# RNase L Induces Expression of A Novel Serine/Threonine Protein Kinase, DRAK1, to Promote Apoptosis
## 2 Results
### Ectopic Expression of DRAK1 Induces Apoptosis by Activating JNK and Cleavage of Caspase-3  
To specifically address the role of DRAK1 and signaling pathways involved in apoptosis, further experiments utilized ectopic expression of DRAK1. Next, we investigated the signaling pathways that mediate DRAK1-induced apoptosis. Previous studies show that JNK is activated in RNase L-induced apoptosis accompanied by cleavage of PARP and caspase-3 [17; 27]. Thus, we examined if ectopic expression of DRAK1 could activate JNK signaling in PC3 cells transfected with DRAK1. Phosphorylation of JNK that is required for its activation was evident 12h after transfection and significant phosphorylation was detected until 36 h. Increase in JNK activity correlated with cleavage of key apoptotic proteins, PARP and caspase 3, which is a hallmark of intrinsic mitochondrial pathway of apoptosis. No cleavage of caspase 8, an extrinsic pathway substrate was observed under similar conditions (Figure 3A). To determine if DRAK1 is required for JNK phosphorylation, we analyzed phospho-JNK and cleavage of caspase 3 in DRAK1 knockdown cells treated with 2-5A. We observed that the cleavage of DRAK1 is not sufficient for the activation of DRAK1. Figure 2: Nuclear localization of DRAK1 and induction of apoptosis (**A**) Fluorescence microscopy of DRAK-GFP or GFP vector expression and bright field images at indicated times in PC3 cells (scale bar, 10 μm, representative images are shown). PC3 cells were transfected with vector or DRAK1 construct and cell viability was determined at indicated times by (**B**) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzolium bromide (MTT) assay and normalized to control, (**C**) uptake of Propidium Iodide (PI) by dying cells as measured by flow cytometry after staining with PI, (**D**) trypan blue dye exclusion assay normalized to control cells. Data shown as % apoptotic cells. PC3 cells were transfected with siRNA targeting DRAK1, DRAK2 or scrambled control siRNA and knock down and JNK phosphorylation and caspase 3 cleavage was determined on immunoblots following transfection with Poly IC (**E**,**F**), and (**G**,**H**) cell viability was analyzed after transfection with 2-5A (10 μmM) by MTT assay and % apoptotic cells was determined by trypan blue exclusion assay. Values shown are mean \(\pm\) s.d of triplicate assays. * \(p<0.01\), *** \(p<0.001\)**, ns: not significant. decrease in both phosphorylation of JNK and caspase 3 cleavage which correlated with increase in cell viability and decrease in apoptosis (Figure 2E-H). To determine whether JNK activity is required for apoptosis, we examined the effect of SP600125, a JNK inhibitor, in PC3 cells over-expressing DRAK1. Treatment of PC3 cells with SP600125 resulted in inhibition of JNK phosphorylation in cells expressing DRAK1, which correlated with absence of caspase 3 cleavage and decrease in PARP cleavage (Figure 3B, lane 3). Treatment with carbobenzoxy-vallyl-alanyl-aspartyl-[O- methyl]- fluoromethylketone (ZVAD-FMK), a pan caspase inhibitor, blocked caspase 3 cleavage, but not JNK phosphorylation indicating that JNK activation by DRAK1 is upstream of caspase 3 function (Figure 3B, lane 4). We further quantitated cell viability in PC3 cells overexpressing DRAK1 following treatment with JNK inhibitor or caspase 3 inhibitor by MTT assay. Consistent with our results, we observed increase in cell viability in DRAK1 expressing cells when we inhibited activity of JNK and cleavage of caspase 3 (Figure 3C). To further demonstrate that caspase 3 cleavage is required for apoptosis in DRAK1 expressing cells, we measured caspase 3 enzyme activity using a luminescent caspase 3 substrate. Cell lysates from DRAK1 over-expressing cells treated with ZVAD-FMK inhibitor showed 2 to 3-fold reduced caspase 3 enzyme activity (Figure 3D).