Human MDM2 isoforms translated differentially on constitutive versus p53-regulated transcripts have distinct functions in the p53/MDM2 and TSG101/MDM2 feedback control loops.
Journal: 2007/January - Molecular and Cellular Biology
ISSN: 0270-7306
Abstract:
Proteins encoded by the mdm2 gene, which has a pivotal role in the regulation of growth and differentiation, exist principally in human and murine cells as two isoforms that migrate in gels as 75-kDa and 90-kDa proteins. There is limited understanding of the respective biological roles of these isoforms, their molecular nature, and their mechanism of formation. We report here that human p75(MDM2) is an N-terminally truncated mixture of protein isoforms produced by the initiation of translation at two distinct internal AUG codons. The p75(MDM2) doublets and p90(MDM2), which is the full-length MDM2 protein, are expressed in approximately equal amounts from transcripts initiated at the constitutive P1 promoter of mdm2. Unlike murine transcripts initiated at the p53-activated P2 promoter, human cell transcripts initiated at the P2 promoter preferentially produce p90(MDM2). The ubiquitin enzyme variant protein TSG101, which interacts functionally with MDM2 in an autoregulatory loop that parallels the p53/MDM2 feedback control loop, interferes with degradation of both isoforms; however, only p90(MDM2) promotes proteolysis of TSG101 and p53. Our results reveal the mechanism of formation of the principal MDM2 isoforms, the differential effects of p53 on the production of these isoforms, and the differential abilities of human MDM2 isoforms as regulators of the MDM2/TSG101 and p53/MDM2 feedback control loops.
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Mol Cell Biol 27(1): 111-119

Human MDM2 Isoforms Translated Differentially on Constitutive versus p53-Regulated Transcripts Have Distinct Functions in the p53/MDM2 and TSG101/MDM2 Feedback Control Loops<sup><a href="#fn2" rid="fn2" class=" fn">▿</a></sup>

Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5120
Corresponding author. Mailing address: Stanford University School of Medicine, Department of Genetics, 300 Pasteur Dr., Stanford, CA 94305-5120. Phone: (650) 723-5315. Fax: (650) 725-1536. E-mail: ude.drofnats@nehocns.
Present address: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, Republic of China.
Received 2006 Feb 8; Revised 2006 Apr 10; Accepted 2006 Oct 9.

Abstract

Proteins encoded by the mdm2 gene, which has a pivotal role in the regulation of growth and differentiation, exist principally in human and murine cells as two isoforms that migrate in gels as 75-kDa and 90-kDa proteins. There is limited understanding of the respective biological roles of these isoforms, their molecular nature, and their mechanism of formation. We report here that human p75 is an N-terminally truncated mixture of protein isoforms produced by the initiation of translation at two distinct internal AUG codons. The p75 doublets and p90, which is the full-length MDM2 protein, are expressed in approximately equal amounts from transcripts initiated at the constitutive P1 promoter of mdm2. Unlike murine transcripts initiated at the p53-activated P2 promoter, human cell transcripts initiated at the P2 promoter preferentially produce p90. The ubiquitin enzyme variant protein TSG101, which interacts functionally with MDM2 in an autoregulatory loop that parallels the p53/MDM2 feedback control loop, interferes with degradation of both isoforms; however, only p90 promotes proteolysis of TSG101 and p53. Our results reveal the mechanism of formation of the principal MDM2 isoforms, the differential effects of p53 on the production of these isoforms, and the differential abilities of human MDM2 isoforms as regulators of the MDM2/TSG101 and p53/MDM2 feedback control loops.

