P53-Independent Functions of MDM2

Vol. 1, 1027–1035, December 2003 Molecular Cancer Research 1027

Subject Review p53-Independent Functions of MDM2

Gitali Ganguli and Bohdan Wasylyk

Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS/INSERM/ULP, Illkirch cedex, France

Abstract In Vivo Evidence The tumor suppressor p53 is inactivated by Human Tumors overexpression of MDM2 in about 10% of human tumors. Human tumors containing both mutant p53 and MDM2 However, p53 is inactivated by other mechanisms in the amplification are rare, but both modifications can occur within majority of tumors, raising the possibility that MDM2 may the same tumor and result in a poorer prognosis (1). The 12% of be irrelevant to transformation in most cases. However, patients with bladder cancer that exhibit high levels of MDM2 MDM2 has been reported to have p53-independent and p53 mutation have a poorer prognosis than patients with functions, in cell cycle control, differentiation, cell fate either MDM2 amplification or p53 mutation (2). That there are determination, DNA repair, basal transcription, and two non-redundant mechanisms to inactivate p53 provides other processes. Furthermore, MDM2 appears to support for a p53-independent function of MDM2. contribute to the transformed phenotype in the absence of wild-type p53. Nevertheless, the number of studies Alternative Splicing of MDM2 is still limited, and the evidence in some cases does Alternative splicing of pre-mRNAs can give rise to proteins not unequivocally show that the functions are p53 with different functions. Interestingly, more than 40 different independent. We will discuss the circuits of regulation splice variants of MDM2 transcripts have been identified both involving MDM2 that do not directly concern p53. in tumors and normal tissues, and the majority of these variants Hopefully, future work will consolidate our understanding do not contain sequences encoding the p53 binding site, of the p53-independent pathological functions of MDM2 suggesting that they have p53-independent functions (for and will lead to useful therapeutic interventions that additional information, see Ref. 3 and the other reviews in this target the majority of tumors. issue). Some of these splice variants may arise from the loss of splicing fidelity during tumorigenesis, and may not have an Introduction 1 important function (4). The functions of the MDM2 splice Although MDM2 has been extensively characterized as a variants lacking the p53 interaction domain need to be regulator of p53, there is considerable evidence that MDM2 has individually evaluated in appropriate model systems. p53-independent functions. We will describe the evidence from in vivo studies, including human tumors, transgenic mice, and cells in culture. We will portray the interactions of MDM2 with Animal Models various factors (Fig. 1) that have effects on functions relevant to Several studies have addressed the effect in vivo in mouse transformation, including cell cycle control, differentiation, DNA models of overexpression of MDM2 in the absence of p53 (see synthesis, RNA biosynthesis, transcription, and cell surface Table 1). Targeted expression to the mammary gland in mice receptor turnover (Fig. 2). We will also consider the regulators of inhibits mammary gland development and uncouples S phase MDM2 that affect its functions, as far as they provide insights from mitosis in a p53- and E2F1-independent manner (5, 6). into the p53-independent functions of MDM2. Finally, we will Overexpression driven by the entire mdm2 gene predisposes to discuss the hopes for tumor therapy. Certain aspects of the p53- spontaneous tumor formation and reveals a p53-independent independent functions of MDM2 are covered in other reviews in role for MDM2 in tumorigenesis. Only one line with the mdm2 this series and will not be discussed in detail here. gene as the transgenic was studied. It displays increased tumorigenesis, with a high incidence of sarcomas that is retained in a p53–/– background (7). Overexpression in the Received 8/28/03; revised 11/11/03; accepted 11/14/03. differentiating compartment of the epidermis inhibits differen- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in tiation in a p53-independent manner, but does not predispose to accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tumor formation (8). Similar results were obtained in transgenic Grant support: Ligue Regionale Contre le Cancer for a fellowship for G.G.; mice in which the involucrin promoter drives Mdm2 expression BioAvenir (Aventis, Rhone-Poulenc); Centre National de la Recherche 2 Scientifique; Institut National de la Sante´ et de la Recherche Me´dicale; to the spinous layer of the epidermis. In the CMV and Hoˆpital Universitaire de Strasbourg; Association pour la Recherche sur le involucrin-mdm2 mice, the mdm2 transgene appears to be Cancer; Fondation pour la Recherche Me´dicale; Ligue Nationale Francaise contre le Cancer (Equipe labellise´e); Ligue Re´gionale (Haut-Rhin) contre le expressed in cells in which p53 is not functional (see Fig. 3, Cancer; Ligue Re´gionale (Bas-Rhin) contre le Cancer; and EU (FP5 project QLK6-2000-00159). Requests for reprints: B. Wasylyk, CNRS/INSERM/ULP, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch cedex, France. Phone: 33-3-88-65-34-11; Fax: 33-3-88- 1The abbreviations used are MDM2, human gene and oncogene; MDM2, human 65-32-01. E-mail: [email protected] protein and isoform; mdm2, mouse gene; Mdm2, mouse protein. Copyright D 2003 American Association for Cancer Research. 2G. Ganguli and B. Wasylyk, unpublished observations.

