Serine Acetylation And Its Effects On CDK8 Function

ObjectiveAccording to the results of LC-MS/MS, we first found theserine acetylated modification in eukaryotic cells. CDK8 is one of important targets whose serine can be acetylated. Our study confirmed that the serine acetylation of CDK8 protein, and to explore its influence on its phosphorylation functions to CTD.Methods1. The detection of serine acetylation modifications of endogenous CDK8 in eukaryotic cells:Obtaining all protein of 293T cell and purify the exogenous CDK8 protein by Immunoprecipitation, and then proteins were detected by LC-MS/MS to understand the serine acetylation modification distribution in functional region.2. The detection of serine acetylation modifications of exogenous CDK8 in prokaryotic cells:Construction of recombinant plasmid vector:PQE-His30-CDK8, induced expression in BL21, and then proteins were detected by LC-MS/MS to analyse the serine acetylation modifications.3. The detection of serine acetylation modifications of exogenous CDK8 in eukaryotic cells:Construction of recombinant plasmid vector:PCDNA3.0-FLAG-CDK8, then expressed in 293T cells by the gene transfection technology, purify the exogenous CDK8 protein by Immunoprecipitation, then proteins were detected by LC-MS/MS to analyse the serine acetylation modifications.4. Detection of serine acetylation modifications and its influence on its phosphorylation functions to CTD:â‘  Construction of the mutation plasmid of PCDNA3.0-Flag-CDK8 [S to Alanine (A), Aspartic acid (D), cysteine (C)]. Then CDK8 expressed in 293T cells by the gene transfection technology, purify the exogenous CDK8 protein by Immunoprecipitation.â‘¡ Construction of the plasmid of PQE30-HIS-CTD. Then CTD expressed in BL21 and purified by Ni column, then incubated with different mutations of CDK8 to see the serine acetylation modifications of CDK8 and its influence on its phosphorylation functions to CTD.Results1. LC-MS/MS results shows that endogenous CDK8 protein has serine acetylation modifications in eukaryotic cells:mainly on the fuctional structure and interaction between sub structure domains.2. LC-MS/MS results shows that exogenous CDK8 protein has serine acetylation modifications in prokaryotic cells.3. LC-MS/MS results shows that exogenous CDK8 protein has serine acetylation modifications in eukaryotic cells, and the rates are different from that in prokaryotic cells.4. Serine acetylated modifications on Serine 10,11 does influence the phosphorylation functions of CDK8.Conclusion:1. We confirmed that serine acetylated modifications are widely exist whether in eukaryotic cells or prokaryotic cells. Also these sites can also be phosphorylated, This reveals a competition between the serine phosphorylation and acetylation.2. The dynamic balance between the phosphorylation and acetylation regulates its phosphorylation functions to CTD.