# RNase L Induces Expression of A Novel Serine/Threonine Protein Kinase, DRAK1, to Promote Apoptosis
## 3 Discussion  
Antiviral activity of RNase L involves apoptotic clearance of virus-infected cells and cleaving diverse RNA substrates to produce IFN, activate inflammasome or autophagy [13; 14; 15; 16]. Our studies have identified DRAK1, a DAP kinase family member, as a component in the apoptotic pathway induced by activation of RNase L. RNase L induces expression of DRAK1 and requires IFN signaling pathway suggesting that DRAK1 is an interferon stimulated gene. The presence of ISRE sequences in DRAK1 promoter further support this conclusion. DRAK1 is localized to the nucleus and ectopically expressing DRAK1 is sufficient to trigger apoptosis in the absence of RNase L activation. Knockdown of DRAK1 levels, but not DRAK2, with siRNAs significantly reduced apoptosis induced by RNase L. Furthermore, ectopic DRAK1 expression triggered apoptosis by activating JNK and translocation of Bax to the mitochondria accompanied by cleavage of caspase 3 and PARP. Finally, we show that kinase activity and nuclear localization is required for DRAK1 induced apoptosis. Together, these data indicate that DRAK1 functions as an effector of RNase L induced apoptosis. Given the complexity of apoptosis pathways activated by RNase L, we overexpressed DRAK1 to study the direct effects in promoting apoptosis in the absence of RNase L activation. We show that ectopic expression of DRAK1 induces cell death involving the activity of JNK and cleavage of caspase 3 and PARP. Inhibiting the activity of JNK using small molecule inhibitor in cells expressing DRAK1 abrogates caspase 3 cleavage and apoptosis. JNK1/2 is required for apoptosis in response to diverse cytotoxic and genotoxic stress stimuli [29; 30; 31; 32; 33]. JNK phosphorylates Bax and activated Bax translocates to the mitochondria to promote mitochondrial outer membrane permeabilization resulting in release of cytochrome C that in turn triggers caspase 9 cascade leading to cleavage of caspase 3 and consequent cellular apoptosis [34; 35]. While we were unable to detect phosphorylation of Bax, here we show that ectopic DRAK1 expression induced translocation of Bax from cytoplasm to mitochondria and release of mitochondrial cytochrome C accompanied by loss of mitochondrial membrane potential (Figure 3,4). Recently, another study showed interaction of DRAK1 with ANT2, a mitochondrial protein and translocation of DRAK1 to the cytosol in response to PKC activation, however, the impact on apoptosis was not explored [24]. In the IFN signaling pathway, Bax has been shown to bind Interferon Regulatory Factor 3 (IRF3) and translocate to mitochondria and induce apoptosis in virally infected cells [36]. Our previous studies show a similar translocation of Bax in response to cleavage of Beclin-1 and insertion of cleaved Beclin-1 in the mitochondria to induce apoptosis [16]. Future studies will test if JNK is phosphorylated directly by DRAK1 and the consequences on cellular localization of both JNK and DRAK1. IFN signaling and apoptosis are complex pathways and apoptosis can be induced by several components of the IFN pathway, including RNase L. It is likely many other factors may contribute to cell death and further studies are required to investigate their interactions. The kinase domain of all members of the DAPK family share significant homology and the regulatory regions are structurally diverse indicating death-inducing roles in response to diverse stimuli [37]. DAPK and Zipper-Interacting Protein Kinase (ZIPK) participate in apoptosis in response to IFN-\(\gamma\), Tumor Necrosis Factor-alpha (TNF-\(\alpha\)), Fas ligand and TGF-\(\beta\), while DRAK2 mediates apoptosis in response to UV treatment and interleukin-2 [38; 39]. In this study, we show that DRAK1 is induced in response to type I IFN and dsRNA. DAPK localizes to the cytoplasm, whereas ZIPK, DRAK1 and DRAK2 localize to the nucleus. Both DRAK1 and DRAK2 share 67% identity in the kinase domain and 24% identity in the regulatory domains suggesting differential regulation.