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

다국어 초록 (Multilingual 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.

국문 초록 (Abstract)

$Ca^{2+}$-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.

참고문헌 (Reference)

1 "proteolysis drives the cell cycle" 274 : 1652-1659, 1996

2 "new perspectives in molecular diversity and physiological-pathological involvement" 814-822, 1994

3 "an upstream regulator of thymocyte apoptosis" 158 : 3690-3697, 1997

4 "an enzyme system active at cellular membranes? FASEB J" 110-115, 1987

5 "Turnover of cyclin E by the ubiquitin proteasome pathway is regulated by cdk2 binding and cyclin phosphorylation" 10 : 1979-1990, 1996

6 "Transferring substrates to the 26S proteasome" 28 : 26-31, 2003

7 "The ubiquitin proteasome pathway" 36 : 285-295, 2004

8 "Structure activation and biology of calpain" 53 : 12-18, 2004

9 "Role of calpain in adipocyte differentiation" 96 : 1279-1284, 1996

10 "Resolution of two forms with different requirements for calcium" 129-133, 1980

11 "Regulation of transcription by proteins that control the cell cycle" 389 : 149-152, 1997

12 "Regulation of cyclin D1 by calpain protease" 272 : 28479-28484, 1997

13 "Proteolytic regulation of the zinc finger transcription factor YY1 a repressor of muscle restricted gene expression" 273 : 6656-6661, 1998

14 "Proteolytic cleavage of human p53 by calpain a potential regulator of protein stability" 17 : 460-468, 1997

15 "Pathways of proteolysis affecting renal cell growth" 11 : 445-450, 2002

16 "On the involvement of calpains in the degradation of the tumor suppressor protein p53" 406 : 17-22, 1997

17 "New era of calpain research Discovery of tissue-specific calpains" 1-5, 1994

18 "Isolation of a Chinese hamster ovary cell clone possessing decreased mu calpain content and a reduced proliferative growth rate" 271 : 15568-15574, 1996

19 "Is calpain activity regulated by membranes and autolysis or by calcium and calpastatin? Bioessays 14" 549-556, 1992

20 "Inhibition of the 3hydroxy 3methylglutaryl coenzyme A reductase pathway induces p53 independent transcriptional regulation of p21" 273 : 10618-10623, 1998

21 "Inactivation of Mphase promoting factor at exit from first embryonic mitosis in the rat is independent of cyclin B1 degradation" 64 : 871-878, 2001

22 "In vivo ubiquitination and proteasome-mediated degradation of p53" 56 : 2649-2654, 1996

23 "Importance of the ATP ubiquitin proteasome pathway in the degradation of soluble and myofibrillar proteins in rabbit muscle extracts" 271 : 26690-26697, 1996

24 "Degradation of transcription factors" 57-61, 1991

25 "Cyclin is degraded by the ubiquitin pathway" 132-349 138, 1991

26 "Cyclin Ddependent kinases" 602 : 73-87, 2002

27 "Cyclin D and CDK inhibitors come of age" 573-582, 1994

28 "Cell cycle-specific effects of lovastatin" 3628-88 3632, 1991

29 "Cell cycle dysregulation in pituitary oncogenesis" 32 : 34-62, 2004

30 "Calpain function in the modulation of signal transduction molecules" 382 : 743-751, 2001

31 "Calpain and calpastatin regulate neutrophil apoptosis" 178 : 311-319, 1999

32 "A novel aspect of calpain activation" 433 : 1-4, 1998