FIGURE 1. MDM2 interacting proteins and functions. MDM2 has a NH2-terminal p53 interacting domain, a nuclear localization signal (NLS), a nuclear export signal (NES), a central acidic domain, a zinc finger (Zn), a RING finger motif, and a nucleolar localization signal (NoLS). The domains of MDM2 that interact with proteins are discussed in the text. The ID1 and ID2 are the G1-arrest domains. Normal cell-specific ID is the growth arrest domain functionally specific in normal diploid cells such as WI38 and MRC5 (discussed in the review by S. P. Deb).

A and B). But when Mdm2 expression is driven by the K14 Fig. 3C). The UV response is impaired by Mdm2, thereby promoter to the basal layer, the mice are predisposed to the decreasing the protective effect of p53. The reduced tumor development of hyperplastic lesions that progress to papillomas suppressor function of p53 results in increased papilloma and squamous cell carcinomas (9), showing that the oncogenic formation after chemical carcinogenesis and spontaneous potential of MDM2 in vivo is revealed when it is targeted to accumulation of genetic lesions resulting in the appearance of cells in which p53 suppresses tumorigenesis. hyperplastic lesions and carcinomas. p53 levels are low in normal epidermis (Fig. 3A). p53 is MDM2 is important for epidermal differentiation. It is activated during the exit from the proliferative state, resulting in highly expressed in normal human and mouse skin (10). Mdm2 inhibition of cell division and stimulation of differentiation. A overexpression in the granular layer perturbs the differentiation consequence of p53 activation could be the MDM2 expression program (8). MDM2 has been implicated in regulation of that is observed in the basal and the suprabasal layers. As differentiation in other systems (11). It may affect differenti- keratinocytes differentiate, they become insensitive to p53- ation through inhibition of p53 and interaction with other induced apoptosis. UV light induces p53 in the proliferative molecules. p53 has been implicated in cell differentiation and compartment of the basal layer, resulting in protection from the development (12). p63, a homologue of p53, is important for tumorigenic effects of DNA damage as a result of p53-induced epidermal differentiation (13, 14). However, it appears unlikely cell cycle arrest or apoptosis. In transgenic mice, p53 is that MDM2 affects p63 activity because the variant expressed inhibited by Mdm2 produced in the basal layer, leading to in the epidermis lacks the MDM2 interaction domain (15). decreased differentiation and increased proliferation (see Mdm2 is expressed in both the basal and suprabasal layers, in

FIGURE 2. Schematic of the interacting factors and modulators of MDM2. These factors are categorized into functional classes that are discussed in the text.

