p53 Stabilization and Accumulation Induced by Human Vaccinia-Related Kinase 1

Abstract

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 ؊/؊ 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 ...

VRK1 Signaling Pathway in the Context of the Proliferation Phenotype in Head and Neck Squamous Cell Carcinoma

“…VRK1 is highly expressed in human tumor cell lines (14) and murine embryos (15), suggesting it might be associated with cell proliferation. This association was further supported by experiments where VRK1 inactivation with small interfering RNA resulted in a block of cell division (16). VRK1 is expressed at very high level in the retina neurons, and its expression drops dramatically the first day after birth (17).…”

“…All these proteins phosphorylated by VRK1 have been associated with cellular responses to stress (25)(26)(27). VRK1 contributes to p53 stability by two mechanisms, one of them dependent on Thr 18 phosphorylation, and also seems to be implicated in the control of normal proliferation in the absence of cellular stress (16). The loss of VRK1 also affects the endocytic transport with a phenotype similar to that induced by silencing of mitogen-activated protein kinase (28).…”

“…The phosphorylation of p53 Thr 18 has been associated with cellular senescence (52) and with the response to Taxol (53). These observations led to the proposal that VRK1 might be a component of a novel mechanism that controls basal p53 levels during normal proliferation, or suboptimal stress situations, and thus permits the cell to respond when situations of severe stress arise (16).…”

“…The substrates thus far identified regarding the enzymatic activity of VRK1 are transcription factors (23,24) and include p53 (16,22). Therefore, it was decided to study if a correlation FIGURE 1.…”

“…VRK1, a new regulator of p53, phosphorylates p53 in Thr 18 (22,45), resulting in its stabilization and favoring its interaction with the transcriptional coactivator p300 (16). Phosphorylated Thr 18 affects the interaction of p53 with hdm2 (46,47), and it is acquiring more relevance lately (48,49), particularly because phosphorylation in other better known residues, such as Ser 15 or Ser 20 , seems to be dispensable for p53 activity (49)(50)(51).…”