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Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells.

ABSTRACT: Murine double minute clone 2 oncoprotein (MDM2) is a key component in the regulation of the tumour suppressor p53. MDM2 mediates the ubiqutination of p53 in the capacity of an E3 ligase and targets p53 for rapid degradation by the proteasome. Stress signals which impinge on p53, leading to its activation, promote disruption of the p53-MDM2 complex, as in the case of ionizing radiation, or block MDM2 synthesis and thereby reduce cellular MDM2 levels, as in the case of UV radiation. It is therefore likely that MDM2, which is known to be modified by ubiquitination, SUMOylation and multi-site phosphorylation, may itself be a target for stress signalling (SUMO is small ubiquitin-related modifier-1). In the present study we show that, like p53, the MDM2 protein is a substrate for phosphorylation by the protein kinase CK2 (CK2) in vitro. CK2 phosphorylates a single major site, Ser(267), which lies within the central acidic domain of MDM2. Fractionation of cellular extracts revealed the presence of a single Ser(267) protein kinase which co-purified with CK2 on ion-exchange chromatography and, like CK2, was subject to inhibition by micromolar concentrations of the CK2-specific inhibitor 5,6-dichlororibofuranosylbenzimidazole. Radiolabelling of cells expressing tagged recombinant wild-type MDM2 or a S267A (Ser(267)-->Ala) mutant, followed by phosphopeptide analysis, confirmed that Ser(267) is a cellular target for phosphorylation. Ser(267) mutants are still able to direct the degradation of p53, but in a slightly reduced capacity. These data highlight a potential route by which one of several physiological modifications occurring within the central acidic domain of the MDM2 protein can occur.

SUBMITTER: Hjerrild M

PROVIDER: S-EPMC1221745 | biostudies-other | 2001 Apr

REPOSITORIES: biostudies-other

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Project description:BackgroundGlioma is the most common and fatal primary cranial tumor. The epidermal growth factor receptor (EGFR) plays an important role in the occurrence and treatment of glioma, which might function through a circular ribonucleic acid (circRNA)-related mechanism. Hsa_circ_0080229 (circ_0080229) has been identified as a circRNA arising from an EGFR gene in gliomas; however, little is known about its molecular mechanism to date.MethodsTo address this question, a series of experiments were conducted to confirm the effect of circ_0080229 in gliomas and identify the downstream mechanism. A quantitative real-time polymerase chain reaction (qRT-PCR) analysis and in-situ hybridization/fluorescence in-situ hybridization (ISH/FISH) testing were performed to identify the expression of circ_0080229 in patient samples. Bioinformatic analysis was carried out to explore the possible mechanism. Next, a series of in-vitro functional assays and in-vivo assays with a xenograft subcutaneous glioma model was carried out to confirm the effect of circ_0080229. Finally, qRT-PCR analysis and a Western Blot analysis were performed to verify the related mechanism.ResultsThe expression of circ_0080229 was upregulated in both glioma tissues and cell lines related to unfavorable clinicopathologic characteristics. The expression of circ_0080229 was found to be inversely correlated with miR-1827, a micro-ribonucleic acid (miRNA) targeting murine double minute-2 (MDM2). The downregulation of circ_0080229 inhibited gliomas in vivo and suppressed U87 and U251 cell lines in vitro, which the transfection of the miR-1827 inhibitor could reverse. Concerning the mechanism, a block of circ_0080229 decreased MDM2 expression, while the inhibition of miR-1827 reversed this effect. Thus, circ_0080229 appears to target the downstream miR-1827/MDM2 signaling pathway.ConclusionsOur results showed that the silencing of circ_0080229 upregulates the expression of miR-1827, which in turn resulted in the suppression of MDM2, and the mediation of the downstream P53 signaling pathway. Circ_0080229 exerted an effect in mediating tumor progression through the MDM2 signaling pathway by sponging miR-1827. Its importance as a potential prognostic biomarker in gliomas has thus been established.

Project description:The ability to separate the isoforms of human tumour suppressor protein p53 expressed in insect cells using heparin-Sepharose correlates with differences in the isoelectric point of p53, demonstrating that p53 can be heterogeneously modified and providing support for the use of insect cells as a model system for identifying novel signalling pathways that target p53. One p53 isoform that was reduced in its binding to the monoclonal antibody DO-1 could be stimulated in its binding to DO-1 by prior incubation with protein phosphatases, suggesting the presence of a previously unidentified N-terminal phosphorylation site capable of masking the DO-1 epitope. A synthetic peptide from the N-terminal domain of p53 containing phosphate at Ser(20) inhibited DO-1 binding, thus identifying the phosphorylation site responsible for DO-1 epitope masking. Monoclonal antibodies overlapping the DO-1 epitope were developed that are specific for phospho-Thr(18) (adjacent to the DO-1 epitope) and phospho-Ser(20) (within the DO-1 epitope) to determine whether direct evidence could be obtained for novel phosphorylation sites in human p53. A monoclonal antibody highly specific for phospho-Ser(20) detected significant phosphorylation of human p53 expressed in insect cells, whereas the relative proportion of p53 modified at Thr(18) was substantially lower. The relevance of these two novel phosphorylation sites to p53 regulation in human cells was made evident by the extensive phosphorylation of human p53 at Thr(18) and Ser(20) in a panel of human breast cancers with a wild-type p53 status. Phospho-Ser(20) or phospho-Thr(18) containing p53 peptides are as effective as the phospho-Ser(15) peptide at reducing mdm2 (mouse double minute 2) protein binding, indicating that the functional effects of these phosphorylation events might be to regulate the binding of heterologous proteins to p53. These results provide evidence in vivo for two novel phosphorylation sites within p53 at Ser(20) and Thr(18) that can affect p53 protein-protein interactions and indicate that some human cancers might have amplified one or more Ser(20) and Thr(18) kinase signalling cascades to modulate p53 activity.

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Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells.

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