Negative regulation of HDM2 to attenuate p53 degradation by ribosomal protein L26

HDM2 is a p53-specific E3 ubiquitin ligase. Its overexpression leads to excessive inactivation of tumor protein p53, diminishing its tumor suppressor function. HDM2 also affects the cell cycle, apoptosis and tumorigenesis through interacting with other molecules, including several ribosomal proteins. To identify novel HDM2 regulators, we performed a yeast two-hybrid screening using HDM2 as bait. Among the candidates, ribosomal protein L26 (RPL26) was characterized as a novel HDM2-interactor. The interaction between HDM2 and RPL26 was further validated by in vivo and in vitro assays. RPL26 modulates the HDM2–p53 interaction by forming a ternary complex among RPL26, HDM2 and p53, which stabilize p53 through inhibiting the ubiquitin ligase activity of HDM2. The ribosomal stress caused by a low dose of Act D enhances RPL26–HDM2 interaction and activates p53. Overexpression of RPL26 results in activating of p53, inhibits cell proliferation and induces a p53-dependent cell cycle arrest. These results provide a novel regulatory mechanism of RPL26 to activate p53 by inhibiting HDM2.     

Analysis of p53 tumor suppressor pathway genes in chronic lymphocytic leukemia

The p53 tumor suppressor gene plays an important role in preventing tumor development. The p53 protein interacts with other p53 signal pathway members to control cell proliferation. In this study, expression of the p53, Human homolog of murine Double Minute 2 (HDM2), p14Alternating Reading Frame (ARF), Zinc Finger and BTB domain containing 7A (ZBTB7A), and B-Cell Lymphoma 6 (BCL6) genes was quantitatively investigated by real-time polymerase chain reaction (PCR) in the peripheral blood of patients with chronic lymphocytic leukemia (CLL) and healthy controls. Plasma fibronectin levels were determined by enzyme-linked immunosorbent assay. Expression of the p53, p14, and HDM2 genes were significantly higher in the patients. However, ZBTB7A and BCL6 gene expression was not detectable in both groups. A positive correlation between p14ARF and HDM2 expression and a negative correlation between p53 and p14ARF expression was observed. Expression of the p14ARF and HDM2 genes were inversely correlated in the control group. Neither HDM2 nor p14ARF gene expression was correlated with p53 expression. The p53 gene was also analyzed for the presence of mutations. A splice-site mutation was found in a single patient. Our findings indicate that expression of the p53, p14ARF, and HDM2 genes are associated with CLL. Elucidation of the mutual interactions at the protein level warrants further studies.     

HDM2-binding partners: interaction with translation elongation factor EF1alpha

To understand the cellular functions of HDM2, we attempted to identify novel HDM2-interacting proteins by proteomic analysis. Along with previously identified interactions with the ribosomal proteins, our analysis reveals interactions of HDM2 with the ribosomal translation elongation factor EF1alpha, 40S ribosomal protein S20, tubulins, glyceraldehyde 3-phosphate dehydrogenase, and a proteolysis-inducing factor dermicidin in the absence of tumor suppressor p53. Because a CTCL tumor antigen HD-CL-08 has high degree of homology with EF1alpha, we confirmed interaction of HDM2 with EF1alpha by immunoprecipitation and Western blot analysis in transformed as well as near normal diploid cells. Endogenous HDM2- EF1alpha complex was detected in cancer cells overexpressing HDM2, suggesting a possible role of this interaction in HDM2-mediated oncogenesis. Consistent with their interaction, colocalization of HDM2 and EF1alpha can be detected in the cytoplasm of normal or transformed cells. Amino acid residues 1-58 and 221-325 of HDM2 were found to be essential for its interaction with EF1alpha, suggesting that the interaction is independent of its other ribosomal interacting proteins L5, L11, and L23. Overexpression of HDM2 did not affect translation. Because EF1alpha has been implicated in DNA replication and severing of microtubules, interaction of HDM2 with EF1alpha may signify a p53-independent cell growth regulatory role of HDM2.