# RNase L Induces Expression of A Novel Serine/Threonine Protein Kinase, DRAK1, to Promote Apoptosis
## 2 Results
### DRAK1 Kinase Activity and Nuclear Localization Are Required for Inducing Apoptosis  
DRAK1 was localized in the nucleus and amino acid sequence analysis identified a highly conserved nuclear localization sequence (NLS) in the N-terminal kinase domain that overlapped with the conserved lysine residue in the ATP-binding site of DRAK1 (Figure 4A). To evaluate the role of kinase activity and nuclear localization of DRAK1 in inducing apoptosis, we constructed three mutants in which (a) a critical lysine in the ATP binding site required for kinase activity was mutated to alanine (K90A), (b) a conserved lysine in the putative NLS sequence was mutated to alanine (K94A) or (c) both lysine residues were replaced with alanine (K90A/K94A). The GFP-tagged mutant proteins were expressed in PC3 cells to determine the subcellular localization and compared to WT DRAK1. The nuclei were simultaneously visualized by staining with 4',6-diamidino-2-phenylindole (DAPI). As shown in Figure 4A, the fluorescent staining of DRAK1 WT overlapped with the nuclear staining, while the kinase-dead (K90A), NLS mutant (K94A), or both kinase and NLS mutant localized to the cytoplasm (Figure 4A). These results suggest that kinase activity of DRAK1 is required for its nuclear localization and NLS mutant which has kinase activity does not localize to the nucleus. Figure 4: Involvement of DRAK1 kinase activity and nuclear localization in inducing apoptosis. (**A**) Schematic representation of DRAK1 kinase ATP binding site and nuclear localization signal (NLS). PC3 cells expressing GFP-WT or DRAK1 mutants analyzed by immunofluorescence microscopy (scale bar, 20 μm, representative images are shown), (**B**) Cell viability analyzed by MTT assay at indicated times following transfection, (**C**) Cell lysates probed with indicated antibodies and normalized to \(\beta\)-actin levels, (**D**) Cells were stained with TMRE and fluorescence intensity was normalized to control cells. (**E,F**) PC3 cells were transfected with GFP-WT or DRAK1 mutants and 24 h later mitochondrial and cytosolic fractions were isolated and analyzed as indicated. Values shown are mean \(\pm\) s.d of triplicate assays. * \(p<0.01\), **\(p<0.001\), *** \(p<0.0001\), n.s. : not significant. To investigate the correlation between kinase activity and nuclear localization with initiation of apoptosis, we expressed DRAK1 or the mutant constructs in PC3 cells and quantitated cell viability in time course experiments. Expression of WT DRAK1 reduced cell viability to 57% when compared to control cells with vector alone. In contrast, viability of cells expressing kinase-dead, NLS mutant and combining both kinase and NLS mutant restored viability to same level as control cells (Figure 4B). Furthermore, DRAK1-induced JNK activity and cleavage of caspase 3 and PARP was reduced in cells expressing the DRAK1 mutants (Figure 4C) which correlated with reduced mitochondrial damage and transmembrane potential (Figure 4D). We have shown that RNase L activates JNK and promotes switch from autophagy to apoptosis involving Bax translocation to the mitochondria and release of cytochrome C to the cytosol [16]. One major mechanism of activation and cleavage of caspase 3 involves release of cytochrome C from the mitochondria. Since ectopic DRAK1 expression activates JNK that is required for apoptosis, we tested Bax translocation to the mitochondria and release of cytochrome C. Following subcellular fractionation, Bax translocation to the mitochondria was detected on immunoblots only in WT DRAK1 expressing cells compared to control cells and this was accompanied by release of mitochondrial cytochrome C to the cytoplasm. Whereas in DRAK1 mutants that lacked apoptosis-inducing function, Bax remained cytosolic along with retention of cytochrome C in the mitochondria (Figure 4E,F). Taken together, our results indicate that DRAK1 activates JNK signaling and induces apoptosis involving translocation of Bax to the mitochondria suggesting that DRAK1 acts upstream of mitochondrial-based pro-apoptotic proteins.