Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Regulation of Cyclin D3 by Calpain Protease in Human Breast Carcinoma MDA-MB-231 Cells

인체 유방암세포에서 calpain protease에 의한 cyclin D3의 발현 조절

  • Choi, Byung-Tae (Department of Anatomy and Biochemistry, Dongeui University College of Oriental Medicine and Department of Biomaterial Control, Dongeui University Graduate School) ;
  • Kim, Gun-Do (Department of Microbiology, College of Natural Sciences, Bukyong National University) ;
  • Choi, Yung-Hyun (Department of Anatomy, Dongeui University College of Oriental Medicine)
  • 최병태 (해부학교실 및 대학원 바이오물질제어학과) ;
  • 김군도 (부경대학교 자연과학대학 미생물학과) ;
  • 최영현 (동의대학교 한의과대학 생화학교실)
  • Published : 2006.07.31
Download PDF
⟨ Previous Next ⟩

Abstract

The $Ca^{2+}-activated$ neutral protease calpain induced proteolysis has been suggested to play a role in certain cell growth regulatory proteins. Cyclin proteolysis is essential for cell cycle progression. D-type cyclins, which form an assembly with cyclin-dependent kinases (cdk4 and cdk6), are synthesized earlier in G1 of the cell cycle and seem to be induced in response to external signals that promote entry into the cell cycle. Here we show that cyclin D3 protein levels are regulated at the posttranscriptional level by calpain protease. Treatment of human breast carcinoma MDA-MB-231 cells with lovastatin and actinomycin D resulted in a loss of cyclin D3 protein that was completely reversible by the peptide aldehyde calpain inhibitor, LLnL. The specific inhibitor of the 26S proteasome, lactacystin, the lysosome inhibitors, ammonium chloride and chloroquine, and the serine protease inhibitor, phenylmethylsulfonylfluoride (PMSF), did not block the degradation of cyclin D3 by lovastatin and actinomycin D. Results of in vitro degradation of cyclin D3 by purified calpain showed that cyclin D3 protein is degraded in a $Ca^{2+}-dependent$ manner, and the half-life of cyclin D3 protein was dramatically increased in LLnL treated cells. These data suggested that cyclin D3 protein is regulated by the $Ca^{2+}-activated$ protease calpain.

$Ca^{2+}$-농도 의존적으로 활성화되는 neutral protease calpain에 의한 단백질 분해는 세포의 성장을 조절하는데 중요한 단백질들의 역할에 매우 중요한 역할을 한다. Cyclin의 분해는 세포주기의 진행을 위한 필연적인 과정이다. D-type cyclins는 외부자극이나 신호에 의하여 세포주기의 G1 초기에 합성이 된 후 cyclin-dependent kinases (cdk4 및 cdk6)와의 결합하여 세포주기 S기 진입을 촉진하는 역할을 한다. 본 연구에서는 MDA-MB-231 인체 유방암세포에서 cyclin D3 단백질이 calpain protease에 의하여 전사 후 수준에서 조절 받고 있음을 제시하였다. 본 실험의 조건에서 lovastatin과 actinomycin D가 처리된 MDA-MB-231 세포에서 cyclin D3 단백질의 발현이 완전히 사라졌지만, calpain inhibitor인 LLnL의 처리에 의하여 정상 수준으로 회복되었음을 알 수 있었다. 그러나 26S proteasome의 선택적 억제제인 lactacystin, the lysosome 억제제인 ammonium chloride 및 chloroquine, serine protease 억제제인 PMSF는 동일 조건에서 lovastatin과 actinomycin D 처리에 의한 cyclin D3의 발현저하를 억제하지는 못하였다. In vitro 조건에서 순수 분리된 calpain은 cyclin D3 단백질을 $Ca^{2+}$ 농도 의존적으로 분해하였으며, cyclin D3 단백질의 half-life는 LLnL 처리에 의하여 매우 유의적으로 증가되었다. 이러한 결과는 cyclin D3 단백질이 $Ca^{2+}$에 의해 활성화 되는 protease calpain에 의해 조절됨을 보여준다.

