Purification and Characterization of the Functional Catalytic Domain of PKR-Like Endoplasmic Reticulum Kinase Expressed in Escherichia coli

  • Yun Jin-A (Department of Biotechnology, Yonsei University) ;
  • Chung Ho-Young (Department of Biotechnology, Yonsei University) ;
  • Kim Seong-Jun (Department of Biotechnology, Yonsei University) ;
  • Cho Hyun-Soo (Department of Biology, Yonsei University) ;
  • Oh Jong-Won (Department of Biotechnology, Yonsei University)
  • 발행 : 2006.09.01

초록

PKR-like endoplasmic reticulum (ER) kinase (PERK) is a type I transmembrane ER-resident protein containing a cytoplasmic catalytic domain with a Ser/Thr kinase activity, which is most closely related to the eukaryotic translation initiation factor-$2{\alpha}$ ($eIF2{\alpha}$) kinase PKR involved in the antiviral defense pathway by interferon. We cloned and expressed the PERK C-terminal kinase domain (cPERK) in Escherichia coli. Like PERK activation in cells under ER stress, wild-type cPERK underwent autophosphorylation when overexpressed in E. coli, whereas the cPERK(K621M) with a methionine substitution for the lysine at amino acid 621 lost the autophosphorylation activity. The activated form cPERK which was purified to near homogeneity, formed an oligomer and was able to trans-phosphorylate specifically its cellular substrate $eIF2{\alpha}$. Two-dimensional phosphoamino acids analysis revealed that phosphorylation of cPERK occurs at the Ser and Thr residues. The functionally active recombinant cPERK, and its inactive mutant should be useful for the analysis of biochemical functions of PERK and for the determination of their three-dimensional structures.

키워드

참고문헌

  1. Boyle, W. J., P. van der Geer, and T. Hunter. 1991. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 201: 110-149 https://doi.org/10.1016/0076-6879(91)01013-R
  2. Chen, J. J. and I. M. London. 1995. Regulation of protein synthesis by heme-regulated eIF-2 alpha kinase. Trends Biochem.Sci. 20: 105-108 https://doi.org/10.1016/S0968-0004(00)88975-6
  3. Clemens, M. J. 1997. PKR -- a protein kinase regulated by double-stranded RNA. Int. J. Biochem. Cell Biol. 29: 945-949 https://doi.org/10.1016/S1357-2725(96)00169-0
  4. Cudna, R. E. and A. J. Dickson. 2003. Endoplasmic reticulum signaling as a determinant of recombinant protein expression. Biotechnol. Bioeng. 81: 56-65 https://doi.org/10.1002/bit.10445
  5. Harding, H. P., Y. Zhang, and D. Ron. 1999. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397: 271 -274 https://doi.org/10.1038/16729
  6. Hinnebusch, A. G. 1993. Gene-specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2. Mol. Microbiol. 10: 215-223 https://doi.org/10.1111/j.1365-2958.1993.tb01947.x
  7. Kaufman, R. J. 1999. Stress signaling from the lumen of the endoplasmic reticulum: Coordination of gene transcriptional and translational controls. Genes Dev. 13: 1211-1233 https://doi.org/10.1101/gad.13.10.1211
  8. Kim, H., S.-J. Kim, S.-N. Park, and J.-W. Oh. 2004. Antiviral effect of amphotericin B on Japanese encephalitis virus replication. J. Microbiol. Biotechnol. 14: 121-127
  9. Koumenis, C., C. Naczki, M. Koritzinsky, S. Rastani, A. Diehl, N. Sonenberg, A. Koromilas, and B. G. Wouters. 2002. Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2alpha. Mol. Cell Biol. 22: 7405-7416 https://doi.org/10.1128/MCB.22.21.7405-7416.2002
  10. Kumar, A., J. Haque, J. Lacoste, J. Hiscott, and B. R. Williams. 1994. Double-stranded RNA-dependent protein kinase activates transcription factor NF-kappa B by phosphorylating 1 kappa B. Proc. Natl. Acad. Sci. USA 91: 6288-6292
  11. Lee, Y.-G, H.-S. Kang, C.-H. Lee, and S.-G. Paik. 2004. Mucosal immune response and adjuvant activity of genetically fused Escherichia coli heat-labile toxin B subunit. J. Microbiol. Biotechnol. 14: 490-497
  12. Ma, K., K. M. Vattem, and R. C. Wek. 2002. Dimerization and release of molecular chaperone inhibition facilitate activation of eukaryotic initiation factor-2 kinase in response to endoplasmic reticulum stress. J. Biol. Chem. 277: 18728-18735 https://doi.org/10.1074/jbc.M200903200
  13. Oh, J.-W, T. Ito, and M. M. Lai. 1999. A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA. J. Virol. 73: 7694-7702
  14. Oh, J.-W., G. T. Sheu, and M. M. Lai. 2000. Template requirement and initiation site selection by hepatitis C virus polymerase on a minimal viral RNA template. J. Biol. Chem. 275: 17710-17717 https://doi.org/10.1074/jbc.M908781199
  15. Park, K.-H., S.-H. Park, H. J. Lee, and B. H. Min. 2004. A probing of inhibition effect on specific interaction between glucose ligand carrying polymer and HepG2 cells. J. Microbiol. Biotechnol. 14: 450-455
  16. Park, S.-Y, J.-H. Kim, and D.-H. Kim. 2005. Purification and characterization of quercitrin-hydrolyzing ${\alpha}$-L-rhamnosidase from Fusobacterium K-60, a human intestinal bacterium. J. Microbiol. Biotechnol. 15: 519-524
  17. Proud, C. G. 1995. PKR: A new name and new roles. Trends Biochem. Sci. 20: 241-246 https://doi.org/10.1016/S0968-0004(00)89025-8
  18. Ron, D. 2002. Translational control in the endoplasmic reticulum stress response. J. Clin. Invest. 110: 1383-1388 https://doi.org/10.1172/JCI0216784
  19. Shi, Y, J. An, J. Liang, S. E. Hayes, G. E. Sandusky, L. E. Stramm, and N. N. Yang. 1999. Characterization of a mutant pancreatic elF-2alpha kinase, PEK, and co-localization with somatostatin in islet delta cells. J. Biol. Chem. 274: 5723-5730 https://doi.org/10.1074/jbc.274.9.5723
  20. Shi, Y, K. M. Vattem, R. Sood, J. An, J. Liang, L. Stramm, and R. C. Wek. 1998. Identification and characterization of pancreatic, eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol. Cell Biol. 18: 7499-7509 https://doi.org/10.1128/MCB.18.12.7499
  21. Tardif, K. D., G. Waris, and A. Siddiqui. 2005. Hepatitis C virus, ER stress, and oxidative stress. Trends Microbiol. 13: 159-163 https://doi.org/10.1016/j.tim.2005.02.004
  22. Thomis, D. C. and C. E. Samuel. 1995. Mechanism of interferon action: Characterization of the intermolecular autophosphorylation of PKR, the interferon-inducible, RNA-dependent protein kinase. J. Virol. 69: 5195-5198
  23. Yan, W., C. L. Frank, M. J. Korth, B. L. Sopher, I. Novoa, D. Ron, and M. G. Katze. 2002. Control of PERK eIF2alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone P58IPK. Proc. Natl. Acad. Sci. USA 99: 15920-15925
  24. Zhuo, S., J. C. Clemens, D. J. Hakes, D. Barford, and J. E. Dixon. 1993. Expression, purification, crystallization, and biochemical characterization of a recombinant protein phosphatase. J. Biol. Chem. 268: 17754-17761