Table 1. Mouse Models of Mdm2 Function

Promoter Organ targeted Phenotype Reference

BLG Mammary epithelium 16% adenocarcinomas and adenocanthomas, p53-independent effect (5) MDM2 Several organs 100% lymphomas and sarcomas, p53-independent effect (7) HCMV Granular layer of the skin Defective differentiation in the skin, p53-independent effect (8) K14 Basal layer of the skin 33% pre-neoplastic lesions, p53-dependent effect (9)

which keratinocytes enter the differentiation program, suggest- of p53 induction are differentiation dependent in the epidermis, ing that Mdm2 is involved in the early steps of differentiation, resulting in either cell cycle arrest or apoptosis in different in which the decision occurs as to whether the basal compartments (25, 26). The basal levels of MDM2 may help proliferating cell will enter the proliferation program. In support determine whether p53 induction results in cell cycle arrest or of this hypothesis, it has been shown that MDM2 levels apoptosis in different keratinocyte populations. increase when keratinocytes are induced to differentiate in vitro Mdm2 overexpression in different layers of the skin has (16). p53 activity could influence MDM2 levels because similar effects on differentiation, proliferation, and apoptosis. MDM2 is one of its target genes. However, p53 may not be However, the molecular mechanisms of these effects are not the major determinant of the basal level of MDM2. p53 levels identical because p53 removal has an effect only when MDM2 are low in uninduced conditions in the epidermis (17). is expressed in the basal layer. These observations are explicable Furthermore, MDM2 expression is independent of p53 in in the light of the known properties of MDM2 and p53. The role keratinocytes (10) and epithelia during mouse development of p53 changes in different states of keratinocyte differentiation. (18). Constitutive MDM2 expression is thought to set the p53 induction results in apoptosis of proliferating keratinocytes, sensitivity of cells to p53 induction (19–24). The consequences whereas p53 is not activated in late-stage suprabasal cells

FIGURE 3. Roles of MDM2 in normal skin and when overexpressed in the granular (HCMV promoter) and basal (K14 promoter) layers of the skin. A. Normal skin has low levels of p53 in the basal layer, which is activated when cells move into the differentiating compartment. MDM2, which is present in the suprabasal layer, inhibits p53 and regulates other unknown factors, thereby controlling differentiation. When normal epidermis is UV-irradiated, p53 levels increase in the basal layer and induce cell cycle arrest and apoptosis. B. In HCMV-MDM2 transgenic skin, overexpression of MDM2 inhibits differentiation of the granular layer independent of p53 but does not induce tumor formation. The p53 protective function after UV irradi- ation in the basal layer is unaltered. C. MDM2 overexpressed in the basal layer inhibits p53, increases proliferation in a p53-dependent manner, and favors tumor formation. When this skin is UV-irradiated, the protective functions of p53 are reduced.

