Functional Analysis Of CiPKZ

In recent years, the kinase PKR-like (PKZ) genes had been reported successively in some teleost fish, such as CaPKZ in Goldfish (Carassius auratus)(Hu et al.,2004), DrPKZ in Zebrafish (Danio rerio)(Rothenburg et al.,2005), SsPKZ in Atlantic salmon (Salmo salar)(Bergan et al.,2008) and GrPKZ in Rare minnow (Gobiocypris rarus)(Su et al.,2008). Because the proteins encoded by these genes contain two Za domains and can bind Z-DNA, so they were designated as "PKZ"(protein kinase containing Z-DNA binding domain)(Rothenburg et al.,2005). It is interested that the C-terminus of PKZ also contain12conserved subdomains which are more closely to fish PKR (dsRNA-activated protein kinase), so Rothenburg et al (2008) considered PKZ as replication of fish PKR and stop at tetrapod (Rothenburg et al.,2008). Hence, PKZ is the special protein kinase, not only is a kind of Z-DNA binding protein but also belongs to an eIF2a protein kinase family.The research on the function of PKZ has a good beginning. The preliminary conclusion is that the PKZ is extremely similar to PKR, so it is likely related to the antivirus of cell (Hu et al.,2004; Rothenburg et al.,2005). For example, the constitutive expression of PKZ is lower, but have a highly up-regulation induced by IFN etc (Hu et al.,2004; Rothenburg et al.,2005; Bergan et al.,2008). Moreover, like the others member of eIF2a kinase, DrPKZ and SsPKZ could inhibit protein synthesis (Rothenburg et al.,2005; Bergan et al.,2008). Despite for all this, we also puzzle about how to regulate and activate the fish PKZ by Z-DNA.Ctenopharyngodon idella Grass carp (Ctenopharyngodon idella) is one of the major fresh fish in china. Because it is very sensitive to many stresses, so the PKZ from grass carp may be beneficial for us to comprehend the function of fish PKZ.On account of it,we constructed the prokaryotic expression plasmids based on grass carp PKZ(CiPKZ, GU299765), expressed in vitro,and purified.We contrasted the yield of protein PKZ between BL21(DE3) and Rosseta, found that Rosseta were higher yield.then we analysed the function of PKZ,and showed that the fusion PKZ protein extracted from E.coli have been activated in the absence of activators. When we added phosphatase,found that PKZ was not able to phosphorylate eIF2a..Moreover, when Z-DNA or Poly I:C were incubated with dephosphorylated PKZ, we found that Z-DNA but not Poly I:C could revive PKZ.In vivo,we found that CiPKZ could restrain the expression of luciferase extremely. Zα and the K198amino acid residue play a key role in its function. Moreover,the analysis of CiPKZ prompter showed us a site that might combine IFN regulatory factor1(I1).So we examined the binding activity of His-tag I1fusion protein and PKZ prompter by agarose gel mobility shift assay. The results demonstrated that I1could combine I1.Our results was a beginning for analysis of I1and CiPKZ prompter in vivo,and exploration the response of CiPKZ transcription to I1.