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The MDM2 oncoprotein has transforming potential that can be activated by overexpression, and it represents a critical regulator of the p53 tumor suppressor protein. To identify other factors with a potential role in influencing the expression and/or function of MDM2, we utilized a yeast two-hybrid screening protocol. Here we report that MDM2 physically interacts with a structurally related protein termed MDMX. The results obtained in these studies provide evidence that C-terminal RING finger domains, contained within both of these proteins, play an important role in mediating the association between MDM2 and MDMX. The interaction of these proteins interferes with MDM2 degradation, leading to an increase in the steady-state levels of MDM2. MDMX also inhibits MDM2-mediated p53 degradation, with subsequent accumulation of p53. Taken together, these data indicate that MDMX has the potential to regulate the expression and function of the MDM2 oncoprotein.An accumulating number of observations have implicated aberrant expression of the MDM2 oncogene in the pathogenesis of human neoplasias. This mammalian gene has transforming potential that can be activated by overexpression (1, 2). Originally identified as a gene amplified and overexpressed in a spontaneously transformed mouse 3T3 cell line (3), MDM2 is now known to be amplified in a variety of human tumors, particularly soft tissue sarcomas (4 -7). Additionally, there are several cases reported of tumor cells having an elevated expression of MDM2 that results from mechanisms other than gene amplification, including enhanced translation of MDM2 transcripts (8 -10).The MDM2 gene encodes a key negative regulator of the p53 tumor suppressor protein, and the role of MDM2 overexpression in cell transformation has been attributed, at least in part, to its disruption of the biological activities of p53 (11,12). MDM2 tightly associates with the N-terminal region of the p53 protein, inhibiting the trans-activation and G 1 growth arrest functions of p53 (13-16). Moreover, binding of MDM2 targets p53 for rapid degradation via the ubiquitin-proteasome pathway (17,18). In recent reports, evidence has been obtained suggesting that MDM2 can function as an E3 1 ubiquitin ligase and is responsible for targeting both itself, and p53, for degradation (19,20). Interestingly, the MDM2 gene itself is a transcriptional target of p53. When activated as a transcription factor, p53 binds to a promoter region within the first intron of the MDM2 gene and up-regulates its expression (21-24). Thus, there is evidence for an autoregulatory feedback loop involving the expression and function of MDM2 and p53 (23). Although the best characterized activities of MDM2 concern its functional interactions with p53, MDM2 also associates with other proteins. Some of these include E2F1 (25), pRb (26), and p300 (27). Such interactions could contribute to the transforming potential of MDM2 or may be concerned with modulating MDM2 function. Recently, a link between MDM2 and yet another tumor suppressor protein, ...

Apoptosis is essential for development, tissue homeostasis, and immune function (1). The key mediators of apoptosis are caspases, a family of cysteine proteases that cleave a critical set of cellular proteins near specific aspartic acid residues (2). Apoptosis-inducing members of the tumor necrosis factor superfamily, such as Fas ligand (FasL) 1 and Apo2L/TRAIL, activate caspases through the cell extrinsic apoptosis signaling pathway by engaging their respective "death" receptors: Fas and DR4 or DR5, leading to assembly of the DISC (3). Upon ligand stimulation, the adaptor protein FADD (Fas-associated death domain) binds to the receptor through homophilic death domain interactions. FADD then recruits the apoptosis initiator proteases caspase-8 and -10, through homophilic death effector domain (DED) interactions. The proximity of caspase molecules in the DISC facilitates their dimerization and thereby stimulates their proteolytic activity (4 -6). Activation of caspase-8 is followed by two self-processing events; the first cleaves a 10-kDa fragment (p10) from the full-length caspase, leaving an intermediate fragment (p43/41) at the DISC, and the second cleaves an 18-kDa fragment (p18) from the p43/41 intermediate, allowing formation of an active caspase-8 (p18/ p10) 2 heterotetramer, which is released into the cytosol. In turn, cytosolic caspase-8 catalyzes the cleavage and activation of downstream effector caspases, such as caspase-3 and -7, which execute the apoptotic death program.c-FLIP (cellular FLICE inhibitory protein) is structurally related to procaspase-8 and -10 but lacks enzymatic activity (7,8). At least 10 splice variants of c-FLIP exist on the mRNA level, but often only two c-FLIP proteins are detected: a 26-kDa short form (c-FLIP S ) and a 55-kDa long form (c-FLIP L ) (9, 10). c-FLIP S resembles its viral counterpart, v-FLIP, consisting of two DEDs and a short C-terminal tail but entirely lacking a caspase-like domain (11,12). In contrast, c-FLIP L contains two N-terminal DEDs and a C-terminal caspase-like domain, similar to caspase-8 and -10. However, c-FLIP L is proteolytically inactive, because it lacks a specific cysteine residue that is critical for caspase activity.Both c-FLIP variants are capable of binding to the Fas DISC (7,8). The presence of c-FLIP S or c-FLIP L in the Fas DISC does not preclude caspase-8 recruitment; rather, DISC-associated caspase-8/c-FLIP complexes are formed (9,(13)(14)(15). Because c-FLIP S lacks caspase cleavage sites, it is not processed by apical caspases in the DISC. In contrast, c-FLIP L is cleaved between its large (p20) and small (p12) subunits by apical caspases in the DISC; however, a second cleavage between the large subunit and the N-terminal DEDs has not been detected, probably because of the lack of a conserved cleavage site (9). Interaction of caspase-8 with c-FLIP S does not support caspase-8 activation; therefore, binding of c-FLIP S to the Fas DISC inhibits Fas-mediated apoptosis (7,8). Indeed, ectopic overexpression of c-FLIP S inhibits apoptosis i...

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