contributed equally to this workThe tumor suppressor p53 is activated in response to many types of cellular and environmental insults via mechanisms involving post-translational modi®cation.Here we demonstrate that, unlike phosphorylation, p53 invariably undergoes acetylation in cells exposed to a variety of stress-inducing agents including hypoxia, anti-metabolites, nuclear export inhibitor and actinomycin D treatment. In vivo, p53 acetylation is mediated by the p300 and CBP acetyltransferases. Overexpression of either p300 or CBP, but not an acetyltransferase-de®cient mutant, ef®ciently induces speci®c p53 acetylation. In contrast, MDM2, a negative regulator of p53, actively suppresses p300/CBPmediated p53 acetylation in vivo and in vitro. This inhibitory activity of MDM2 on p53 acetylation is in turn abrogated by tumor suppressor p19 ARF , indicating that regulation of acetylation is a central target of the p53±MDM2±p19 ARF feedback loop. Functionally, inhibition of deacetylation promotes p53 stability, suggesting that acetylation plays a positive role in the accumulation of p53 protein in stress response. Our results provide evidence that p300/CBP-mediated acetylation may be a universal and critical modi®-cation for p53 function. Keywords: acetylation/CBP/MDM2/p300/p53 IntroductionThe tumor suppressor p53 plays a critical role in human cancer formation. In response to a variety of stress signals, often associated with the progression of neoplastic diseases, p53 becomes activated and induces cell cycle arrest and/or programmed cell death (apoptosis). By eliminating damaged and potentially dangerous cells that might otherwise become cancerous, p53 suppresses tumor formation. In unstressed cells, p53 is latent and is maintained at low levels by targeted degradation mediated by its negative regulator, MDM2 (reviewed in Freedman et al., 1999). The critical role of MDM2 in regulating p53 is best illustrated by a study carried out in mice where inactivation of p53 was shown to completely rescue the embryonic lethality caused by the loss of MDM2 function (Montes de Oca Luna et al., 1995). MDM2 counteracts p53 tumor suppressor activity by physically binding to p53 and suppressing its transcriptional activity. MDM2 also functions as the p53 ubiquitin ligase and triggers its degradation (reviewed in Freedman et al., 1999). This latter activity requires the Ring ®nger domain located at the C-terminus of MDM2 (Fang et al., 2000), and may also involve the acetyltransferase p300, which binds both MDM2 and p53 (Grossman et al., 1998). Therefore, MDM2 negatively regulates p53 by at least two independent mechanisms.The activation and stabilization of p53 are thought to be mediated by speci®c protein modi®cations, with phosphorylation being the major focus of earlier studies (reviewed in Giaccia and Kastan, 1998;Appella and Anderson, 2000). Although the exact functions of speci®c phosphorylation events remain controversial, evidence indicates that they probably contribute to both the stabilization and activation of p53. For ex...

The ubiquitin-protein ligase (E3), hRPF1/Nedd4, is a component of the ubiquitin-proteasome pathway responsible for substrate recognition and specificity. Although previously characterized as a regulator of the stability of cytoplasmic proteins, hRPF1/Nedd4 has also been suggested to have a role in the nucleus. However, in light of the cytoplasmic localization of hRPF1/Nedd4, it is unclear whether bona fide nuclear substrates of hRPF1/Nedd4 exist, and if so, what mechanism may allow a cytoplasmic ubiquitin ligase to manifest nuclear activity. Our search for nuclear substrates led to the identification of the human proline-rich transcript, brain-expressed (hPRTB) protein, the ubiquitination and degradation of which is regulated by hRPF1/Nedd4. Interestingly, hPRTB colocalizes with the splicing factor SC35 in nuclear speckles. Finally, we demonstrate that hRPF1/Nedd4 is indeed capable of entering the nucleus; however, the presence of a functional Rev-like nuclear export sequence in hRPF1/Nedd4 ensures a predominant cytoplasmic localization. Cumulatively, these findings highlight a nuclear role for the ubiquitin ligase hRPF1/Nedd4 and underscore cytoplasmic/nuclear localization as an important regulatory component of hRPF1/Nedd4-substrate recognition.The posttranslational modification of proteins by ubiquitination has been shown to play an important role in the regulation of cell cycle progression, signal transduction, and transcriptional events within the cell. Covalent attachment of the 76-amino acid polypeptide