The Conserved CPH Domains of Cul7 and PARC Are Protein-Protein Interaction Modules That Bind the Tetramerization Domain of p53

Abstract

Cul7 is a member of the Cullin Ring Ligase (CRL) family and is required for normal mouse development and cellular proliferation. Recently, a region of Cul7 that is highly conserved in the p53-associated, Parkin-like cytoplasmic protein PARC, was shown to bind p53 directly. Here we identify the CPH domains (conserved domain within Cul7, PARC, and HERC2 proteins) of both Cul7 and PARC as p53 interaction domains using size exclusion chromatography and NMR spectroscopy. We present the first structure of the evolutionarily conserved CPH domain and provide novel insight into the Cul7-p53 interaction. The NMR structure of the Cul7-CPH domain reveals a fold similar to peptide interaction modules such as the SH3, Tudor, and KOW domains. The p53 interaction surface of both Cul7 and PARC CPH domains was mapped to a conserved surface distinct from the analogous peptide-binding regions of SH3, KOW, and Tudor domains, suggesting a novel mode of interaction. The CPH domain interaction surface of p53 resides in the tetramerization domain and is formed by residues contributed by at least two subunits.The ubiquitin-proteosome system plays an important role in controlling diverse biological processes, ranging from signal transduction to cell cycle control (1, 2). These complex processes are controlled via specific degradation of individual or groups of proteins. Protein degradation via the ubiquitin pathway involves two successive steps: tagging of the substrate by covalent attachment of multiple ubiquitin molecules (ubiquitylation) and degradation of the tagged protein by 26 S proteosome complex with release of free and reusable ubiquitin (3).Ubiquitylation is the ultimate result of coordinated activity of an enzymatic cascade, which includes a ubiquitin-activating enzyme (E1), 3 a ubiquitin-conjugating enzyme (E2), and ubiquitin-ligating (E3) enzymes. The E3 ligases are the "brain" of this process and determine substrate specificity (4, 5).Cul7, is a recently identified member of the Cullin family of ubiquitin E3 ligases, localizes predominantly in the cytoplasm (6), and forms a unique Skp1-Cul7-Fbx29-like complex with FBXW8, a WD40 containing F-box protein (7-9). Although a target substrate for FBXW8 has not been yet identified, Cul7 recruits RBX1 to form a Skp1-Cul7-Fbx29-like E3 ubiquitin ligase complex (7,8). The biological function of Cul7 is unclear.However, Cul7 appears to play an important role in development (8, 9), and overexpression of Cul7 accelerates the rate of cell proliferation (6).Cul7 has significant sequence similarity (see Fig. 1) with the p53-associated, Parkin-like cytoplasmic protein, PARC (10). Both proteins contain CPH (domain that is conserved in Cul7, PARC, and HERC2 proteins) (11), DOC (DOC1/APC10), and Cullin homology domains (see Fig. 1A) that are linked with E3 ligase function, suggesting that PARC and Cul7 may both function as E3 ubiquitin ligases. PARC has been shown to sequester p53 in the cytoplasm via interaction between the N terminus of PARC and the C terminus of p53 (10). Cul7 may perform f...

Induction of Cullin 7 by DNA damage attenuates p53 function

“…While this manuscript was in preparation, a direct interaction between ectopic p53 and Cul7 was reported by others (24)(25)(26), which confirms our findings. Unlike the interaction between PARC and p53 (19), the Cul7/ p53 interaction does not seem to influence p53 localization (24).…”

“…Because several E3 ligases have been shown to mono-and di-ubiquitinate p53 (11,(13)(14)(15), it is possible that immunoprecipitates of Cul7/p53 contain other E3-ligases responsible for the in vitro ubiquitination of p53 observed by Andrews et al (24). The association of Cul7 with p53 was shown to occur with oligomeric forms of p53 (26) and tetrameric p53 is less efficiently imported into the nucleus than its monomeric form (31). Ectopic Cul7 interfered with p53 activation in response to DNA damage when cytoplasmic and nuclear p53 levels are increased.…”

The E3 Ubiquitin Protein Ligase HERC2 Modulates the Activity of Tumor Protein p53 by Regulating Its Oligomerization

“…The HERC2-binding domain of p53 was located in its carboxyl terminus, similar to other p53-binding proteins with CPH domains, such as PARC and CUL7 (6 -8). Structural studies of the CPH domain of PARC and CUL7 indicated that this domain interacts with the tetramerization domain of p53 (residues 310 -360) (49). In agreement with these data, deletion of the last 43 amino acid residues of p53 (residues 350 -393) impaired the interaction with HERC2 (Fig.…”

Solution structure of the carboxy‐terminal Tudor domain from human Coilin

“…This diverse fold bears many similarities to KOW [24], CPH [25] and SH3 domains [26], of which some are shown in Fig. 4 for comparison.…”