Structure And Activation Regulation Mechanism Of Calcium-dependent Protein Kinases(CPKs) In Arabidopsis

In eukaryotes,protein kinases are involved in many key aspects of regulating cell function,including cell division,cell metabolism,and response to external signals.Calcium-dependent protein kinases CPKs/CDPKs(Ca2+-dependent protein kinases)are a class of Ca2+-dependent protein kinases,which are the main transmitters of Ca2+signaling in plants and certain protozoa,and can recognizes Ca2+signals triggered by development and environmental stimuli and converts them into protein phosphorylation events.In plants,CPKs/CDPKs are a multi-gene family,and their regulatory mechanisms are very complex.In recent years,there have been many studies on the function of different CPKs/CDPKs in plants to trigger a variety of downstream responses in the immune and stress signal network,but the regulatory mechanisms of their activation are elusive.In this paper,a preliminary study was carried out on the regulation mechanism of activition of Arabidopsis Thaliana CDPKs(also referred to as AtCPKs)and protozoa Toxoplasma Gondii CDPK1(TgCDPK1).CPKs/CDPKs are mostly composed of an N-terminal variable domain,a Ser/Thr kinase domain KD(Kinase Domain),a junction region J(Junction)and a C-terminal calmodulin-like domain CLD(Calmodulin-like Domain).AtCPK genes of different subgroups were obtained by cloning the Arabidopsis cDNA library,and the TgCDPK1 gene was synthesized after codon optimization.On this basis,we cloned the full-length and different fragments of multiple AtCPKs and TgCDPK1 and expressed them in E.coli system.After a series of purification steps,high-concentration and high-purity recombinant proteins were obtained.Using NADH-LDH coupled enzyme assays and Western blotting analyses,we obtained the following preliminary results:1)AtCPKs and TgCDPK1 recombinant proteins obtained from the E.coli expression system had protein kinase activity and had phospho-Ser/Thr signals;2)AtCPKs in different subgroups displayed different dependence on Ca2+in Arabidopsis;3)comparing the kinase activity of AtCPKs in phosphorylated and dephosphorylated states,it was found that phosphorylation had little effect on the activity of AtCPKs;4)The activity of different fragments of AtCPK3,AtCPK4 and TgCDPK1 was determined,and it was found that CPK-KD was active while KD+J was inactive in Arabidopsis,and both KD and KD+J of TgCDPK1 showed no activity,which suggested different regulation mechanisms for AtCPKs and TgCDPK1;5)structural study of AtCPKs to elucidate the regulatory mechanism.