Studies On The Effects Of Casein Kinase 1ε/δ Activity On The Circadian Rhythms

The mammalian circadian clock includes the core circadian clock(SCN)and the peripheral circadian clock(liver,spleen and kidneys)to maintain the body’s operation.CK1ε and CK1δ proteins in mammalian cells belong to the casein kinase family.CKlε/CK1δ transfers the y-phosphate from ATP to serine and threonine residues on the substrate,and generates phosphorylated substrate proteins and ADP.CK1ε/CK1δplays an important role in the transcription-translation feedback loop of the circadian clock,and the phosphorylation mechanism of proteins,involved in the circadian clock such as PER,CRY,BMAL1,regulates protein activity and nuclear localization,and then regulates the circadian clock pathways.The circadian protein mutation may cause the diseases associated with the circadian rhythm.For example,the mutation of S662G of PER causes the mammals to go to bed early and early to rise.The mutation of CK1ε-R178C and the mutation of CK15-T44A can cause the advance of sleep phase.However,there is a controversy over the change of enzyme activity after the mutation of CK1ε protein.In order to further study the effect of CK1ε protein R178C mutation on enzyme activity and the role of CK1ε/CK1δ in cells,we purified the CK1εTAU catalytic domain protein(CK1ε-ΔC-TAU)and screened the crystallization conditions.It is prepared to analyze the effect of CK1ε protein R178C mutation on the structure of enzymes at the molecular three-dimensional level,but unfortunately that no protein crystals have been obtained.Purification of CK1ε/CK1δ/CK1εTAU N-terminal catalytic domain protein and GST-hP53 protein,and investigating the activity of CK1ε/CK1δ/CK1εTAU N-terminal catalytic domain protein on GST-hP53,The results showed that the activity of catalytic domain protein was CK1ε-AC-WT,CK1δ-ΔC-WT and CK1ε-AC-TAU in sequence from high to low.In UPLC experiment,the concentration of ATP/ADP at a certain peak area was calculated by using the standard curve of ATP/ADP concentration and peak area,and the decrease of ATP concentration was detected in a certain time.The enzyme activity of CK1ε-ΔC-WT/CK1δ-ΔC-WT/CK1ε-ΔC-TAU on substrate was compared.The results of three protease activities were consistent with those of phosphorylation.Auto-phosphorylation found that CK1ε protein had the highest phosphorylation activity and the CK1εTAU protein was the lowest with purified CK1ε/CK1δ/CK1εTAU full-length protein,which was consistent with the results of the catalytic domain protein kinase experiment.In the CK1ε/CK1δ cells encoding protein gene(csnkle/d)knockout cell circadian clock was lengthened to 0.5 h and 2 h respectively,indicating that CK1ε/CK1δ regulates the cell cycle,and CK1ε/CK1δ knockout makes the circadian clock longer.It also confirms that CK1εtAU full-length protein activity is higher than that of CK1ε protein.From the molecular level,it was revealed that CK1ε protein R178C mutated with decreased activity of catalytic domain protein and decreased auto-phosphorylation,and that the cycle of circadian clock became longer after knockout of this gene.This gives us a better understanding of sleep phase advance and circadian clock regulation mechanism.