Precise mapping of the tms1 binding site on p53

Wagner, P.; Fuchs, Annette; Prowald, Alexandra; Montenarh, Mathias; Nastainczyk, Wolfgang

doi:10.1016/0014-5793(95)01329-6

Cited by 11 publications

(12 citation statements)

References 32 publications

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“…This is in good agreement with our identi®ed p34 cdc2 binding domain spanning from amino acid 334 ± 339. Interestingly, the same domain has been identi®ed previously as binding domain of the ®ssion yeast protein tms1 (Wagner, 1994;Wagner et al, 1995b) which had been isolated as a multi copy suppressor of a p53-induced growth arrest in ®ssion yeast (Wagner et al, 1991(Wagner et al, , 1993. In addition, the corresponding region was found to be critical for the association of the b-subunit of protein kinase CK2 with p53 suggesting the identi®cation of an important new functional domain on the p53 protein (Wagner et al, 1994a;Appel et al, 1995).…”

Section: Discussionmentioning

confidence: 75%

“…This method allows the rapid solid phase synthesis of peptides in equal molar quantities immobilised on a cellulose membrane in a format suitable for systematic analysis of proteinprotein interactions or antibody epitope mapping. This method had already proven successful for the mapping of the p53 binding site on the tms1 protein (Wagner, 1994;Wagner et al, 1995a) and vice versa (Wagner et al, 1995b) as well as the precise de®nition of the epitopes of three dierent monoclonal antibodies raised against recombinant tms1 protein (Wagner et al, 1994b;Schneider et al, 1995). First we synthesized a set of 11 overlapping peptides of 10 amino acid long sections of the p53 primary amino acid sequence spanning the p34 cdc2 binding region from amino acid 285 ± 344.…”

Section: The P34mentioning

confidence: 99%

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Fine mapping and regulation of the association of p53 with p34cdc2

et al. 1998

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In vivo p53 is multiply phosphorylated by dierent protein kinases suggesting a central role for phosphorylation in modulating p53 function. In addition, p53 was found to be associated with two protein kinases, p34 cdc2 and protein kinase CK2. Here we report the precise mapping of the interaction sites of p53 ± p34 cdc2 complexes. The p34 cdc2 binding site on human p53 maps to one distinct C-terminal site LQIRGRERFE (aa 330 ± 339) close to the corresponding phosphorylation site at serine 315. In order to test whether phosphorylation of p53 might in¯uence the binding of p53 to p34 cdc2 phosphorylation mutants of the C-terminus of p53, which mimick permanent phosphorylation, were tested on their ability to bind to p34 cdc2 in vitro. Substitution of serine 315 (the p34 cdc2 phosphorylation site) with aspartic acid had only little eect on complex formation whereas an exchange of serine 392 (the protein kinase CK2 phosphorylation site) to aspartic acid resulted in a signi®cant reduced relative binding anity of p53 to p34 cdc2 . The same result was obtained when the Cterminus of p53 was phosphorylated by puri®ed protein kinase CK2 prior to examination of complex formation. In addition, the speci®city of the complex formation has been checked by competition experiments with full length p53 proteins and the in¯uence of cyclin B on complex formation was examined.

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Section: Discussionmentioning

confidence: 75%

Section: The P34mentioning

confidence: 99%

Fine mapping and regulation of the association of p53 with p34cdc2

et al. 1998

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“…In addition, these residues are hydrophobic (Fig. 5, C and D) and have been proposed to be a potential site for interaction with other proteins (33,34). Residues Gly 334 and Arg 335 are also surface-exposed, and they belong to the turn that links the ␣-helices to the ␤-sheets.…”

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confidence: 99%

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

Kaustov

Lukin

Lemak

et al. 2007

Journal of Biological Chemistry

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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...

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“…For example, Lomax et al (1998) have shown that both L344P and R337C have reduced ability to bind to MDM2 which forms a negative feedback regulatory loop with p53. Other proteins such as tms1 (Wagner et al, 1995) and p34 cdc2 (Wagner et al, 1998) interact with residues in the tet domain spanning the R337C mutation site. The occurrence of these two mutations in familial cancers underlines the important role the tet domain plays in p53 function.…”

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confidence: 99%

Characterization of the oligomerization defects of two p53 mutants found in families with Li–Fraumeni and Li–Fraumeni-like syndrome

et al. 1998

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Recently two germline mutations in the oligomerization domain of p53 have been identi®ed in patients with Li ± Fraumeni and Li ± Fraumeni-like Syndromes. We have used biophysical and biochemical methods to characterize these two mutants in order to better understand their functional defects and the role of the p53 oligomerization domain (residues 325 ± 355) in oncogenesis. We ®nd that residues 310 ± 360 of the L344P mutant are monomeric, apparently unfolded and cannot interact with wild-type (WT) p53. The full length L344P protein is unable to bind sequence speci®cally to DNA and is therefore an inactive, but not a dominant negative mutant. R337C, on the other hand, can form dimers and tetramers, can hetero-oligomerize with WTp53 and can bind to a p53 consensus element. However, the thermal stability of R337C is much lower than that of WTp53 and at physiological temperatures more than half of this mutant is less than tetrameric. Thus, the R337C mutant retains some functional activity yet leads to a predisposition to cancer, suggesting that even partial inactivation of p53 oligomerization is su cient for accelerated tumour progression.

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Precise mapping of the tms1 binding site on p53

Cited by 11 publications

References 32 publications

Fine mapping and regulation of the association of p53 with p34cdc2

Fine mapping and regulation of the association of p53 with p34cdc2

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

Characterization of the oligomerization defects of two p53 mutants found in families with Li–Fraumeni and Li–Fraumeni-like syndrome

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