(25, 27). MDM2 produced in the basal layer has effects on p53 activity of pRb, as well as p53 (32). MDM2 forms a complex and can thereby indirectly target downstream molecules such as with pRb in vivo, and perturbs pRb-mediated G1 arrest (32). E2F and pRb. MDM2 produced in late differentiating Conversely, pRb impairs certain functions of MDM2 through keratinocytes cannot do this and can only target downstream the formation of a trimeric complex with p53. pRb thereby molecules directly, resulting in a p53-independent phenotype. overcomes the ability of MDM2 to inhibit p53-mediated apoptosis (33). Mdm2 has been shown to confer a growth advantage to cells lacking p53 and pRb, and to overcome a G1 Cell Line Models cell cycle arrest induced by p107, a member of the pRb family. Mdm2 confers a growth advantage to cells that lack p53 and The minimum transforming and p107-inhibiting region of pRb, and can overcome a G1 cell cycle arrest induced by p107 Mdm2 corresponds to its p53-binding domain (28). It remains (28), a member of the pRb tumor suppressor family (29). The to be seen if the other member of the pRb family, p130, is a minimum transforming and p107-inhibiting region of MDM2 target for MDM2. corresponds to its p53 binding domain. p53 inhibits transformation by MDM2, apparently without requiring transcription. E2F1/DP1 p53 can thus be considered to be a suppressor of MDM2, a MDM2 has been shown to interact with E2F1/DP1, a pRb- positive effector of the cell cycle. MDM2 overexpression in regulated transcription factor involved in S-phase progression. tumors is reminiscent of p53 mutations with gain of function, in There is a striking similarity between a domain of E2F-1 that MDM2 both transforms cells and inhibits p53 activity. (amino acids 390–406) and the MDM2 binding domain of p53 Muscle cell differentiation is controlled by a complex set of (34). MDM2 stimulates E2F1/DP1-dependent activation of the interactions between tissue restricted transcription factors, E2F promoter, and DNA synthesis in cooperation with E2F1/ ubiquitously expressed transcription factors, and cell cycle DP1. MDM2 also increases degradation of the heterodimer, regulatory proteins. One obvious phenotype of tumor cells is thereby inhibiting the pro-apoptotic effect of E2F1/DP1. the lack of terminal differentiation. Rhabdomyosarcoma cell Increased degradation may serve to reduce the amounts of lines have a recessive or a dominant nondifferentiating E2F1/DP1 to levels appropriate for the G -S phase transition phenotype. To study the genetic basis of the dominant non- 1 (35). The growth-promoting and anti-apoptotic activities of differentiating phenotype, microcell fusion was used to transfer MDM2 on E2F1 could be crucial for the p53-independent chromosomes from rhabdomyosarcoma cells into C2C12 oncogenic activities. However, various studies suggest that the myoblasts. Amplification of MDM2 in rhabdomyosarcoma effects of MDM2 on E2F1 could be p53 dependent. MDM2 has cells was found to interfere with MyoD activity and been shown to inhibit E2F1-induced apoptosis, apparently by consequently inhibits overt muscle cell differentiation (11). inhibiting E2F1 induction of p53 (36). In addition, E2F1 has Recently, it was found that MDM2 inhibition of MyoD can be been shown to be up-regulated in response to DNA damage in a overcome by expression of pRb or Sp1, thereby overcoming the p53-dependent manner (37). Microinjection of an antibody block of muscle cell differentiation by amplified MDM2. There against a domain of MDM2 required for MDM2-p53 is a circuit of regulation involving MDM2, Sp1, and pRb. interaction or an antisense MDM2 oligonucleotide increases MDM2 interacts with Sp1 and inhibits Sp1-dependent tran- E2F-1 protein levels, suggesting that endogenous E2F1 may be scription, and pRb can restore Sp1 activity by displacing sensitive to MDM2 protein because of its effects on p53. In MDM2 from Sp1. The COOH-terminal domain of pRb another study, a set of cell lines with differing p53 and pRb was interacts with MDM2, and is both necessary and sufficient to used to show that overexpression of MDM2 stimulates E2F1/ restore muscle cell differentiation in cells with amplified DP1 transactivation by a p53-dependent mechanism (38). MDM2, and to stimulate premature differentiation of prolifer- MDM2 induction of E2F1 transactivation was concluded to ating myoblast cells. These results suggest that the pRb-MDM2 result from inhibition of p53-dependent transcription of p21. interaction modulates normal muscle cell differentiation (30). The consequent increased cyclin-dependent kinase activity and Rather surprisingly, expression of MDM2 can inhibit the G - 1 phosphorylation of pRb results in increased E2F1/DP1 trans- S phase transition of NIH 3T3 and normal human diploid cells activation (38). These different studies are not necessarily (31, 31a). Two cell cycle inhibitory domains have been contradictory. Similar to the conclusions from studies in the identified in MDM2, ID1 and ID2, which do not overlap with epidermis previously mentioned, they may indicate that the p53 interaction domain (Fig. 1). Some tumor-derived cells depending on the cellular background and the activity of p53, are relatively insensitive to the growth-inhibitory effects of MDM2 can affect E2F1/DP1 transactivation by p53-dependent MDM2, suggesting that inactivation of the MDM2-induced G 1 and -independent mechanisms. arrest may contribute to tumorigenesis. MDM2 may have p53- independent functions as a tumor suppressor, and loss of these functions in tumors could unmask the positive effects of MDMX MDM2 on tumorigenesis. MDMX (MDM4) is structurally and functionally related to MDM2 (39). They are nearly identical in size, yet only share some common domains. They are similar within the The Cell Cycle p53-binding domain at their amino termini and both The Rb Family contain a central zinc finger domain and a carboxyl- pRb is a tumor suppressor that has functions similar to p53, terminal ring finger domain (40). Like MDM2, MDMX is in cell cycle inhibition and cell death. MDM2 modulates the an essential negative regulator of p53, because loss of

