# RNase L Induces Expression of A Novel Serine/Threonine Protein Kinase, DRAK1, to Promote Apoptosis
## 3 Discussion
Consistent with other studies, DRAK1 kinase-dead mutant (K90A) localized to the cytoplasm in PC3 cells. An earlier study reported that kinase activity of both DRAK1 and DRAK2 is not required for nuclear localization but is required for inducing apoptosis [19]. However, a recent study showed that DRAK2 nuclear localization and increase in kinase activity is required for cell death [38; 40]. Our study also identified a short stretch of basic residues RKRRK (aa residues 93-97) in the DRAK1 kinase domain that functions as a nuclear localization signal as K94A mutation resulted in cytoplasmic retention of DRAK1. A corresponding region in DRAK2 (KKRRR, aa 65-69) functions as a NLS for DRAK2 in Jurkat cells [41]. Another putative NLS was identified SKRFK (aa residues 395-399) in the C-terminus of DRAK1 that overlapped with PKC phosphorylation site at S395 [24]. Similarly, a second NLS in the C-terminal of DRAK2 was identified which was regulated by phosphorylation of Ser350 in the C-terminal region [40]. Kinase activity and nuclear localization of DRAK1 is required for inducing apoptosis as DRAK1 kinase-dead, NLS mutant or kinase-dead and NLS double mutant showed reduced apoptosis when ectopically expressed in PC3 cells. The kinase-dead mutant had similar ability to rescue cell viability as NLS mutant or double mutant suggesting that both nuclear localization and kinase activity of DRAK1 is perhaps integral to its apoptosis-inducing activity. While we have observed variability in levels of apoptosis in comparing DRAK1 WT and mutants, we consistently observed lack of JNK phosphorylation in all mutants. It is unclear if DRAK1 phosphorylates JNK in the nucleus and if there are additional substrates in the nucleus that are phosphorylated and needed for signaling. In that regard, we have not explored if DRAK1 is autophosphorylated and if trans-phosphorylation of JNK is compromised by the mutants we have studied. It is also likely that other substrates may be phosphorylated in the nucleus that promotes apoptosis. In Human osteosarcoma (U2 OS) cells, DRAK1 bound to p53 in the nucleus and cisplatin-induced phosphorylation of p53 was reduced in DRAK1 knockdown cells [24]. Further investigations will reveal if DRAK1 directly or indirectly regulates p53 and its apoptotic activities. Many tumor cells, including PC3, have mutant p53 protein so the effects we observed are p53-independent. Future studies will be aimed at resolving the distinct roles of DRAK1 as a kinase and as a nuclear localized apoptosis-inducing protein. In addition to IFN and dsRNA, DRAK1 is induced in a p53-dependent manner in response to DNA damage agents in testicular, ovarian and pancreatic cell lines [20]. Cells with increased expression of DRAK1 were more susceptible to cell death with chemotherapeutic drugs [21]. In testicular cancer cells, DRAK1 is induced in response to cisplatin and induces cell death by modulating Reactive Oxygen Species (ROS) activity. However, in Head and neck squamous cell carcinoma (HNSCC) cell lines, where DRAK1 is overexpressed, DRAK1 in the cytoplasm binds to Smad3 and sequesters it in the cytoplasm, thereby preventing Smad3/Smad4 complex formation and inhibiting TGF-\(\beta\)1 tumor suppression [23]. These results suggest that DRAK1, predominantly, has anti-tumor functions and agents like IFN and dsRNA that can induce DRAK1 expression can potentially be combined for effective cancer therapy.