Expression screening using a Medaka cDNA library identifies evolutionarily conserved regulators of the p53/Mdm2 pathway

Abstract

BackgroundThe p53 tumor suppressor protein is mainly regulated by alterations in the half-life of the protein, resulting in significant differences in p53 protein levels in cells. The major regulator of this process is Mdm2, which ubiquitinates p53 and targets it for proteasomal degradation. This process can be enhanced or reduced by proteins that associate with p53 or Mdm2 and several proteins have been identified with such an activity. Furthermore, additional ubiquitin ligases for p53 have been identified in recent years. Nevertheless, our understanding of how p53 abundance and Mdm2 activity are regulated remains incomplete. Here we describe a cell culture based overexpression screen to identify evolutionarily conserved regulators of the p53/Mdm2 circuit. The results from this large-scale screening method will contribute to a better understanding of the regulation of these important proteins.MethodsExpression screening was based on co-transfection of H1299 cells with pools of cDNA’s from a Medaka library together with p53, Mdm2 and, as internal control, Ror2. After cell lysis, SDS-PAGE/WB analysis was used to detect alterations in these proteins.ResultsMore than one hundred hits that altered the abundance of either p53, Mdm2, or both were identified in the primary screen. Subscreening of the library pools that were identified in the primary screen identified several potential novel regulators of p53 and/or Mdm2. We also tested whether the human orthologues of the Medaka genes regulate p53 and/or Mdm2 abundance. All human orthologues regulated p53 and/or Mdm2 abundance in the same manner as the proteins from Medaka, which underscores the suitability of this screening methodology for the identification of new modifiers of p53 and Mdm2.ConclusionsDespite enormous efforts in the last two decades, many unknown regulators for p53 and Mdm2 abundance are predicted to exist. This cross-species approach to identify evolutionarily conserved regulators demonstrates that our Medaka unigene cDNA library represents a powerful tool to screen for these novel regulators of the p53/Mdm2 pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0208-y) contains supplementary material, which is available to authorized users.

Hypoxia‑induced internalization of connexin 26 and connexin�43 in pulmonary epithelial cells is involved in the occurrence of non‑small cell lung cancer via the P53/MDM2 signaling pathway

“…Evidence has been previously presented that indicates that, if activated under hypoxic conditions, P53 is able to function as an extraordinary tumor suppressor regarding progression and prognosis through its negative interaction with MDM2 (36). The function of P53 is largely linked to cell proliferation and apoptosis, along with cell cycle distribution, as P53 can promote cell apoptosis (37). The follow-up in vivo experiments further verified that downregulated CX26 and CX43 promoted tumorigenicity.…”

Fam83F induces p53 stabilisation and promotes its activity

“…We previously identified Fam83F in a cell culture-based expression screen for novel regulators of p53 where it was shown to regulate p53 levels in an Mdm2-dependent manner. 9 We performed a more detailed Fam83F overexpression titration, both in the presence and absence of Mdm2, and looked at the effect this has on p53. In the presence of co-expressed Mdm2, Fam83F overexpression increased p53 abundance in a dose-dependent manner without altering Mdm2 levels ( Figure 1A, lanes 1-3).…”

N-Glycosylation of LRP6 by B3GnT2 Promotes Wnt/β-Catenin Signalling

“…Due to the evolutionarily conserved nature of Wnt signalling, a variety of different cell lines and tools from different species may be used to study the pathway, an approach we have taken advantage of previously [ 17 , 45 , 46 , 47 ]. Here, an arrayed and annotated medaka (Oryzias latipes) cDNA library [ 43 ] was used as a source of genes to screen for functional regulators of human LRP6 in a human cell line (HEK293T) ( Figure 1 a).…”