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J. Biol. Chem., Vol. 275, Issue 47, 37240-37245, November 24, 2000

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The Super Anti-apoptotic Factor Bcl-xFNK Constructed by Disturbing Intramolecular Polar Interactions in Rat Bcl-x_L^*

Sadamitsu Asoh, Takashi Ohtsu, and Shigeo Ohta^§

From the Department of Biochemistry and Cell Biology, Institute of Gerontology, Nippon Medical School, 1-396, Kosugi-cho, Nakahara-ku, Kawasaki-city, Kanagawa-ken 211-8533, Japan

A powerful artificial anti-apoptotic factor will be useful for medical applications of the future therapies for many diseases by prolonging survival of sick cells. For constructing it, we designed the super anti-apoptotic factor by disturbing three intramolecular polar interactions among -helix structures of Bcl-x_L. The resultant mutant Bcl-x_L, named Bcl-xFNK, was expected to make the pore-forming domain more mobile and flexible than the wild-type. When overexpressed in Jurkat cells, Bcl-xFNK was markedly more potent in prolonging survival following apoptosis-inducing treatment with a kind of cell death cytokines (anti-Fas), a protein kinase inhibitor (staurosporine), cell cycle inhibitors (TN-16, camptothecin, hydroxyurea, and trichostatin A), or oxidative stress (hydrogen peroxide and paraquat) than wild-type Bcl-x_L. Furthermore, the transfectants of bcl-xFNK became more resistant against a calcium ionophore and even a heat treatment than wild-type Bcl-x_L. In addition, Bcl-xFNK showed marked anti-apoptotic activity in Chinese hamster ovary and Jurkat cells deprived of serum. Thus, Bcl-xFNK may be the first mutant generated by site-directed mutagenesis of Bcl-x_L with a gain-of-function phenotype. Interestingly, Bcl-xFNK was found to allow interleukin-3-dependent FDC-P1 to grow without interleukin-3, but not BaF/3. In Bcl-xFNK transfectants of FDC-P1 and Jurkat, the p42/p44 mitogen-activated protein kinase was activated by 2 to 5 times, but not in those of BaF/3 and Chinese hamster ovary. Bcl-xFNK might gain a new function to activate the mitogen-activated protein kinase in a cell-type specific manner. The findings of this study suggest that the central 5-6 pore-forming region of anti-apoptotic factor Bcl-x_L has a pivotal role in suppressing apoptosis.

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