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. A powerful synthetic anti-apoptotic factor would be useful for future therapeutic applications in medicine. Analysis of the crystal structure of renatured human BclxL has suggested that it forms an ion channel (1). Additionally, Bcl-xL has been shown to form an ion channel in synthetic lipid membranes (2). We have independently determined the x-ray structure of native rat Bcl-xL with a 2.5 A resolution and found it to have nine intramolecular polar interactions to stabilize the central putative pore-forming domain (α5- α6 helices) (3). We hypothesized that these hydrogen bonds may disturb the anti-apoptotic effect by inhibiting the insertion of the pore-forming domain into membranes. In the present study we designed a mutant Bcl-xL, named Bcl-xFNK to make the α5- α6 helices more mobile or flexible by disturbing the formation of three hydrogen bonds which stabilize the tip of the putative poreforming domain. METHODS. To construct Bcl-xFNK, three amino acid substitutions (Tyr 22 to Phe [F], Gln 26 to Asn [N] and Arg 165 to Lys [K]) were introduced into rat Bcl-xL by a two-step PCR mutagenesis. Chinese hamster CHO K1 cells were cultured in D-MEM/F-12 medium containing 10 % fetal bovine serum. Human Jurkat cells and IL-3 dependent murine cells, FDC-P1 and BaF/3, were cultured in RPMI 1640 medium containing 10 % FBS. For FDC-P1 and BaF/3 cells, IL-3 was added in the medium. Plasmids were introduced into Jurkat, FDC-P1 and BaF/3 cells by electroporation and into CHO cells using Superfect. Stable transfectants Jurkat cells were treated with various death-inducing stimuli. CHO cell transfectants were deprived of serum. FDC-P1 and BaF/3 transfectant cells were incubated in the absence of IL-3. Surviving cells were counted by the trypan blue exclusion or by the WST-1 assay. BIOTRAK p42/p44 MAP kinase enzyme assay system was used to measure the p42/p44 MAP kinase activity of cells. RESULTS. When over expressed in Jurkat or CHO cells, Bcl-xFNK was markedly more potent than wild-type Bcl-xL in prolonging survival against anti-Fas (CH-11), staurosporine, TN-16, camptothecin, hydroxyurea, trichostatin A, hydrogen peroxide, paraquat, a calcium ionophore (A23187), heat treatment and serum deprivation. Interestingly, Bcl-xFNK allowed IL-3-dependent FDC-P1 but not BaF/3 cells to grow without IL-3. In Bcl-xFNK transfectants of FDC-P1 and Jurkat cells, the p42/p44 MAP kinase was activated by 2 to 5 times, but not in BaF/3 and CHO cells. Thus, Bcl-xFNK might acquire a new function to activate the MAP kinase in a cell-type specific manner.

The Super Anti-Apoptotic Factor Bcl-xFNK: A Novel Mutant of Rat Bcl-Xl with a Gain-of-Function Phenotype