Keywords

References

  1. Ciechanover, A. 1994. The ubiquitin-proteasome proteolytic pathway. Cell 79, 13-21 https://doi.org/10.1016/0092-8674(94)90396-4
  2. Clurman, B. E., R. J. Sheaff, K. Thress, M. Groudine and J. M. Roberts. 1996. Turnover of cyclin E by the ubiquitin- proteasome pathway is regulated by cdk2 binding and cyclin phosphorylation. Genes Dev. 10, 1979-1990 https://doi.org/10.1101/gad.10.16.1979
  3. Choi, Y. H., S. J. Lee, P. Nguyen, J. S. Jang, J. Lee, M. L. Wu, E. Takano, M. Maki, P. A. Henkart and T. B. Trepel. 1997. Regulation of cyclin D1 by calpain protease. J. Biol. Chem. 272, 28479-28484 https://doi.org/10.1074/jbc.272.45.28479
  4. Dynlacht, B. D. 1997. Regulation of transcription by proteins that control the cell cycle. Nature 389, 149-152 https://doi.org/10.1038/38225
  5. Franch, H. A. 2002. Pathways of proteolysis affecting renal cell growth. Curr Opin Nephrol. Hypertens. 2002 11: 445-450 https://doi.org/10.1097/00041552-200207000-00012
  6. Glotzer, M., A. W. Murray and M. W. Kirschner. 1991. Cyclin is degraded by the ubiquitin pathway. Nature 349, 132-138 https://doi.org/10.1038/349132a0
  7. Goll, D. E., V. F. Thompson, R. G. Taylor and T. Zalewska. 1992. Is calpain activity regulated by membranes and autolysis or by calcium and calpastatin? Bioessays 14, 549-556 https://doi.org/10.1002/bies.950140810
  8. Gonen, H., D. Shkedy, S. Barnoy, N. S. Kosower and A. Ciechanover. 1997. On the involvement of calpains in the degradation of the tumor suppressor protein p53. FEBS Lett. 406, 17-22 https://doi.org/10.1016/S0014-5793(97)00225-1
  9. Hartmann-Petersen, R., M. Seeger and C. Gordon. 2003. Transferring substrates to the 26S proteasome. Trends Biochem. Sci. 28, 26-31 https://doi.org/10.1016/S0968-0004(02)00002-6
  10. Hirai, S., H. Kawasaki, M. Yaniv and K. Suzuki. 1991. Degradation of transcription factors, c-Jun and c-Fos, by calpain. FEBS Lett. 287, 57-61 https://doi.org/10.1016/0014-5793(91)80015-U
  11. Hunter, T. and J. Pines. 1994. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell 79, 573-582 https://doi.org/10.1016/0092-8674(94)90543-6
  12. Jakobisiak, M., S. Bruno, J. S. Skierski, Z. Darzynkiewicz. 1991. Cell cycle-specific effects of lovastatin. Proc. Natl. Acad. Sci. USA 88, 3628-3632
  13. Josefsberg, L. B., O. Kaufman, D. Galiani, M. Kovo and N. Dekel. 2001. Inactivation of M-phase promoting factor at exit from first embryonic mitosis in the rat is independent of cyclin B1 degradation. Biol. Reprod. 64, 871-878 https://doi.org/10.1095/biolreprod64.3.871
  14. King, R. W., R. J. Deshaies, J. M. Peters and M. W. Kirschner. 1996. How proteolysis drives the cell cycle. Science 274, 1652-1659 https://doi.org/10.1126/science.274.5293.1652
  15. Kubbutat, M. H. and K. H. Vousden. 1997. Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability. Mol. Cell. Biol. 17, 460-468 https://doi.org/10.1128/MCB.17.1.460
  16. Lee, S. J., M. J. Ha, J. Lee, P. Nguyen, Y. H. Choi, F. Pirnia, W. K. Kang, X. F. Wang, S. J. Kim and J. B. Trepel. 1998. Inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase pathway induces p53-independent transcriptional regulation of p21(WAF1/CIP1) in human prostate carcinoma cells. J. Biol. Chem. 273, 10618-10623 https://doi.org/10.1074/jbc.273.17.10618
