Assay Of Phosphorylation Activity Of Casein Kinase 2 For Hyperthetical Protein MP24

Protein kinase CK2 (casein kinase 2) is a widespread and highly conservative courier-dependent serine / threonine protein kinase in eukaryotic cells, it plays an important function in cell survival, growth, proliferation and apoptosis, also it influent on the tumors. mp24 gene is a new gene of mouse, its function is unknown. Compared with the human gene hp23, whose hyperthetical protein shows kinase activity of CK2 in preliminary study in our lab, mP24 shown 78% homology with hP23 on the amino acids sequences. To determine whether the hyperthetical protein mP24 also shows CK2 kinase activity, the phosphorylation activity of mP24 immunoprecipitation product was detected, which is from the the cell transfected with the recombinant pcDNA3.1/mp24, thus, the potential function of mP24 will be expored.In this study, the mp24 sequence from NCBI was confirmed firstly. There are two difference bases between the sequence amplified from mouse NIH3T3 cells and NCBI, further analysis shown that one of them is SNP of mouse. Bioinformatics analysis revealed that mP24 has four CK2 phosphorylation sites, a PKC phosphorylation sites and two N-myristoylation sites.The eukaryotic expression vector pcDNA3.1/mp24 was constructed, which express foreign mP24-V5 fusion protein. The cell transfected with mP24-V5 fusion protein was obtained with the method of transient transfection. mP24-V5 fusion protein was detected to localize in the cytoplasmic matrix, mitochondria and nucleus by using immunofluorescence of V5 antibody.This experiment detect phosphorylation activity of mP24 from two aspects: it shows the role of CK2 kinase or function as a substrate of CK2 kinase by using the kinase CK2 activity detection kit of UpState and immunoprecipitation product of mouse NIH3T3 cells which express mP24-V5 fusion protein. The results confirme that mP24 is mostly to be the substrate of CK2 kinase. Meanwhile, the second CK2 prediction phosphorylation sites of mP24 was mutant by site-directed mutagenesis technology and the result of the phosphorylation activity experiment shown that this prediction phosphorylation sites may affect the phosphorylation activity of mP24. In a conclusions, it could be deduced that mP24 may affect the biological function of mouse cells as a new CK2 kinase substrate.