Anti-Phosphoserine/Phosphothreonine/Phesphotyrosine Antibody Immunoaffinity Column Chromatography를 이용한 Streptomyces griseus의 인산화 단백질 동정

Identification of Protein Kinases by Anti-phosphoserine/Phosphothreonine/Phosphotyrosine Antibody Immunoaffinity Column Chromatographies in Streptomyces griseus.

  • 정용훈 (명지대학교 이과대학 생명과학정보학부) ;
  • 김종희 (서일대학 식품영양과)
  • 발행 : 2007.06.28

초록

Protein kinase는 진핵생물과 원핵생물을 포함하는 모든 생명체에서 세포생존에 절대적으로 중요한 조절 기능을 담당한다. 일반적으로 원핵생물은 histidine 과 aspartic acid kinase로 구성된 bacterial two-component regulatory system에 의하여 환경변화에 따른 유전자의 발현이 조절되지만, 방선균을 비롯한 고등 원핵생물에서는 진핵생물성의 serine/threonine kinase들이 세포분화와 같은 분화과정을 조절하고 있다. Streptomycin 생산균인 Streptomyces griseus 균주에서도 다양한 serine/threonine kinase들이 존재하는 것으로 추정되며, 이들의 기능을 밝히는 것은 생명현상을 이해하는 중요한 열쇠를 제공해 줄 것으로 기대된다. 따라서, S. griseus로부터 protein kinase 를 동정하는 연구를 실시하였으며, 기존의 복잡한 chromatography법의 단점을 보완하기 위해 anti-phosphothreonine, anti-phosphoserine, anti-phosphotyrosine antibody를 이용한 immunoaffinity column chromatography 방법을 도입하였다. 실험 결과 약 14, 29, 31, 35, 40, 52, 56, 60 kDa의 단백질을 효과적으로 동정 할 수 있었으며, nonradioactive protein kination assay 방법으로 이들의 인산화능을 확인하였다.

Protein kinases play very important role for maintaining viability in prokaryote and eukaryote. The metabolism of prokaryotic cell is generally regulated by bacterial two-component regulatory systems that are composed of histidine and asparitic acid kinases, however, some eukaryotic signal transduction system such as, serine and threonine kinases, have been also found to be involved in the regulation of morphogenesis and physiological differentiation in Streptomyces. Streptomyces griseus, a streptomycin producer, was expected to have varlous types of eukaryotic-type serine/threonine protein kinases, controlling morphogenesis. Thus, many steps of chromatographies were applied to isolate serine and threonine kinases from S. griseus IFO13350. The immunoaffinity steps using anti-phosphoserine, anti-phosphothreonine, and anti-phosphotyrosine agarose column chramatographies were successfully introduced to identify eukaryotic protein kinases from S. griseus IFO13350. Eight proteins with the expected molecular weight of 14, 29, 31, 35, 40, 52, 56, and 60 kDa, were identified on SDS-PAGE, and the their kination activity was confirmed by nonradioactive protein kination assay using FITC-labeled peptide as the substrate.

키워드

참고문헌

  1. Babcock, M. J. and K. E. Kendrick. 1990. Transcriptional and translational features of a sporulation gene of Streptomyces griseus. Gene 95: 57-63 https://doi.org/10.1016/0378-1119(90)90413-L
  2. Beppu, T. and S. Horinouchi. 1991. Molecular mechanisms of the A-factor-cependent control of secondary metabolism in Streptomyces. Planta Medica 57: S44-S47 https://doi.org/10.1055/s-2006-960228
  3. 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
  4. 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
  5. Distler, J., K. Mansouri, G. Mayer, M. Stockmann, and W. Piepersberg. 1992. Streptomycin biosynthesis and its regulation in Streptomycetes. Gene 115: 105-111 https://doi.org/10.1016/0378-1119(92)90547-3
  6. 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: 235-240
  7. Hong, S.-K. and S. Horinouchi. 1998. Effects of protein kinase inhibitors on in vitro protein phosphorylation and on secondary metabolism and morphogenesis in Streptomyces coelicolor A3(2). J. Microbiol. Biotechnol. 8: 325-332
  8. 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
  9. Hong, S.-K., M. Kito, T. Beppu, and S. Horinouchi. 1991. Phosphorylation of the AfsR product, a global regulatory protein for secondary-metabolite formation in Streptomyces coelicolor A3(2). J. Bacteriol. 173: 2311-2318 https://doi.org/10.1128/jb.173.7.2311-2318.1991
  10. Horinouchi S. 2002. A microbial hormone, A-factor, as a master switch for morphological differentiation and secondary metabolism in Streptomyces griseus. Front Biosci. 7: 2045-2057 https://doi.org/10.2741/horinouc
  11. Horinouchi, S., K. Miyake, S.-K. Hong, D. Vujaklija, K. Ueda, and T. Beppu. 1991. Regulation by A-factor and afsR of secondary metabolism and morphogenesis in Streptomyces. Actinomycetologica 5: 119-125 https://doi.org/10.3209/saj.5_119
  12. Kang, S. S., T Kwon, D. Y. Kwon, and S. I. Do. 1999. Akt protein kinase enhances human telomerase activity through phosphorylati.on of telomerase reverse transcriptase subunit. J. Biol. Chem. 274: 13085-13090 https://doi.org/10.1074/jbc.274.19.13085
  13. 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
  14. Ohnishi Y., H. Yamazaki, J. Y. Kato, A. Tomono, and S. Horinouchi. 2005. AdpA, a central transcriptional regulator in the A-factor regulatory cascade that leads to morphological development and secondary metabolism in Streptomyces griseus. Biosci. Biotechnol. Biochem. 69: 431-439 https://doi.org/10.1271/bbb.69.431
  15. Park, U. and S.-K. Hong. 1998. Regulatory factors involved in physiological differentiation of Streptomyces coelicolor. Actinomycetologica 12: 134-140 https://doi.org/10.3209/saj.12_134
  16. Sawai R., A. Suzuki, Y. Takano, P. C. Lee, and S. Horinouchi. 2004. Phosphorylation of AfsR by multiple serine/threonine kinases in Streptomyces coelicolor A3(2). Gene 334: 53-62 https://doi.org/10.1016/j.gene.2004.02.046