Cloning and functional analysis of PKZ (PKR-like) from grass carp (Ctenopharyngodon idellus).
The new teleost fish PKZ (PKR-like) full-length cDNA (GU299765) had been cloned and identified from grass carp (Ctenopharyngodon idellus). The cDNA of grass carp PKZ (CiPKZ) has 2185 bp in length with a largest open reading frame (ORF) encoding 513aa. CiPKZ possesses a conserved C-terminal catalytic domain of eIF2α kinase family. Within its N-terminal there are two binding domain (Zα) named Zα1 (1-67aa) and Zα2 (81-152aa). BLAST homologous search reveals that CiPKZ has a high-level homology with other fish PKZs and PKRs. Like other fish PKZs and PKRs, CiPKZ is a ubiquitous tissue expression gene that had a very low level of constitutive expression but up-regulated in response to Poly I:C or hot stress (34 °C). For the purpose of searching for the potential function of CiPKZ, we obtained CiPKZ polypeptide via Escherichia coli Rosetta prokaryotic expression and purified with Ni-NTA His-Bind Resin affinity chromatography. CiPKZ polypeptide was used for the test of phosphorylating eIF2αin vitro. The results demonstrated that CiPKZ could be activated by Z-DNA but not by Poly I:C, and with subsequent could phosphorylate eIF2α. Meanwhile, four pcDNA3.1/PKZ recombinant plasmids, including pcDNA3.1/PKZ-wet, pcDNA3.1/PKZ-wet-K198R, pcDNA3.1/PKZ-wet-C, pcDNA3.1/PKZ-wet-C-K198R had been constructed, respectively. Mouse Myeloma cells (Sp2/0) and Human Umbilical Vein Endothelial Cells (HUVEC) were transiently cotransfected with pcDNA3.1/PKZ recombinant plasmid and PGL-3-promoter plasmid. The results revealed that CiPKZ could greatly decrease luciferase level in these cells. Zα and the K198 amino acid residue may play a key role in its function.
Department of Bioscience, College of Life Science and Food Engineering, Nanchang University, Nanchang 330031, China.
Peng-Jie
PJ
Yang
Chu-Xin
CX
Wu
Wen
W
Li
Li-Hua
LH
Fan
Gang
G
Lin
Cheng-Yu
CY
Hu
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002347:Carps / Q000235:genetics*; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D043542:DNA, Z-Form / Q000378:metabolism*; D040461:Gene Components; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology*; D006358:Hot Temperature; D006801:Humans; D008156:Luciferases; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011070:Poly I-C / Q000276:immunology / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D014162:Transfection; D019892:eIF-2 Kinase / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D043542:DNA, Z-Form; D008156:Luciferases; D011070:Poly I-C; D019892:eIF-2 Kinase