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J. Biol. Chem., Vol. 280, Issue 16, 16383-16392, April 22, 2005

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Disruption of Mcl-1·Bim Complex in Granzyme B-mediated Mitochondrial Apoptosis^*

Jie Han, Leslie A. Goldstein, Brian R. Gastman¶, Asaf Rabinovitz, and Hannah Rabinowich||**

From the Departments of Pathology and ^¶Plastic Surgery, the University of Pittsburgh School of Medicine and the ^||University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213

Recently, we reported the identification of a novel mitochondrial apoptotic pathway for granzyme B (GrB) (Han, J., Goldstein, L. A., Gastman, B. R., Froelich, C. J., Yin, X. M., and Rabinowich, H. (2004) J. Biol. Chem. 279, 22020–22029). The newly identified GrB-mediated mitochondrial cascade was initiated by the cleavage and subsequent degradation of Mcl-1, resulting in the release of mitochondrial Bim from Mcl-1 sequestration. To investigate the biological significance of Mcl-1 cleavage by GrB, we mapped the major GrB cleavage sites and evaluated the apoptotic potential of the cleavage products. GrB cleaves Mcl-1 after aspartic acid residues 117, 127, and 157, generating C-terminal fragments that all contain BH-1, BH-2, BH-3, and transmembrane domains. These fragments accumulate at an early apoptotic phase but are eliminated by further degradation during the apoptotic process. The major Mcl-1 C-terminal fragment generated by GrB (residues 118–350) was unable to induce or enhance apoptosis when transfected into tumor cells. Instead, this Mcl-1 C-terminal fragment maintained a partial protective capability against GrB-mediated apoptosis via its lower affinity to Bim. In comparison with ectopically expressed full-length Mcl-1, the stably transfected C-terminal fragments of Mcl-1 were less efficiently localized to the mitochondria. Knockdown of Mcl-1, as achieved by transfection with Mcl-1-specific short interfering RNA, resulted in a significant level of apoptosis in the absence of external apoptotic stimulation and, in addition, enhanced the susceptibility of breast carcinoma cells to GrB cytotoxicity. The significance of Bim in this GrB apoptotic cascade was indicated by the marked protection against GrB-mediated apoptosis endowed on these cells through Bim knockdown. Our studies suggest that the disruption of the Mcl-1·Bim complex by GrB initiates a major Bim-mediated cellular cytotoxic mechanism that requires the elimination of Mcl-1 following its initial cleavage.

Received for publication, October 5, 2004 , and in revised form, January 25, 2005.

^* This work was supported by grants from National Institutes of Health Grant RO1 CA 109285 (to H. R.) and Department of Defense Grant DAMD17-02-1-0552 (to H. R.). 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.

These authors contributed equally to this work.

^** To whom correspondence should be addressed: University of Pittsburgh Cancer Institute, The Hillman Cancer Center, Research Pavilion, Rm. G17c, 5117 Centre Ave., Pittsburgh, PA 15213. Tel.: 412-623-3212; Fax: 412-623-1119; E-mail: rabinow{at}pitt.edu.

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