p19 Interacts with and Activates p73 by Involving the MDM2 Protein

Abstract

p73␤ is a structural and functional homologue of p53, a tumor suppressor gene. In this study, we identified a novel p73␤-binding protein, p19ras , by the yeast two-hybrid screening method. Alternative splicing of the proto-oncogene H-ras pre-mRNA has led to two distinct transcripts, p19 ras and p21 ras . In both endogenous and overexpressed systems, we confirmed that p19 ras binds to full-length p73␤ in vivo and in vitro. Coexpression of p19 ras with p73␤ stimulated the transcriptional activity of p73␤. Ras proteins are known to be small membrane-localized guanine nucleotide-binding proteins. However, unlike other Ras proteins, p19 ras is localized in the nucleus and the cytosol and its interaction with p73␤ occurred exclusively in the nucleus. Oncogenic MDM2 (mouse double minutes 2) is a known repressor of p73 transcriptional activity. In this study, when p19 ras was bound to MDM2, it further inhibited the association of MDM2 to the p73␤ protein. In addition, p19 ras abolished MDM2-mediated transcriptional repression of p73␤. Therefore, this study presents a novel pathway of Ras signaling that occurs in the nucleus, involving p19 ras and p73␤. Furthermore, a p19 rasmediated novel regulatory mechanism of p73 involving the MDM2 protein is described.More than a decade after the recognition of p53 as a major tumor suppressor, two p53 homologues, p73 and p63, were identified (1). The significant sequence homology between p73 and p53 suggests that the two proteins have similar functions as transcriptional factors. p73 not only activates the transcription of p53 target genes, but it also induces apoptosis and cell cycle arrest, like p53 (2, 3). However, a number of differences between p73 and p53 have been reported. p73 transactivates only certain p53-responsive genes (4, 5). Viral oncoproteins that bind to p53 and inhibit its activity do not interact with p73 (6, 7). Furthermore, the expression level of p73 is not affected by exposure to DNA-damaging agents that increase p53 levels, indicating that the two proteins have distinct cellular functions (8).MDM2 (mouse double minutes 2) is an oncoprotein that is overexpressed in many human cancers (9). It is well established that MDM2 inactivates p53 activity through monomeric ubiquitination of lysine residues of p53 (10 -12). In contrast, although p73 binds to MDM2 as well, their interaction does not lead to the proteosomal degradation of p73 (13). Nevertheless, the binding of p73 to MDM2 results in a dramatic inhibition of p73 transactivating activity (14). It has been shown that the binding to a cofactor, p300/CREB-binding protein (CBP), 3 is required for the transcriptional activity of p73 and that MDM2 competes with p73 for the p300/CBP (15). In a previous report, we demonstrated that Amphiphysin IIb-1 sequesters nascent p73 proteins in the cytoplasm and thus inhibits its transcriptional activity (16).Using the yeast two-hybrid screening method, we identified a novel p73␤-binding protein, p19ras , which is an alternative splicing variant of c-H-ras (17). Proto-oncogen...

Analysis of K-Ras Interactions by Biotin Ligase Tagging

“…However, H-Ras has been shown to localize to cell nuclei in a cell cycle-dependent fashion (93). Moreover, p19 H-Ras has been shown to bind to both the tumor suppressor protein p73β and its repressor, MDM2, enhancing p73β signaling (94). While current data concerning K-Ras nuclear localization are unclear , it would seem worthwhile to examine its potential nuclear activities.…”

Ras proteins: paradigms for compartmentalised and isoform-specific signalling

“…Finally, an H-Ras splice variant, p19 ras , is localised to the cytosol and nucleus because it lacks the C-terminal HVR [107]. Nuclear p19 ras inhibits the transcriptional activity of the p53 homologue p73 through modulation of MDM2-p73 interactions [108].…”

TIP60 Represses Transcriptional Activity of p73β via an MDM2-bridged Ternary Complex

“…These PCR products were introduced into the pCRII-TOPO vector (Invitrogen) and subcloned into the pCDNA-HA vector between EcoRI and XbaI and then verified by DNA sequencing. pCDNA-MDM2 and pCDNA3-HA-p73␤ have been described previously (28,29).…”