The Studyof CaMk2Serine Acetylated Modificationandfunction

ObjectiveAccording to the result of Preliminary test, we hypothesis:In eukaryotic cells exist extensively serine acetylated modification, but CaMk2β is one of important target protein serine acetylation. Our study confirmed that the serine acetylated modification of CaMk2protein, and to explore the influence of serine acetylated modification on the CaMk2β activity. Methods1.To confirm of eukaryotic cells endogenous CaMk2beta protein serine acetylated: According to the result of preliminary test:In Renal clear cell carcinoma may be exist extensively serine acetylated modification, to obtain all protein of293T cell and purify the exogenous CaMk2β protein by Immunoprecipitation, the protein are resolved by SDS-PAGE according to the size of molecular weight, Through a series of processing before are analyzed by LC-MS/MS, To Confirm CaMk2beta protein serine acetylated, To understand the distribution of CaMk2beta protein serine acetylated modification distribution in functional region.2. To confirm the exogenous CaMk2beta protein serine acetylation in prokaryotic cells:①Construction of recombinant plasmid vector:PQE-His30-CaMk2β,Total RNA of293T cells as templates, RT-PCR amplification precursor sequence of CaMk2β genes. The CaMk2β genes and PQE-His30plasmid are cut by double enzymes, Then connect and transform to construct the recombinant plasmid. the invitrogen company to detect base sequence of the recombinant plasmid.②The expression of exogenous CaMk2beta protein in prokaryotic cells:by IPTG to induce the expression of exogenous CaMk2beta plasmid in prokaryotic cells.③LC-MS/MS technology detect CaMk2beta protein serine acetylation in prokaryotic cells, the exogenous CaMk2beta plasmid expressed, then purify CaMk2β protein by Immunoprecipitation, to incubate the CaMk2β and the protein of293T cell, without incubation CaMk2beta protein as contrast, To detect both CaMk2beta protein serine acetylated modification.3. To confirm the exogenous CaMk2beta protein serine acetylation in eukaryotic cells:①Construction of recombinant plasmid vector:PCDNA-Flag3.0-CaMk2P, PQE-His30-CaMk2β as templates, RT-PCR amplification precursor sequence of CaMk2β genes. The CaMk2β genes and PCDNA-Flag3.0plasmid are cut by double enzymes, Then connect and transform to construct the recombinant plasmid. the invitrogen company to detect base sequence of the recombinant plasmid.②To detect the expression of PCDNA-Flag3.0-CaMk2P:Frist the invitrogen company to detect Whether or not the recombinant plasmid base sequence, then the recombinant plasmid enter293T cells by the gene transfection technology, after forty hours to ontain the protein of293T cells. The last to detect the expression of recombinant plasmid by Westerning Bloting.③To purify the exogenous CaMk2P protein by Immunoprecipitation and analyze the content of serine acetylated modification by LC-MS/MS technology:First to purify the exogenous CaMk2β protein by Immunoprecipitation, the protein are resolved by SDS-PAGE according to the size of molecular weight, then cut down the brand of CaMk2β protein from SDS-PAGE, use some technology analyze the content of serine acetylated modification.④To detect the expression of CaMk2β protein serine acetylated modification by Western blotting:Frist the Tian Jin San Jian company synthesis the antibody of serine acetylated modification, To detect the expression of CaMk2β protein serine acetylated modification.4. To confirm the effect of CaMk2beta protein serine acetylated modification on the function:①To construct the mutation plasmid of PCDNA-Flag3.0-CaMk2β and detect the expression of mutation plasmid:Frist the PCDNA-Flag3.0-CaMk2β as templates, synthesis the mutation primers, CaMk2β protein ser276were mutate Alanine(A)、 Aspartic acid (D)、cysteine (C), Immunoprecipitation to detect the expression of mutation plasmid.②To detect the CaMk2β protein acetylated modification on ser276and its influence no the activity of protein kinase:CaMk2β protein acetylated modification on ser276Whether or not influence the Phosphorylated modification on thr287,namely the activity of protein kinase.Results1. In eukaryotic cells endogenous CaMk2beta protein exist extensively serine acetylated modification:LC-MS/MS analyze discover CaMk2beta protein many serines was acetylated, and Serine acetylated modification mainly focus on the adjust structure and interaction between sub structure domains.2. In prokaryotic cells exogenous CaMk2beta protein exist extensively serine acetylated modification:②Through enzyme identification and invitrogen company to detect base sequence of the recombinant plasmid PQE30-His-CaMk2β.to induce the expression PQE30-His-CaMk2β plasmid in prokaryotic cells.③LC-MS/MS technology detect CaMk2beta protein serine acetylation in prokaryotic cells, the exogenous CaMk2beta plasmid expressed, then purify CaMk2β protein by Immunoprecipitation, to incubate the CaMk2β and the protein of293T cell, without incubation CaMk2beta protein as contrast, To detect both CaMk2beta protein serine acetylated modification.3. In eukaryotic cells exogenous CaMk2beta protein exist extensively serine acetylated modification:①Through enzyme identification and the invitrogen company detect the recombinant plasmid base sequence is correct.②Western Bloting detect the expression of PCDNA-Flag3.0CaMk2β recombinant plasmid is correct.③To purify the exogenous CaMk2β protein by Immunoprecipitation, the protein was deal with before Mass spectrometry, then was analyzed by some software, detect the content of every acetylated modification on serine,as follow:S51accounted for11%、 S71accounted for16.7%、S235accounted for16.7%、S276accounted for41.8%、 S280accounted for41.5%、S315accounted for50%、S327accounted for50%、S331accounted for40%、S343accounted for40%、S362accounted for20%、S367accounted for10%、S451accounted for2%、S456accounted for7%、S494accounted for1%、S504accounted for1.5%、S513accounted for8.5%、S522accounted for13%、S533accounted for22%.④By means of Tian Jin San Jian company synthesis the antibody of serine acetylated modification to detect the serine of exogenous CaMk2β protein kinase were acetylated modification.4. CaMk2beta protein serine acetylated influence the phosphorylation site of287:①The invitrogen company detect the recombinant mutation plasmid base sequence is correct, the PCDNA-Flag3.0-CaMk2β mutation plasmid was transfect into293T cells, Western Bloting detect the expression of recombinant plasmid is correct.②IP-WB technology detect the acetylated modification on ser276had an influence on the activity of CaMk2β protein kinase, when the Serine was mutate into Alanine, it could reduce the content of Phosphorylated modification on thr287,namely reduce the activity of CaMk2β protein kinase, when the Serine was mutate into Aspartic acid and cysteine, it enhance the Phosphorylated modification of thr287, namely the activity of protein kinase.Conclusion1. To confirm exogenous CaMk2beta protein exist extensively serine acetylated modification in eukaryotic cells.2. To confirm exogenous CaMk2beta protein exist extensively serine acetylated modification In prokaryotic cells.3. CaMk2β multiple serine sites was acetylated modification, Confirm the acetylated modification sites can occur phosphorylated modification before, This reveal the serine phosphorylation and acetylation may exist competition, That may be regulate CaMk2beta protein kinase activation process.4. CaMk2beta276acetylation and phosphorylated modification may affect the protein287threonine phosphorylation. That may be regulate CaMk2beta protein kinase activation process.