Biochemical And Genetic Study On The Ser/Thr Protein Kinase SiRe₂056 In Sulfolobus Islandicus REY15A

Ser/Thr protein kinase can transfer the y-Phosphate groups onto the amino acid residues of the protein and is important in the signal transduction.SiRe₂056 in Sulfolobus islandicus was a predicted Ser/Thr protein kinase.It was found in the study of the Holliday Junction resolving enzyme Hje.Hje is a kind of structure-specific endonuclease,which can cut HJ substrates and is involved in DNA double strand breaks repair and the restart of replication forks.Hje may be modified after translation and overexpression of Hje can induce the up-regulation of SiRe₂056.Based on this,we suspect that SiRe₂056 may participate in the modification of Hje.Therefore,the analysis of the functional and in vitro biochemical properties of SiRe₂056 was carried out using the unmarked gene knockout and the expression systems established in Sulfolobus islandicus.Firstly,a series of pET22b vectors were constructed.Because the full length of SiRe₂056 protein can not be expressed in E.coli,we constructed vectors to purify a series of C-terminal His-tag protein kinase domains of wild type and mutant proteins in E.coli.In vitro assay using a PKC kinase assay Kit and[?-32P]ATP demonstrated that the SiRe 2056 did have protein kinase activity and autophosphorylated and phosphorylated Hje and BSA.SiRe₂056 exhibited this activity at high temperature?60? and 65??and prefers Mn2 + as the cofactor.After D476 and D510 were mutanted into alanine,SiRe₂056 lost the ability of autophosphorylation and phosphorylation on the substrates,confirming that the D476 and D510 are two key active sites.Secondly,we tried to use the unmarked gene knockout method to delete the target gene.After got pure integrated strains,the mutant propagation assay was performed.The results showed that the Sis/pMID-SiRe 2056 can grow up as similar to Sis/pMID-Hjc.This indicated that the SiRe₂056 gene can be knocked out.On the other hand,after five rounds counter-selection,PCR verification indicated that there were few wild type cells remaining in the strain.DNA damage agent analysis found that the growth of Sis/pMID 2056 was better than SisE233S in the presence of HU and MMS,indicative of decrease in the sensitivity to DNA damage agents.All the results indicated SiRe₂056 may be involved in DNA damage-related signal pathways.This requires further validation later.Finally,we tried to analyze the phenotype of SiRe₂056 overexpression strain.Overexpression of the full length protein SiRe₂056-C-His and protein kinase domain PKC-C-His inhibited the growth of strains,while overexpression of the mutants D476A and D510A did not.After the mutation of the critical sites,D476 and D510,the mutant protein lost the kinase activity and,even if expressed,does not inhibit the growth.The results suggest that the inhibitory was dependent on the kinase activity.In order to explore the function of the three auto-phosphorylation sites T525,T592,and T606,two different kinds of mutations were overexpressed Sulfolobus islandicus.Overexpression of PKC-T525E-C-His,PKC-T592A-C-His,PKC-T592E-C-His,PKC-T606A-C-His,and PKC-T606E-C-His can inhibit the growth of the strains and enlarged cells were observed,while overexpression of PKC-T525E-C-His did not.Western blot results showed that T525 may be the key site for the protein stability.Mutation of T525 into alanine can cause the protein unstable leading to lower expression of the target protein.The inhibitory can be removed.Mutation of T592,T606 into alanine did not make the protein kinase denatured indicating the two sites may be not the key sites for protein kinase.At the same time,enlarged cells were observed in the strains overexpression 2056-C-His,PKC-C-His,and T525E-C-His.These results suggested that the gene SiRe₂056 may be involved in the regulation of cell growth and metabolism.