p53 Downregulates Its Activating Vaccinia-Related Kinase 1, Forming a New Autoregulatory Loop

Abstract

The stable accumulation of p53 is detrimental to the cell because it blocks cell growth and division. Therefore, increases in p53 levels are tightly regulated, mainly by its transcriptional target, mdm2, that downregulates p53. Elucidation of new signaling pathways requires the characterization of the members and the nature of their connection. Vaccinia-related kinase 1 (VRK1) contributes to p53 stabilization by partly interfering with its mdm2-mediated degradation, among other mechanisms; therefore, it is likely that some form of autoregulation between VRK1 and p53 must occur. We report here the identification of an autoregulatory loop between p53 and its stabilizing VRK1. There is an inverse correlation between VRK1 and p53 levels in cell lines, and induction of p53 by UV light downregulates VRK1 in fibroblasts. As the amount of p53 protein increases, there is a downregulation of the VRK1 protein level independent of its promoter. This effect is indirect but requires a transcriptionally active p53. The three most common transcriptionally inactive mutations detected in hereditary (Li-Fraumeni syndrome) and sporadic human cancer, p53(R175H), p53(R248W), and p53(R273H), as well as p53(R280K), are unable to induce downregulation of VRK1 protein. The p53 isoforms ⌬40p53 and p53␤, lacking the transactivation and oligomerization domains, respectively, do not downregulate VRK1. VRK1 downregulation induced by p53 is independent of mdm2 activity and proteasome-mediated degradation since it occurs in the presence of proteasome inhibitors and in mdm2-deficient cells. The degradation of VRK1 is sensitive to chloroquine, an inhibitor of the late endosome-lysosome transport, and to serine protease inhibitors of the lysosomal pathway.

VRK1 interacts with p53 forming a basal complex that is activated by UV‐induced DNA damage

“…VRK1 downregulation is mediated by p53 transcriptional activation of DRAM (damage-regulated autophagic modulator) [57]. DRAM targets VRK1 to enter in the autophagic pathway [33] and thus be degraded in the lysosome [9,15]. This autoregulatory loop is altered in tumours with p53 mutations [34], resulting in an accumulation of VRK1 protein, which has been detected in human lung [34] and head and neck squamous cell carcinomas [37].…”

“…VRK1 mutants were generated using the Quick-Mutagenesis system (Stratagene, San Diego, CA) and specific primers (indicated in Table S1) following the manufacturer's instructions and using plasmid pCEFL-HA-VRK1 as template. Plasmids expressing full-length human p53 and the mutants R273H, R248H and R280K were obtained from B. Vogelstein (John Hopkins University, Baltimore, USA) and we have used them previously [9,33]. Plasmids expressing GST-p53N-terminal transactivation domains for bacterial expression were obtained from D. Meek (University of Dundee, Scotland) and have been reported before [7] and human GST-p53 fusion proteins 1-390, 90-290 and 290-390 were from T. Kouzaridis (Cancer Research UK, Cambridge).…”

“…All culture reagents were from Invitrogen-GIBCO. In experiments in which plasmid constructs were transiently transfected, cells were in exponential growth phase [9,15]. Twenty-four hours before transfection cells were plated in P100 dishes at 60-70% confluence at the time of transfection as described previously [15,28].…”

A role for p53 in the regulation of extracellular matrix metalloproteinase inducer in human cancer cells

“…27,28 Recently, the p53-regulated lysosomal degradation of other protein has been reported by Valbuena et al 23 In addition, the role of p53 in the regulation of the lysosomal function has also been reported. 29,30 However, the precise mechanism by which how p53 regulates protein degradation remains unclear.…”

“…It has been reported that p53 is able to modulate degradation of certain proteins via either proteasomal 22 or lysosomal 23 pathway. Therefore, we next sought to determine whether the downregulation of EMMPRIN by p53 occured at protein level.…”