  17. Maki, C. G., J. M. Huibregtse and P. M. Howley. 1996. In vivo ubiquitination and proteasome-mediated degradation of p53. Cancer Res. 56, 2649-2654
  18. Mellgren, R. L. 1980. Canine cardiac calcium-dependent proteases: Resolution of two forms with different requirements for calcium. FEBS Lett. 109, 129-133 https://doi.org/10.1016/0014-5793(80)81326-3
  19. Mellgren, R. L. 1987. Calcium-dependent proteases: an enzyme system active at cellular membranes? FASEB J. 1, 110-115 https://doi.org/10.1096/fasebj.1.2.2886390
  20. Mellgren, R. L., Q. Lu, W. Zhang, M. Lakkis, E. Shaw and M. T. Mericle. 1996. Isolation of a Chinese hamster ovary cell clone possessing decreased mu-calpain content and a reduced proliferative growth rate. J. Biol. Chem. 271, 15568-15574 https://doi.org/10.1074/jbc.271.26.15568
  21. Murachi, T. 1989. Intracellular regulatory system involving calpain and calpastatin. Biochem. Int. 18, 263-294
  22. Musat, M., V. V. Vax, N. Borboli, M. Gueorguiev, S. Bonner, M. Korbonits and A.B. Grossman. 2004. Cell cycle dysregulation in pituitary oncogenesis. Front. Horm. Res. 32, 34-62 https://doi.org/10.1159/000079037
  23. Ortega, S., M. Malumbres and M. Barbacid. 2002. Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim. Biophys. Acta. 602, 73-87
  24. Patel, Y. M. and M. D. Lane. 1996. Role of calpain in adipocyte differentiation. Proc. Natl. Acad. Sci. USA 96, 1279-1284
  25. Roos-Mattjus, P. and L. Sistonen. 2004. The ubiquitin- proteasome pathway. Ann. Med. 36, 285-295 https://doi.org/10.1080/07853890310016324
  26. Saido, T. C., H. Sorimachi and K. Suzuki. 1994. Calpain: new perspectives in molecular diversity and physiological- pathological involvement. FASEB J. 8, 814-822 https://doi.org/10.1096/fasebj.8.11.8070630
  27. Sato, K. and S. Kawashima. 2001. Calpain function in the modulation of signal transduction molecules. Biol. Chem. 382, 743-751 https://doi.org/10.1515/BC.2001.090
  28. Solomon, V. and A. L. Goldberg. 1996. Importance of the ATP-ubiquitin-proteasome pathway in the degradation of soluble and myofibrillar proteins in rabbit muscle extracts. J. Biol. Chem. 271, 26690-26697 https://doi.org/10.1074/jbc.271.43.26690
  29. Sorimachi, H., T. C. Saido and K. Suzuki. 1994. New era of calpain research. Discovery of tissue-specific calpains. FEBS Lett. 343, 1-5 https://doi.org/10.1016/0014-5793(94)80595-4
  30. Squier, M. K. and J. J. Cohen. 1997. Calpain, an upstream regulator of thymocyte apoptosis. J. Immunol. 158, 3690-3697
  31. Squier, M. K., A. J. Sehnert, K. S. Sellins, A. M. alkinson, E. Takano and J. J. Cohen. 1999. Calpain and calpastatin regulate neutrophil apoptosis. J. Cell. Physiol. 178, 311-319 https://doi.org/10.1002/(SICI)1097-4652(199903)178:3<311::AID-JCP5>3.0.CO;2-T
  32. Suzuki, K. and H. Sorimachi. 1998. A novel aspect of calpain activation. FEBS Lett. 433, 1-4 https://doi.org/10.1016/S0014-5793(98)00856-4
  33. Suzuki, K., S. Hata, Y. Kawabata and H. Sorimachi. 2004. Structure, activation, and biology of calpain. Diabetes 53, S12-18 https://doi.org/10.2337/diabetes.53.2007.S12
  34. Walowitz, J. L., M. E. Bradley, S. Chen and T. Lee. 1998. Proteolytic regulation of the zinc finger transcription factor YY1, a repressor of muscle-restricted gene expression. J. Biol. Chem. 273, 6656-6661 https://doi.org/10.1074/jbc.273.12.6656