ubiquitin to a substrate protein is a catastrophic signal, targeting the substrate for rapid degradation (1, 2). The specific enzymes involved in this process, E1, 1 E2, and E3, have been studied in great detail (3). A human ubiquitin-activating enzyme (E1) is responsible for the ATPdependent activation of the ubiquitin polypeptide. Activated ubiquitin is subsequently transferred to a downstream ubiquitin carrier protein (E2), and in many cases to a ubiquitinprotein isopeptide ligase (E3), which mediates the final transfer of activated ubiquitin to a substrate protein. Evidenced by the numerous examples of cellular dysregulation resulting from aberrant ubiquitination (4, 5), this ultimate enzyme-substrate recognition step is crucial for cellular homeostasis. Accordingly, there is of late a heightened level of interest in defining the mechanisms that govern the target specificity of the various E3 ligases and how this event is regulated in target cells.The experiments that have formed the foundation of our understanding of the role of E3 ligases were those that describe E6-associated protein and its ability to cooperate with the viral E6 protein in ubiquitinating p53 following human papillomavirus infection (6). This work led to the discovery of a family of proteins with sequence homology to E6-associated protein, the homology to E6-associated protein at the carboxyl terminus (hect) family of proteins (7), and the observation that the amino terminus is the primary determinant of target specificity. Recent w...

Heterotrimeric G proteins transduce signals from activated transmembrane G protein-coupled receptors to appropriate downstream effectors within cells. Signaling specificity is achieved in part by the specific ␣, ␤, and ␥ subunits that compose a given heterotrimer. Additional structural and functional diversity in these subunits is generated at the level of posttranslational modification, offering alternate regulatory mechanisms for G protein signaling. Presented here is the identification of a variant of the ␥ 2 subunit of G protein heterotrimer purified from bovine brain and the demonstration that this RDTASIA ␥2 variant, containing unique amino acid sequence at its N terminus, is a substrate for ubiquitylation and degradation via the N-end rule pathway. Although N-end-dependent degradation has been shown to have important functions in peptide import, chromosome segregation, angiogenesis, and cardiovascular development, the identification of cellular substrates in mammalian systems has remained elusive. The isolation of RDTASIA ␥2 from a native tissue represents identification of a mammalian N-end rule substrate from a physiological source, and elucidates a mechanism for the targeting of G protein ␥ subunits for ubiquitylation and degradation.T he G protein heterotrimer, composed of an ␣, ␤, and ␥ subunit, is complexed with the nucleotide GDP in its inactive state. Upon activation, GDP is exchanged for GTP, resulting in subunit dissociation, and the GTP-bound ␣ or the ␤␥ dimer can both subsequently modulate the activity of a variety of downstream effectors (1, 2). The ␥ subunit, although generally associated in the cell with a ␤ subunit, is the smallest and most variable heterotrimeric G protein subunit (3, 4) and has been the least well understood with regard to its specific biological function. Several well established lipid modifications of ␥ subunits include farnesylation or geranylgeranylation of the C terminus (5, 6), and these modifications play an important role in membrane association, receptor coupling, and effector regulation (7-9). Recent work suggests a role for the ␥ subunit of transducin in ubiquitylation and destabilization of G t ␤␥ dimers (10). Although it is unclear whether such observations can be extended to other distinct ␥ isoforms, the crucial question of how a ␥ subunit could be targeted to the ubiquitylation machinery in a regulated fashion also remains unanswered. The amino terminal region of ␥ subunits is the region most divergent between the 12 known ␥ isoforms (11), an observation which suggests that the first 20 amino acids may be crucial for a specific function or subcellular localization. Indeed, presented here is the identification of an N-terminal variant of the G␥ 2 subunit isolated from bovine brain. Our discovery that this RDTASIA ␥ 2 variant undergoes N-end rule degradation highlights regulated degradation as an important function for the ␥ subunit of the G protein heterotrimer, offering a mechanism by which G protein subunits can be targeted to enzymatic components of the...