MDMX expression results in p53-dependent embryonic lethality toward hNumb and that it induces its ubiquitination and in mice (41, 42). Although the current evidence suggests that degradation in cells (48). The possibility that the interaction the only role of MDMX in control cell proliferation and the between Numb and MDM2 contributes to the oncogenic cell cycle is through its regulation of p53, the possibility properties of MDM2 through effects on differentiation exists that MDMX may affect the p53-independent functions requires further investigation. of MDM2. It may also have p53-independent functions that are not revealed in the mouse studies because of the early DNA Synthesis embryonic lethality of the inactivation. DNA Polymerase e The physical interaction between human DNA polymerase PML q and human MDM2 has been established by a yeast two- The tumor suppressor protein PML and MDM2 have hybrid screen, an in vitro binding assay, and in vivo co- opposing effects on p53. PML stimulates p53 activity by immunoprecipitation (49). MDM2 expressed in and purified recruiting it to nuclear foci termed PML-nuclear bodies (PML- from either Escherichia coli or insect cells stimulates the NBs). An in vivo interaction between MDM2 and PML has activity of DNA polymerase q in vitro. Moreover, the COOH- been reported recently that is independent of p53 (43). Two terminal domain of DNA polymerase q, to which MDM2 regions of MDM2 interact with PML; the central region with binds, and the NH2-terminal 166 amino acids of MDM2, to the COOH-terminal half of PML (300–633) and the RING which DNA polymerase q binds, are both essential for the finger with the RING finger of PML (1–200). Interestingly, stimulation (50). The proposed roles of DNA polymerase q sumoylation of PML inhibits the interaction. MDM2 inhibits include DNA repair, recombination, replication, damage PML by exclusion from the nucleus, similar to one of the sensing, and chromatin remodeling. In response to DNA mechanisms by which MDM2 inhibits p53. MDM2 inhibits the damage, MDM2 may mediate a reconfiguration process that ability of PML to stimulate the transcriptional activity of a allows DNA polymerase q to associate with repair/recombi- GAL4-CBP fusion protein. The importance of MDM2 nation proteins. regulation of the activity and localization of PML in the tumor suppressor function of PML remains to be established. Ribosome Biosynthesis Ribosomal Protein L5 and 5S RNA MTBP A possible function for MDM2 in ribosome biosynthesis or A novel MDM2binding protein (MTBP) has been in translational regulation is suggested by the specific identified using two-hybrid screens (44). The central region interaction of the central domain of MDM2 domain with L5 of MDM2 (amino acids 167–304) binds to the carboxyl- (a component of the large ribosomal subunit) and the COOH- terminal 380 amino acids of MTBP. MTBP induces G1 arrest in terminal domain with 5S rRNA (51–53). MDM2 has a a p53-independent manner, and this arrest can in turn be nucleolar localization signal and is found in the nucleolus inhibited by MDM2. Further studies are required to establish (54), the site of ribosome biosynthesis and assembly. These the significance of the interactions of MDM2 and MTBP observations raise the interesting speculation that MDM2 has a regulation of the cell cycle. role in ribosome assembly, transport, or RNA synthesis.

