title: A truncated Danio rerio PKZ isoform functionally interacts with eIF2α and inhibits protein synthesis.
abstract: A protein kinase containing Z-DNA binding domains (PKZ), which resembles protein kinase R (PKR) in domain organization, was recently discovered to be a member of the eIF2α kinase family in fish. PKR has roles in antiviral immunity through inhibiting protein synthesis and activating NF-κB; therefore, it is thought that PKZ may have a similar role in fish antiviral immunity. In the present study, the roles of two Danio rerio PKZ isoforms (DrPKZ-A and DrPKZ-B) in eIF2α phosphorylation and protein synthesis regulation were explored. DrPKZ-A and DrPKZ-B possess N-terminal Z-DNA binding domains and a conserved eIF2α kinase domain; however, they have domains of differing lengths inserted between kinase subdomains IV and V. DrPKZ-A has an insert domain of 73 amino acids (aa), whereas DrPKZ-B has an insert sequence of only 10 aa, suggesting that DrPKZ-B could be a dysfunctional isoform or could interact with different substrates. Our results show that both DrPKZ-A and DrPKZ-B functionally interact with eIF2α and inhibit protein synthesis, although DrPKZ-B possesses attenuated kinase activity. Our results also show that deletion of the insert in either isoform results in the complete abrogation of kinase activity, suggesting that the insert is critical for PKZ kinase activity. Kinase activity appears to be independent of insert length but may depend on the presence of specific amino acids within the insert domain. Furthermore, the effects of the N-terminal regulatory domain on kinase activity were analyzed. Deletion of the N-terminus results in reduced kinase activity of these isoforms relative to the wild-type forms, indicating that the isolated kinase domain is sufficient for eIF2α phosphorylation and that DrPKZ-A and DrPKZ-B may be regulated in a similar manner. Overall, our results show that DrPKZ-B is a functional kinase in zebrafish and contribute to our understanding of the function of PKZ in fish.
authors.affiliation: MOE Key Laboratory of Aquatic Product Safety/State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou 510275, PR China.
authors.forename: Zhao-Yu
authors.initials: ZY
authors.lastname: Liu
authors.forename: Kun-Tong
authors.initials: KT
authors.lastname: Jia
authors.forename: Chuan
authors.initials: C
authors.lastname: Li
authors.forename: Shao-Ping
authors.initials: SP
authors.lastname: Weng
authors.forename: Chang-Jun
authors.initials: CJ
authors.lastname: Guo
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: He
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D015027:Zebrafish / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015852:Eukaryotic Initiation Factor-2; D007527:Isoenzymes; D029961:Zebrafish Proteins; C583881:PKZ protein, zebrafish; D011494:Protein Kinases
keywords: ADAR1; GAPDH; GCN2; HRI; ISKNV; PERK; PKR; PKZ; Protein kinase; RNA-regulated protein kinase (PKR)-like endoplasmic reticulum kinase; Z-DNA binding domain; ZBD; Zebrafish; a protein kinase containing Z-DNA binding domains; co-IP; co-immunoprecipitation; double-stranded RNA adenosine deaminase; eIF2α; eukaryotic initiation factor 2α; general control nonrepressed-2; glyceraldehyde-3-phosphate dehydrogenase; heme-regulated eIF2α kinase; infectious spleen and kidney necrosis virus; protein kinase R