Abstract

mdm2 initially was identified in a screen for genes amplified on double minute chromosomes found in spontaneously transformed mouse 3T3 fibroblasts (14). Subsequent work, which has shown that elevated expression of the MDM2 protein can promote neoplastic transformation of cells in culture and tumor formation in animals (24) and that amplification of the human ortholog of mdm2 occurs in a wide variety of cancers (39, 41), suggests that mdm2 is an oncogene of major importance. Perhaps the most extensively studied biological function of MDM2 is regulation of the activity and abundance of p53, a tumor suppressor protein that modulates the expression of genes involved in DNA repair, cell cycle progression, and apoptosis (13). By binding to p53, MDM2 inhibits p53's transcription-promoting actions and additionally functions as an E3 ubiquitin ligase to accelerate degradation of the p53 protein (15, 21, 40); conversely, transcription of mdm2 is activated by p53 (3). Thus, MDM2 and p53 participate in an autoregulatory (“feedback control”) loop in which p53 modulates the production of a protein that inhibits its function (53).

The actions of other cellular genes implicated in tumorigenesis affect the workings of the MDM2/p53 feedback control loop (13). Among these is tumor susceptibility gene tsg101, which initially was identified in a genetic screen for mouse fibroblasts that undergo neoplastic transformation as a result of random chromosomal gene inactivation (28). Deficiency of TSG101 in NIH 3T3 cells reversibly results in colony formation in soft agar, focus formation in monolayer cultures, and the ability to form metastatic tumors in nude mice (28). The steady-state level of TSG101, which is an essential cellular protein (45, 52), is regulated posttranslationally within a narrow range (16), and overexpression of TSG101 from an adventitious promoter can also result in neoplastic transformation (28). Through its ubiquitin-conjugating E2 variant (UEV) domain, TSG101 interacts with MDM2, inhibits MDM2 ubiquitination, and prolongs the half-life of MDM2 protein; conversely, elevation of MDM2 promotes proteolysis of TSG101, as occurs for p53 (29). As the MDM2/TSG101 regulatory loop modulates the cellular levels of both proteins and consequently affects MDM2 control of p53 (29), TSG101 is both a regulator of and target of p53/MDM2 circuitry. TSG101 has also been shown to be a key component of complexes that mediate endocytic trafficking of cell surface receptors and the budding of medically important pathogenic viruses (18, 30, 32).

More than 40 splice variants of mdm2 mRNA and also multiple isoforms of MDM2 protein that interact differentially with p53 have been identified in tumors and normal tissues (4). However, two particular isoforms, which migrate in sodium dodecyl sulfate (SDS) polyacrylamide gels as 90-kDa and 75-kDa proteins, predominate in both mouse and human cells (6, 46). Earlier experiments from our laboratory have shown that ubiquitination and stability of at least one of these human isoforms, p90, is affected by TSG101 (29). There have been differing conclusions as to whether this isoform or p75 is the full-length protein (7, 37, 55). Here we report the results of investigations of the molecular nature and mechanism of production of these two principal human MDM2 isoforms and their respective roles in the MDM2/TSG101 and MDM2/p53 feedback control loops. Our findings confirm that p90 is an unconjugated full-length human MDM2 (HDM2) protein, demonstrate that this human MDM2 isoform is preferentially synthesized on transcripts initiated at the p53-regulated P2 promoter, and establish that p75 is a mixture of truncated proteins produced by the initiation of translation at two different internal AUG codons within transcripts encoded largely by P1. We further show that only p90 promotes proteolysis of either p53 or TSG101, but that TSG101 stabilizes both the p75 and p90 isoforms. Our results identify p90 as the human MDM2 isoform that controls the protein levels of p53, TSG101, and MDM2 itself through the actions of p53/MDM2 and MDM2/TSG101 feedback control loops.

Acknowledgments

We thank A. Levine and L. Gerace for MDM2 and RanGAP1 antibodies, J. Ford for HCT116 cells, and C. Contag for the pEYFP-N1 plasmid. We acknowledge the helpful comments and advice of L. Li as well as members of the Cohen laboratory.

These studies were supported by grants from the National Foundation for Cancer Research and the California Breast Cancer Research Program to S.N.C. and in part by a grant from the National Science Council of Taiwan (NSC-93-2320-B-010-063) to T.H.C.

Acknowledgments

Footnotes

Published ahead of print on 23 October 2006.

Footnotes

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