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J. Biol. Chem., Vol. 278, Issue 22, 19732-19742, May 30, 2003

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Deletion of the BH1 Domain of Bcl-2 Accelerates Apoptosis by Acting in a Dominant Negative Fashion^*

Makoto Kawatani and Masaya Imoto 

From the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan

To investigate the exact biochemical functions by which Bcl-2 regulates apoptosis, we established a stable human small cell lung carcinoma cell line, Ms-1, overexpressing wild-type human Bcl-2 or various deletion and point mutants thereof, and examined the effect of these Bcl-2 mutants on apoptosis induced by antitumor drugs such as camptothecin. Cytochrome c release, caspase-3-(-like) protease activation, and apoptosis induced by antitumor drugs were accelerated by overexpression of Bcl-2 lacking a Bcl-2 homology (BH) 1 domain (Bcl-2/ BH1), but not by that of BH2, BH3, or BH4 domain-deleted Bcl-2. A similar result was obtained upon the substitution of glycine 145 with alanine in the BH1 domain (Bcl-2/G145A), which failed to interact with either Bax or Bak. Pro-apoptotic Bax and Bak have been known to be activated in response to antitumor drugs, and Bcl-2/G145A as well as Bcl-2/BH1 also accelerated Bax- or Bak-induced apoptosis in HEK293T cells. These two mutants still retained the ability to interact with wild-type Bcl-2 and Bcl-x_L, and abrogated the inhibitory effect of wild-type Bcl-2 or Bcl-x_L on Bax- or Bak-induced apoptosis. In addition, immunoprecipitation studies revealed that Bcl-2/BH1 and Bcl-2/G145A interrupted the association between wild-type Bcl-2 and Bax/Bak. Taken together, our results demonstrate that Bcl-2/BH1 or Bcl-2/G145A acts as a dominant negative of endogenous anti-apoptotic proteins such as Bcl-2 and Bcl-x_L, thereby enhancing antitumor drug-induced apoptosis, and that this dominant negative activity requires both a failure of interaction with Bax and Bak through the BH1 domain of Bcl-2 and retention of the ability to interact with Bcl-2 and Bcl-x_L.

Received for publication, December 20, 2002 , and in revised form, March 3, 2003.

^* This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and was performed using Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyohi, Kohoku-ku, Yokohama 223-8522, Japan. Tel. and Fax: 81-45-566-1557; E-mail: imoto{at}bio.keio.ac.jp.

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