Studies On The Effects Of Casein Kinase 1?/? 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.CK1? and CK1? proteins in mammalian cells belong to the casein kinase family.CKl?/CK1? transfers the y-phosphate from ATP to serine and threonine residues on the substrate,and generates phosphorylated substrate proteins and ADP.CK1?/CK1?plays 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 CK1?-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 CK1? protein.In order to further study the effect of CK1? protein R178C mutation on enzyme activity and the role of CK1?/CK1? in cells,we purified the CK1?TAU catalytic domain protein(CK1?-?C-TAU)and screened the crystallization conditions.It is prepared to analyze the effect of CK1? 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 CK1?/CK1?/CK1?TAU N-terminal catalytic domain protein and GST-hP53 protein,and investigating the activity of CK1?/CK1?/CK1?TAU N-terminal catalytic domain protein on GST-hP53,The results showed that the activity of catalytic domain protein was CK1?-AC-WT,CK1?-?C-WT and CK1?-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 CK1?-?C-WT/CK1?-?C-WT/CK1?-?C-TAU on substrate was compared.The results of three protease activities were consistent with those of phosphorylation.Auto-phosphorylation found that CK1? protein had the highest phosphorylation activity and the CK1?TAU protein was the lowest with purified CK1?/CK1?/CK1?TAU full-length protein,which was consistent with the results of the catalytic domain protein kinase experiment.In the CK1?/CK1? cells encoding protein gene(csnkle/d)knockout cell circadian clock was lengthened to 0.5 h and 2 h respectively,indicating that CK1?/CK1? regulates the cell cycle,and CK1?/CK1? knockout makes the circadian clock longer.It also confirms that CK1?tAU full-length protein activity is higher than that of CK1? protein.From the molecular level,it was revealed that CK1? 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.