TGF-b1 Transcription Resistance to transforming growth factor h (TGF-h) TBP and TAFII250 inhibition of cell proliferation is linked to tumorigenesis. MDM2 interacts in vivo and in vitro with the general h MDM2 was isolated in a screen for factors that rescue TGF- transcription factor TFIID but not with TFIIB, another sensitivity of mink lung epithelial cells. Rescue results from the general factor (55–58). Different domains of MDM2 contact effects of MDM2 on pRb/E2F and does not involve p53 (45). two subunits of the complex, the acidic domain contacts TBP Increased MDM2 expression correlates with TGF-h resistance and the Ring finger contacts TAFII250 (59; Fig. 1). MDM2 in human breast tumor cells. These observations raise the activates the promoter of cyclin A, a gene that is important h possibility that MDM2 may confer TGF- resistance through its for S-phase entry (59). Expression of MDM2 activates the effects on pRb, at least in certain cell types (46). Further studies cyclin A gene promoter but not c-fos, suggesting that the are needed to consolidate and extend these observations. effects of MDM2 are specific. Overexpression of MDM2 inhibits the cyclin A promoter, possibly by titration of Differentiation general transcription factors. The mechanisms of repression Numb of the cyclin A and fos promoters appear to be different. The Numb protein was isolated in a screen for factors Cyclin A repression is lost by deleting the COOH terminus, that interact with MDM2 (47). Drosophila Numb antago- whereas that of c-fos is lost by removal of the acidic domain. nizes Notch signaling and is involved in neural cell These observations link the activity of MDM2 with cyclin A, differentiation and cell fate determination. The NH2-terminal a regulator of the cell cycle. Further studies are required to region of MDM2 binds to Numb, the same region that determine whether MDM2 acts directly, through for example interacts with p53. MDM2 alters the subcellular localization effects on E2F/pRb family members that regulate this of Numb and promotes its degradation. Recent evidence promoter, or whether the effects are less direct, through strongly suggests that MDM2 functions as a ubiquitin ligase effects on the cell cycle.

