Abstract
BackgroundThe vaccinia-related kinase 1 (VRK1) protein, an activator of p53, can be proteolytically downregulated by an indirect mechanism, which requires p53-dependent transcription.Principal FindingsIn this work we have biochemically characterized the contribution of several p53 transcriptional cofactors with acetyl transferase activity to the induction of VRK1 downregulation that was used as a functional assay. Downregulation of VRK1 induced by p53 is prevented in a dose dependent manner by either p300 or CBP, but not by PCAF, used as transcriptional co-activators, suggesting that p53 has a different specificity depending on the relative level of these transcriptional cofactors. This inhibition does not require p53 acetylation, since a p53 acetylation mutant also induces VRK1 downregulation. PCAF can not revert the VRK1 protection effect of p300, indicating that these two proteins do not compete for a common factor needed to induce VRK1 downregulation. The protective effect is also induced by the C/H3 domain of p300, a region implicated in binding to several transcription factors and SV40 large T antigen; but the protective effect is lost when a mutant C/H3Del33 is used. The protective effect is a consequence of direct binding of the C/H3 domain to the transactivation domain of p53. A similar downregulatory effect can also be detected with VRK2 protein.Conclusions/SignificanceSpecific p53-dependent effects are determined by the availability and ratios of its transcriptional cofactors. Specifically, the downregulation of VRK1/VRK2 protein levels, as a consequence of p53 accumulation, is thus dependent on the levels of the p300/CBP protein available for transcriptional complexes, since in this context this cofactor functions as a repressor of the effect. These observations point to the relevance of knowing the cofactor levels in order to determine one effect or another.
Highlights
The vaccinia-related kinases (VRK) form a group of three proteins in the human kinome that diverged early from the casein kinase I branch [1]
The direct mechanism implicated in the downregulation of vaccinia-related kinase 1 (VRK1) protein is mediated by a p53-dependent gene, since different p53 mutants, including the most common mutations detected in human cancers, which are not able to induce transcription does not cause downregulation of VRK1 protein [12]
A mechanism that was confirmed in human squamous cell lung carcinomas, where those cases harboring p53 mutations presented very high levels of VRK1 protein [33]
Summary
The vaccinia-related kinases (VRK) form a group of three proteins in the human kinome that diverged early from the casein kinase I branch [1]. Several lines of evidence suggest that VRK1 contributes to cell division. VRK1 is highly expressed in proliferating cell lines [2], and in embryonic development during the expansion of the hematopoietic system [3]. In human biopsies VRK1 is mainly detected in the amplifying compartment of epithelial surfaces, where it co-localizes with several proliferation markers [4]. Loss of human VRK1 by siRNA reduces cell division [5], and in C. The vaccinia-related kinase 1 (VRK1) protein, an activator of p53, can be proteolytically downregulated by an indirect mechanism, which requires p53-dependent transcription
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