Expression and Activation of Akt/PKB Protein Kinase using Escherichia coli

대장균을 이용한 Akt/PKB Protein Kinase의 발현 및 활성화

  • Lee, Jae-Hag (Department of Food and Nutrition, Seoil University)
  • Received : 2009.03.13
  • Accepted : 2009.04.16
  • Published : 2009.06.28

Abstract

Among signal transduction systems by protein phosphorylation Akt/PKB protein kinase which is one of serine/threonine kinases, is known to regulate the survival and death of the cell and glucose metabolism. Thus, Akt/PKB protein kinase has been used as one of the target proteins to find anti-cancer agents from natural products. In this study, human Akt/PKB protein kinase was expressed in Escherichia coli expression system for the mass production. Human Akt/PKB protein kinase expressed in E. coli formed inclusion body under the general condition. However, most of the expressed protein was solubilized under the culture temperature at $27^{\circ}C$ and 0.01-0.09 mM of IPTG for induction of the protein expression. The expressed protein was purified using $Ni^{2+}$-NTA agarose column and confirmed by using anti-Akt antibody. Subsequently, the purified human Akt/PKB protein kinase was activated by in vitro phosphorylation using cellular extract containing kinases. The activated protein was confirmed to phosphorylate the specific fluorescent peptide specially designed as the artificial substrate for Akt/PKB protein kinase.

Keywords

Akt/PKB;Escherichia coli expression system;purification;Akt/PKB specific fluorescent peptide;anti-cancer agent

Acknowledgement

Supported by : 서일대학

References

  1. Her, J.-H., Y. H. Cheong, J.-H. Kim, S.-K. Sin, C. G. Hyun, J. Chun, S. S. Kang, D.-K. Kang, and S.-K. Hong. 2002. Identification of a protein kinase using a FITC-labelled synthetic peptide in Streptomyces griseus IFO13350. Kor. J. Microbiol Biotechnol. 30(3): 235-240
  2. Takeuchi, H., Y. Kondo, K. Fujiwara, T. Kanzawa, H. Aoki, G. B. Mills, and S. Kondo. 2005. Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors. Cancer Res. 65: 3336-46
  3. Kang, S. S., T. Kwon, D. Y. Kwon, and S. I. Do. 1999. Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase V subunit. J. Biol. Chem. 274: 13085-13090 https://doi.org/10.1074/jbc.274.19.13085
  4. Paz, K., Y.-F. Liu, H. Shorer, R. Hemi, D. LeRoith, M. Quan, H. Kanety, R. Seger, and Y. Zick. 1999. Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function. J. Biol. Chem. 274: 28816-28822 https://doi.org/10.1074/jbc.274.40.28816
  5. Yang, H. Y., H.-S. Lee, J.-H. Ko, S.-W. Yeon, T.-Y. Kim, B. Y. Hwang, S. S. Kang, J. Chun, and S.-K. Hong. 2006. Identification of 3'-hydroxymelanetin and liquiritigenin as Akt protein kinase inhibitors. J. Microbiol. Biotechnol. 16: 1384-1391
  6. Sharma, S. V., T. Agatsuma, and H. Nakano. 1998. Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol. Oncogene 16: 2639-2645 https://doi.org/10.1038/sj.onc.1201790
  7. Ko, J. H., S. W. Yeon, H. S. Lee, T. Y. Kim, D. Y. Noh, K. S. Shim, S.-K. Hong, and S. S. Kang. 2004. Inhibitory effects of dithiolo-thione derivative SWU-20009 on Akt activity. J. pharmaceutical society of Korea 48(2): 105-110
  8. Lee, H. Y., S. H. Oh, J. K. Woo, W. Y. Kim, C. S. Van Pelt, R. E. Price, D. Cody, H. Tran, J. M. Pezzuto, R. M. Moriaty, and W. K. Hong. 2005. Chemopreventive effects of deguelin, a novel Akt inhibitor, on tobacco-induced lung tumorigenesis. J. Natl. Cancer Inst. 97: 1695-1699 https://doi.org/10.1093/jnci/dji377
  9. Kase, H., K. Iwahashi, Y. Matsuda, M. Takahashi, C. Murakata, A. Sato, and M. Kaneko. 1987. K-252a compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem. Biophys. Res. Commun. 142: 436-440 https://doi.org/10.1016/0006-291X(87)90293-2
  10. Kondapaka, S. B., M. J. Zarnowski, D. R. Yver, E. A. Sausville, and S. W. Cushman. 2004. 7-Hydroxystaurosporine (UCN-01) inhibition of Akt t$thr^{308}$ but not $ser^{473}$ phosphorylation: a basis for decreased insulin-stimulated glucose transport. Clinical Cancer Res. 10: 7192-7198 https://doi.org/10.1158/1078-0432.CCR-04-0772
  11. Bourret, R. B., K. A. Borkovich, and M. I. Simon. 1991. Signal transduction pathways involving protein phosphorylation in prokaryotes. Annu. Rev. Biochem. 60: 401-441 https://doi.org/10.1146/annurev.bi.60.070191.002153
  12. Fayard, E., L. A. Tintignac, A. Baudry, and B. A. Hemmings. 2005. Protein kinase B/Akt at a glance. J. Cell Sci. 118: 5675-5678 https://doi.org/10.1242/jcs.02724
  13. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  14. Hong, S.-K., A. Matsumoto, S. Horinouchi, and T. Beppu. 1993. Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus. Mol. Gen. Genet. 236: 347-354 https://doi.org/10.1007/BF00277132
  15. Uehara, Y. and H. Ogawara. 1991. Use and selectivity of herbimycin A as inhibitor of protein tyrosine kinases. Methods Enzymol. 201: 370-379 https://doi.org/10.1016/0076-6879(91)01033-X
  16. Kwon, T., D. Y. Kwon, J. Chun, J. H. Kim, and S. S. Kang. 2000. Akt protein kinase inhibits Rac1-GTP binding through phosphorylation at serine 71 of Rac1. J. Biol. Chem. 275: 423-428 https://doi.org/10.1074/jbc.275.1.423
  17. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, U. S. A