have not yet been known. NtCDPK1 but not NtCDPK2 phosphorylates Ser-114 of RSG, indicating RSG is an NtCDPK1-specific substrate. To clarify the mechanisms of substrate specificity of NtCDPK1, we performed pull-down assay using deletion mutant series of NtCDPK1 and identified Arg-10 in N-terminal variable domain as a key residue for binding to RSG. Furthermore, mutation of Arg-10 by alanine substantially decreased its kinase activity for phosphorylation of RSG Ser-114.