p300 The ARF (p19 ARF/p14ARF)-MDM2-p53 Pathway MDM2 can also directly interact with the transcriptional ARF is a central player in tumor surveillance due to its ability coactivator p300 (60, 61). The consequences of the to protect p53 from MDM2-mediated degradation (67–70). interaction are complex, and appear to involve effects of Human ARF sequesters MDM2 in the nucleolus and blocks p300 on p53 and MDM2 protein turnover and MDM2 nuclear export of MDM2 and p53 (71, 72). However, ARF can synthesis. p300 is required for MDM2 gene induction by also affect the p53-independent functions of MDM2. In p53, and inhibition of p300 results in stabilization of p53 and transgenic mice, MDM2 expression directed to the mammary apoptosis (61). Interestingly, inhibition of TAFII250 similarly epithelium disrupts the cell cycle, resulting in multiple rounds of inhibits MDM2 induction by p53 and causes apoptosis, DNA synthesis without proper cell division and poor mammary suggesting that the p53-MDM2 loop is a sensor of tran- gland development. These effects are p53 and E2F1 indepen- scriptional defects (62). p300 overexpression increases dent, because MDM2 transgenic mice that are null for these MDM2 protein levels without effects on MDM2 mRNA. genes exhibit the same defects (5). Interestingly, MDM2 p300 associates with MDM2 in nuclear body-like structures transgenic mice that are null for ARF have enhanced defects, where MDM2 might be protected from proteasomal degra- as if more MDM2 is free to exert the phenotype (73). dation (63). The functional consequences of the p300-MDM2 interaction on the p53-independent functions of MDM2 The TSG101/MDM2 Regulatory Loop remain to be further elucidated. TSG1001 is a tumor suppressor, the loss in fibroblasts of which, results in cellular transformation and the ability to form NF-jB/p65 metastatic tumors in nude mice. tsg101À/À embryos fail to MDM2 has been shown to induce the expression of the develop beyond day 6.5 of embryogenesis (E6.5). Mutant p65 subunit of NF-nB through effects on the p65 promoter embryos have decreased cellular proliferation, and increased (64). NF-nB has various roles, including inhibition of p53 protein accumulation by a posttranscriptional mechanism. apoptosis in response to chemotherapy in certain neoplastic Elimination of p53 in null animals prolongs survival, showing cells. MDM2 overexpression in leukemic bone marrow cells that there is a functional connection between TSG101 and the of patients with BCP-ALL or in an ALL cell line (EU-4) p53 pathway in vivo (74). TSG101 participates with MDM2 in has been associated with elevated expression of p65 and an autoregulatory loop that modulates the levels of both in vitro resistance to doxorubicin. MDM2 expression in the proteins and p53, by affecting protein decay (75). TSG101 absence of p53 increases p65 promoter activity, apparently inhibits MDM2 ubiquitination, and the stabilized MDM2 by direct binding of MDM2 to the Sp1 site. Further studies negatively regulates p53 and accelerates decay of TSG101. are required to establish whether the induction of p65 by The effects of TSG101 on MDM2 may also influence the p53- MDM2 is an important p53-independent role of MDM2 in independent functions of MDM2 (75). tumorigenesis. p53-Independent Activation of MDM2 Expression B2-Adrenergic Receptor and B-Arrestin MDM2 gene expression is regulated by mechanisms that are h-Arrestin regulates the internalization of G protein- independent of p53. MDM2 expression during development is coupled receptors (GPCRs) away from the cell membrane. tissue-specific and is independent of p53 in different organs Internalized receptors are either recycled back to the surface (18). High levels of MDM2 expression are observed in human after dephosphorylation, or are degraded. MDM2 has been tumor cell lines with little or no functional p53 (76). MDM2 shown to be a key factor in the sequestration of the cell expression increases following FGF-2 treatment of cells (77), surface h2-adrenergic receptor (h-AR) through interactions and in cells harboring a chimeric M-CSF/platelet-derived with h-arrestin (65). Agonist stimulation of h-AR leads to growth factor (PDGF) receptor (77, 78). The MDM2 promoter rapid ubiquitination of both the receptor and the receptor has been shown to be a target of the Ras/mitogen-activated regulatory protein, h-arrestin. Abrogation of h-arrestin protein (MAP) kinase pathway, and hence activation of Ras ubiquitination, either by expression in MDM2-null cells or during normal cell signaling or through mutation in neoplastic by dominant-negative forms of MDM2 lacking E3 ligase transformation can increase MDM2 functions (76). MDM2 activity, inhibits receptor internalization with marginal effects promoter regulation in T47D breast cancer cells has been on receptor degradation. However, a h-AR mutant lacking shown to be independent of p53 and Ras/MAP kinase lysine residues, which was not ubiquitinated, was internalized signaling, but dependent on the composite AP1-ETS motif normally but was degraded ineffectively. These findings show and a nonconserved upstream (nnGGGGC; 5) repeat sequence. that h-arrestin is a substrate for MDM2 ubiquitination, and MDM2 promoter activity is increased by the ETS2 expression that MDM2 is involved in h-AR trafficking. MDM2 may through the AP1-ETS element (79). Further studies are required connect extracellular signals mediated through GPCRs to p53 to determine how altered MDM2 expression through these or to other cellular effectors (66). different pathways affects the p53-dependent and -independent functions of MDM2. Regulators of MDM2 A variety of factors other than p53 regulate MDM2 activity, AKT/PKB and they may have effects on the p53-independent functions of PKB phosphorylates MDM2 on several residues, resulting in MDM2. translocation of MDM2 to the nucleus, where it can regulate

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Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. p53-Independent Functions of MDM21 1 Ligue Regionale Contre le Cancer for a fellowship for G.G.; BioAvenir (Aventis, Rhone-Poulenc); Centre National de la Recherche Scientifique; Institut National de la Santé et de la Recherche Médicale; Hôpital Universitaire de Strasbourg; Association pour la Recherche sur le Cancer; Fondation pour la Recherche Médicale; Ligue Nationale Francaise contre le Cancer (Equipe labellisée); Ligue Régionale (Haut-Rhin) contre le Cancer; Ligue Régionale (Bas-Rhin) contre le Cancer; and EU (FP5 project QLK6-2000-00159).

Gitali Ganguli and Bohdan Wasylyk

Mol Cancer Res 2003;1:1027-1035.

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