Mechanisms Of Nemo-like Kinase (Nlk)-Mediated Regulation Of P53Stabilization In Response To DNA Damage

The integrity and stability of the DNA are essential to life. However, DNA is subject to assaults from exogenous and endogenous factors, and any damage or change will lead to genetic information change and can influence the survival of cells. Cells develop a set of efficient and complex DNA damage response (DDR) mechanisms to maintain the integrity and stability of the DNA. p53, as one of the most important factor in DDR, plays a pivotal role in DNA damage response.p53, a tumor suppressor gene, plays an important role in tumorigenesis. It is reported that p53mutations or dysfunctions have been found in more than half of human tumors. When DNA injury occurs, p53is activated and reduces cell cycle arrest to repair damage DNA. If DNA damage is excessive, p53could induce cell appotosis to reduce the probability of gene deletion or mismatch and altimately decrease tumorigenesis. The mRNA level of p53is high in normal cells, but the protein level remains low due to ubiquitin-mediated proteasomal degradation. MDM2, an ubiquitin ligase of p53, binds to p53and catalyzes its ubquitination. Activation of p53in response to DNA damage is associated with a rapid increase of its protein level and activity, which can regulate cell cycle and cell apoptosis. However, it has still not yet been determined how p53is activated after DNA damage. When we study the role of NLK protein in DNA damage, we observed that the NLK protein levels concomitant with p53protein induction after DNA damage. Interestingly, the resultant increase in p53levels was dramatically lower in the NLK-deficient cells compared to NLK-wild type cells after DNA damage. p53protein level is elevated when NLK is overexpressed, and p53 activity is also increased in a dose-dependent manner. Similarly, NLK kinase mutant expression also increased p53protein level and p53activity. Taken together, these findings demonstrate that NLK regulates p53stability and increases its activity, and these effects are not associated with NLK kinase activity. Ubiquitination experiments showed that NLK blocks MDM2-mediated p53ubiquitination and degradation to stabilize p53. Coimmunoprecipitations indicated that NLK interacts with p53, and the association was increased after stimulation with DNA damage-agents. We constructed NLK and p53truncation mutants, and performed coimmunoprecipitation experiments using these truncation mutants. The results demonstrated that the structural integrity of p53and NLK is important for their interaction. In our study, when NLK was overexpressed, interaction of p53with MDM2was dramatically decreased. By contrast, NLK deficiency enhanced the interaction between p53and MDM2.NLK also involved in the β-catenin signaling pathway and regulates a number of transcription factors, including Myb, therefore NLK may have more comprehensive role in tumorigenesis. Nevertheless, our study provides the first evidence for the role of NLK in regulation of p53stability after DNA damage.