# RNase L Induces Expression of A Novel Serine/Threonine Protein Kinase, DRAK1, to Promote Apoptosis
## 2 Results  
### Induction of DRAK1 by RNase L Requires Interferon Signaling  
Microarray analysis of DU145 cells transfected with 2-5A to activate RNase L identified changes in mRNA levels of antiviral and antitumor genes including death-associated protein kinase (DAPK)-related apoptosis-inducing protein kinase 1 (DRAK1), a member of the death-associated protein (DAP) family of serine/threonine protein kinases. To determine if DRAK1 is induced in prostate cancer cells in response to RNase L activation, PC3 and DU145 cells were transfected with 2-5A or synthetic dsRNA, Poly IC, which activates oligoadenylate synthetase to produce 2-5A. After 6 h, 6- or 7-fold increase in mRNA in PC3 cells and 11- or 4.5-fold in DU145 cells was observed that correlated with protein expression (Figure 1A-D). However, DRAK2, a DRAK1 homolog, was not induced by 2-5A or Poly I:C treatment. RNase L activity produces cleaved dsRNAs that amplify interferon signaling [13]. Compared to wild-type (2 fTGH, fibrosarcoma) cells, DRAK1 induction was significantly reduced in STAT1-defective cells (U3A, derived from 2 fTGH and defective in type I IFN signaling), while DRAK2 levels remained unchanged (Figure 1E,F). Cells lacking RNase L (inset Figure 1G) or STAT1 signaling showed reduced DRAK1 mRNA induction by 2-5A (Figure 1G). Further, treatment of cells with both Poly I:C and IFN induced DRAK1 mRNA levels 6-fold compared to 2- to 3-fold by either Poly I:C or IFN (Figure 1H). These results suggest the requirement of the IFN-signaling pathway in DRAK1 transcriptional induction by RNase L.  
Figure 1: Induction of DRAK1 by RNase L activation and type I IFN signaling. DU145 and PC3 cells were transfected with Poly I:C (2 ug/mL) or 2–5A (10 uM) for indicated times and (**A**,**B**) mRNA expression of DRAK1 and DRAK2 were analyzed by qPCR, (**C**,**D**) protein expression was determined by immunoblot analysis. (**E**,**F**) DRAK1 and DRAK2 expression on immunoblots in HT1080 and U3A (STAT1-defective) cells, (**G**) mRNA levels in indicated cells were compared to untreated cells and analyzed by qPCR. Inset shows immunoblot analysis of RNase L KO cells (**H**) DRAK1 mRNA levels in PC3 levels transfected with Poly I:C (2 ug/mL), interferon (1000 U/mL) or combined treatment for 6 h, (**I**) Schematic of DRAK1 promoter region showing IFN-stimulated response element (ISRE) sequences (in green) and Octamer-Binding Protein-2 (Oct-2) binding sites (in red). DRAK1 promoter driven luciferase activity normalized to _renilla_ luciferase in PC3 treated with Poly I:C (2 ug/mL) or 2–5A (10 uM) or interferon (1000 U/mL). Values shown are mean \(\pm\) s.d of triplicate assays. * \(p\) \(<\) 0.01, **_p_ \(<\) 0.001, **_p_ \(<\) 0.0001, n.s: not significant, WT: Wild Type.  
To determine if RNase L-induced expression of DRAK1 was due to transcriptional activation of the DRAK1 promoter, the 5' flanking region corresponding to \(-\)2718/+1 of DRAK1 promoter was inserted upstream of a luciferase reporter gene and induction by 2-5A, Poly I:C or IFN was monitored. To define the minimum promoter region necessary for induction, promoter deletion constructs was generated. A 1336 bp promoter fragment conferred greater responsiveness to 2-5A, Poly I:C and IFN and harbored perfect consensus sequences for IFN-stimulated response element (ISRE) typically found in the promoter regions of IFN-stimulated genes (Figure 11) [25]. The \(-\)2718 bp and \(-\)2496 bp fragments harbored ISRE sequences in addition to Oct-2 binding sites that function as transcriptional repressors [26]. Deletion of the Oct-2 binding sites in \(-\)1336 bp fragment conferred greater inducibility. However, further studies will be needed to demonstrate if Oct-2 or other negative regulatory elements regulate DRAK1 promoter activity. These findings suggest that DRAK1 is transcriptionally induced by RNase L activation and more directly by IFN.