TI - Discussion . AB - The direct mechanism implicated in the downregulation of 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] . Furthermore , as shown in this work analysis of p53 PHOSphorylation mutants in residues implicated in responses to stress , which are not essential for transcription [23] , [24] , have a similar effect to that of wild-type p53 on VRK1 downregulation . This mechanism induces the targeting of VRK proteins to enter in a pathway that result in their proteolytic downregulation ; but the mechanism by which VRK1 or VRK2 proteins are targeted is not yet known . This downregulation of VRK1 or VRK2 , induced by p53 requires transcriptional activation of an intermediate gene that is responsible for targeting VRK proteins to enter the endocytic-lysosomal pathway [12] , as shown by its sensitivity to chloroquine . VRK1 plays an early role in cell cycle progression [34] ; and VRK2 regulates signal transduction in response to hypoxia [32] or interleukin-1beta [35] by interaction with components of MAP kinase pathways . In this work we have analyzed the contribution of acetyl transferases cofactors to regulate the induction by p53 of this VRK1 downregulatory effect . The relevance of p53 transcriptional specificity is manifested by the observation that overexpression of p300 or CBP can block the effect of p53 , but the PCAF coactivator does not . This might be due to the promoter specificity of different p53-transcriptional cofactor complexes [36] . But PCAF and p300 do not compete for the same cofactor ; since the p300 protective effect is detected even in the presence of excess PCAF . This p300 inhibitory competition of VRK1 downregulation is independent of acetyl transferase activity , since it is blocked by proteins that do not have the acetyl transferase activity as is the case for the C/H3 region of p300 . Also the downregulatory mechanism does not require p53 acetylation since it is also induced by a p53 protein containing all its acetylation sites replaced . The inhibitory mechanism can be explained by a competition for binding to p53 by proteins containing a C/H3 domain , such as p300/CBP . Other acetyl transferase such as PCAF , lacking a C/H3 domain , has no effect . The C/H3 domain depletes the p53 molecules needed for induction of VRK1 downregulation . The C/H3 domain of p300/CBP proteins is an interaction region known to bind to several transcription factors such as MyoD , Fos , c-Jun or E2F and to viral proteins such as adenovirus E1A or SV40 large antigen [18] , and also to the transactivation domain of p53 . Functionally , these interactions have been shown to have competing effects , thus C/H3 mediates a stimulation of c-Fos that is blocked by binding to E1A [37] . The inhibitory effect of p300/CBP , or their isolated C/H3 domain , is the consequence of a successful competition for p53 , because of its direct interaction , which is also required for the transcriptional complex of p53 needed to induce the indirect downregulation of VRK1 . This competition effect is lost in the presence of the C/H3Del33 defective domain , or by PCAF that does not have a C/H3 region . Mechanistically these observations are consistent with a dominant negative role for the C/H3 domain [38] -[40] . The interaction of p53 with these cofactors is affected by the residue phosphorylaTED [41] ; for example , Ser15 PHOSphorylation , or its aspartic substitution , favors binding to p300 ; while its substitution by alanine results in a much weaker interaction [42] . The association with these cofactors in transcriptional complexes is necessary for specific gene expression . Differential protein associations determine p300 and CBP specificity [43] in processes such as in myogenesis [44] ; p300/CBP modulates the BRCA1 inhibition of estrogen receptor [45] and downregulation of p300/CBP activates a senescence checkpoint in melanocytes [46] . PCAF acetylates the transcription factor Fetal-Kruppel-like factor ( FKLF2 ) [47] . PCAF acetylates PTEN and reduces its ability to down-regulate phosphatidylinositol 3-kinase signaling and to induce G1 cell cycle arrest [48] . But also p300 and PCAF can cooperate in activation in Notch responses [49] ; and both p300/CBP and PCAF can acetylate p53 in response to DNA damage [50] . Also CBP and PCAF can acetylate MyoD increasing its transcriptional activity [51] . Differential effects of p53 cofactors have also been reported in the apoptotic response that requires p300 in response to ionizing radiation [53] . In the context of cancer , it is important to note that , in the presence of activated H-Ras or N-Ras oncogenes , an active degradation of p300 is induced [54] , and this change will permit the activation and subsequent degradation of VRK1 by the new complexes of p53 . Among the genes regulated by p53 there is a clear candidate to be implicated in this process . The expression of DRAM is positively regulated by p53 , and encodes a lysosomal protein implicated in degradation of stable proteins [55] , as is the case of VRK1 [34] . VRK1 degradation is promoted by DRAM ( unpublished results ) . Inducible active degradation of stable proteins is an important step in biological processes such as autophagy [55] . Target selection by transcriptional complexes , in which proteins such as p53 or transcriptional cofactors with acetyl transferase activity are implicated , are very likely to be affected by the relative intracellular concentrations of these proteins ; and depending on their intracellular concentrations , one or another group of genes may be stimulated . However , the role of relative changes in factor concentration has so far received little attention , in comparison with all or none effects , despite their very important physiological relevance . The effect of changing levels of p300/CBP on the induction of VRK1 proteolytic downregulation , requiring p53 dependent transcription as an intermediate step , can be considered in this context of factor competition . Thus the downregulation of VRK1 is not only determined by the level of p53 , but VRK1 downregulation is also conditioned by the relative levels of different acetyl transferases present in the cell . It is highly likely that the heterogeneity of effects frequently observed in response to common types of stimulation is precisely reflecting differences in the intracellular balance of the proteins implicated .