Abstract
14-3-3 proteins play a pivotal role in intracellular localization of transcription factor RSG involving synthesis of gibberellins. We characterized protein kinase activity in tobacco which phosphorylates Ser-114 of RSG involved in its association with 14-3-3 proteins usingin-gel kinase assay. GST-fusion proteins of wild and S114A of RSG were prepared by E.coli cells. Ser-114 of RSG was phosphorylated by protein kinases at 50-60 kDa in the tobacco microsomal fraction in Ca2+-dependent manner. Thus, it was suggested that CDPKs phosphorylate Ser-114 of RSG. Tobacco CDPK1 cDNA was isolated from tobacco cDNA library and GST-fused CDPK1 was prepared. CDPK1 preferentially phosphorylates Ser-114 of RSG in vitro. Pull-down assay demonstrated that in vitro phosphorylated RSG binds to 14-3-3 proteins and that CDPK1 interacts with 14-3-3 proteins by itself. These results suggest that CDPK regulates interaction between RSG and 14-3-3 protens via phosphorylation of Ser-114, leading to modulation of intracellular localization of RSG.
