The C/H3 Domain of p300 is required to protect VRK1 and VRK2 from their downregulation induced by p53

Background: The vaccinia-related kinase 1 (VRK1) protein, an activator of p53, can be proteolytically downregulated by an indirect mechanism, which requires p53-dependent transcription. Principal Findings: In 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/Significance: Specific 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.A.V. and S.B. have predoctoral fellowships from Ministerio de Educacio´n y Ciencia. F.M.V. has a predoctoral fellowship from Fundacio´n Ramo´n Areces. This work was funded by grants from Ministerio de Educacio´n y Ciencia (SAF2007-60242), Consolider-Ingenio-2010 (CSD-2007-0017), Junta de Castilla y Leo´n (CSI05A05), Fundacio´n de Investigacio´n Me´dica MM and Federacio´n de Cajas de Ahorro de Castilla y Leo´ n.Peer reviewe