Variations in intracellular levels of p53 regulate many cellular functions and determine tumor susceptibility. Major mechanisms modulating p53 levels include phosphorylation and interaction of p53 with specific ubiquitin ligases that promote its degradation. N-terminal phosphorylation regulates the interaction of p53 with several regulatory molecules. Vaccinia-related kinase 1 (VRK1) is the prototype of a new Ser-Thr kinase family in the human kinome. VRK1 is located in the nucleus outside the nucleolus. Overexpression of VRK1 increases the stability of p53 by a posttranslational mechanism leading to its accumulation by a mechanism independent of the Chk2 kinase. Catalytically inactive VRK1 protein (a K179E mutant) does not induce p53 accumulation. VRK1 phosphorylates human p53 in Thr18 and disrupts p53-Mdm2 interaction in vitro, although a significant decrease in p53 ubiquitination by Mdm2 in vivo was not detected. VRK1 kinase does not phosphorylate Mdm2. VRK1-mediated p53 stabilization was also detected in Mdm2 Ű/Ű cells. VRK1 also has an additive effect with MdmX or p300 to stabilize p53, and p300 coactivation and acetylation of p53 is enhanced by VRK1. The p53 stabilized by VRK1 is transcriptionally active. Suppression of VRK1 expression by specific small interfering RNA provokes several defects in proliferation, situating the protein in the regulation of this process. VRK1 might function as a switch controlling the proteins that interact with p53 and thus modifying its stability and activity. We propose VRK1 as the first step in a new pathway regulating p53 activity during cell proliferation.Regulation of p53 levels plays a major role in determining the fate of a cell and its susceptibility to tumor development (17,19). The p53 protein is at a crossroad in the pathways implicated in the cellular response to many different types of stresses, such as genotoxic damage by UV or ionizing irradiation, hypoxia, and heat shock (36), and it has been implicated in different cell cycle checkpoints (7, 48). Several cell protection mechanisms are based on the ability of the p53 protein to regulate the progression of the cell cycle, the induction of apoptosis, or replicative senescence (6,46,50). These responses are controlled by the p53 protein level. p53 is a shortlived protein that is maintained at low levels in the cell under normal conditions and increases in response to stress. The levels and stability of the p53 protein in the cell are mainly controlled by its interactions with the negative regulator Mdm2 (hdm2 in humans) (3) or with other E3 ubiquitin ligases, such as COP1 (14) or Pirh2 (24). Mdm2 targets p53 for degradation by the ubiquitination pathway, promotes its nuclear export, and thus allows cell cycle progression (3). The new ubiquitin ligases COP1 and Pirh2 are also negative regulators of p53 function, targeting p53 for degradation by the proteasome in a ubiquitin-dependent fashion, and like Mdm2 are encoded by p53-inducible genes (14,24). Interactions with stabilizing proteins, such as p300 (53), MdmX ...