Natural killer (NK) cells target and kill tumor cells by direct anti-tumor cytotoxicity. NK lytic-associated molecule (NKLAM) is a protein involved in this cytolytic function. Acting as an E3 ubiquitin ligase, NKLAM binds to and ubiquitinates a novel protein, uridine-cytidine kinase like-1 (UCKL-1), targeting it for degradation. However, UCKL-1's function in tumor cell survival and NK cell cytotoxicity is unknown. UCKL-1's homology to uridine kinases and over expression in tumor cells suggests a role for UCKL-1 in tumor growth and/or survival. We propose that NKLAM and UCKL-1 interact in the tumor cell, where degradation of UCKL-1 leads to increased tumor cell apoptosis. Here we use RNA interference to downregulate UCKL-1 expression in K562 erythroleukemia cells. It was seen that downregulation of UCKL-1 initiated apoptosis and slowed the cell cycle, resulting in lower growth in the small interfering UCKL-1 RNA treated K562 cell culture. In addition, the chemotherapeutic agent staurosporine was seen to be more effective in inducing cell death by apoptosis in UCKL-1 depleted K562 cells compared with controls. We also found that UCKL-1 depleted K562 cells were more susceptible to NK mediated cytolysis than controls. These results indicate a role for UCKL-1 in tumor cell survival and suggest possible therapeutic potential of UCKL-1 inhibitors in cancer treatment.
Uridine-cytidine kinase like-1 (UCKL-1) is a largely uncharacterized protein with high sequence similarity to other uridine-cytidine kinases (UCKs). UCKs play an important role in the pyrimidine salvage pathway, catalyzing the phosphorylation of uridine and cytidine to UMP and CMP, respectively. Only two human UCKs have been identified, UCK1 and UCK2. Previous studies have shown both enzymes phosphorylate uridine and cytidine using ATP as the phosphate donor. No studies have evaluated the kinase potential of UCKL-1. We cloned and purified UCKL-1 and found that it successfully phosphorylated uridine and cytidine using ATP as the phosphate donor. The catalytic efficiency (calculated as kcat/KM) was 1.2 x 104 s-1, M-1 for uridine and 0.7 x 104 s-1, M-1 for cytidine. Previously, our lab determined UCKL-1 is upregulated in tumor cells, providing protection against natural killer (NK) cell killing activity. We utilized small interfering RNA (siRNA) to downregulate UCKL-1 in vitro and in vivo to determine the effect of UCKL-1 on tumor growth and metastasis. The downregulation of UCKL-1 in YAC-1 lymphoma cells in vitro resulted in decreased cell counts and increased apoptotic activity. Downregulation of UCKL-1 in K562 leukemia cells in vivo led to decreased primary tumor growth and less tumor cell dissemination and metastasis. These results identify UCKL-1 as a bona fide pyrimidine kinase with the therapeutic potential to be a target for tumor growth inhibition and for diminishing or preventing metastasis.
Uridine cytidine kinase like-1 (UCKL-1) is a largely uncharacterized protein over-expressed in many tumor cells, especially in highly malignant, aggressive tumors. Sequence analysis indicates that UCKL-1 has homology to uridine kinases, enzymes that play a role in DNA and RNA synthesis and that are often up-regulated in tumor cells. Previous studies have shown that UCKL-1 is a substrate for natural killer lytic-associated molecule (NKLAM), an E3 ubiquitin ligase found in NK cell cytolytic granules. Ubiquitination of UCKL-1 by NKLAM leads to its degradation. Increased expression of NKLAM enhances NK-mediated tumoricidal activity. The fact that UCKL-1 is a substrate for NKLAM suggests that UCKL-1 may provide resistance to NK killing in tumor cells. Here we show that UCKL-1 over-expression protects tumor cells from NK killing and enhances tumor survival in vivo. UCKL-1 also has a much broader role, protecting tumor cells from spontaneous and drug-induced apoptosis and increasing tumor cell proliferation. Nuclear factorkappa B (NF-κB) activity is higher in tumor cells transfected with UCKL-1 compared to control transfected cells, suggesting at least one possible mechanism by which UCKL-1 influences tumor growth and survival.
NK lytic-associated molecule (NKLAM) plays a critical role in NK-mediated tumor cytotoxicity. Our hypothesis is that NKLAM and other granule proteins are released from NK cells upon interaction with tumor targets. Acting as a ubiquitin ligase, NKLAM ubiquitinates uridine cytidine kinase like-1 protein (UCKL-1) in tumor cells, leading to its degradation. Downregulation of UCKL-1 by RNA interference was previously found to increase tumor susceptibility to NK killing. Here we show that UCKL-1 over-expression protects tumor cells from NK killing and from apoptosis mediated by chemotherapeutic agents. Gene array analyses and protein expression studies indicate an involvement of UCKL-1 with the NF-κB and death receptor (DR) 3 pathways. Studies were performed to evaluate the role of UCKL-1 in tumor survival in vivo. Wild type and NKLAM-deficient mice were injected intravenously with RMA-S tumor cells expressing various levels of UCKL-1. Results to date indicate that RMA-S cells expressing the highest levels of UCKL-1 have the greatest survival in wild type mice. Tumor survival is potentiated in NKLAM-deficient mice, suggesting that the NKLAM/UCKL-1 interaction contributes to eradication of tumor cells in vivo. These findings indicate an involvement of UCKL-1 in tumor cell survival. Findings suggest an important role for UCKL-1 and its interaction with NKLAM in cancer control mediated by NK cells.
NK lytic associated molecule (NKLAM), which is upregulated in NK cells by activation, has been shown to play a critical role in NK-mediated tumor cytotoxicity. We have found that NKLAM interacts with uridine kinase-like 1 protein (UCKL-1) and causes its ubiquitination and subsequent degradation. This suggests that NKLAM may play a role in tumor killing by decreasing UCKL-1 expression in target cells. In support of this is the finding that UCKL-1 mRNA is most abundant in tumor cell lines, indicating that it may be important in tumor cell growth and/or survival. In order to determine the function of UCKL-1 in tumor cell killing, K562 target cells were treated with UCKL-1 RNAi or control RNAi. Selective knockdown of UCKL-1 resulted in increased apoptosis and decreased proliferation of K562. In addition, decreased UCKL-1 expression increased the susceptibility of K562 to the apoptosis inducers staurosporine and etoposide. This finding indicates that UCKL-1 plays a role in the proliferation of K562 cells and protects them from apoptosis. By ubiquitinating and degrading UCKL-1 in target cells, NKLAM released from NK cells would therefore enhance tumor killing. This research is supported by a merit review award from the Department of Veterans Affairs.
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