Characterizing Class I WW domains defines key specificity determinants and generates mutant domains with novel specificities

Kasanov, Jeremy; Pirozzi, Gregorio; Uveges, Albert J.; Kay, Brian K.

doi:10.1016/s1074-5521(01)00005-9

Cited by 97 publications

(83 citation statements)

References 65 publications

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“…The fact that neither p53 nor JNK was identified in our experiments may indicate that the cDNA clones encoding these proteins are not represented in the high-density protein array. It is worth noting, however, that the p53 proline-rich region does not contain any PPXY motifs, and it has been well documented that ligand preference by the various known WW domains is 'highly specific' (Kay et al, 2000;Sudol and Hunter, 2000;Kasanov et al, 2001;Otte et al, 2003). Indeed, we have presented evidence that the WWOX WW domain specifically binds the PPXY motif.…”

Section: Discussionmentioning

confidence: 59%

“…Indeed, we have presented evidence that the WWOX WW domain specifically binds the PPXY motif. Moreover, WWOX binding to two candidate interacting proteins was demonstrated to have preference for one of two PPXY motifs within their sequence, demonstrating that a higher level of specificity also exists, as has been observed for many other Group I WW domains (Kasanov et al, 2001). It remains to be determined which p53 and JNK1 amino acids directly interact with WWOX as they may represent novel WW domain interaction motifs.…”

Section: Discussionmentioning

confidence: 84%

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WWOX binds the specific proline-rich ligand PPXY: identification of candidate interacting proteins

et al. 2004

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WWOX, the gene that maps to common chromosomal fragile site FRA16D, is frequently affected by aberrations in multiple types of cancers. WWOX encodes a 46 kDa protein that contains two WW domains and a short-chain oxidoreductase (SDR) domain. We recently demonstrated that ectopic expression of WWOX inhibits xenograft tumor growth of tumorigenic breast cancer cells. Little is known of the biochemical function(s) of WWOX. The SDR domain is predicted to be involved in sex-steroid metabolism and the WW domains are likely involved in protein-protein interactions. In this report, we identify the specific proline-rich ligand for WWOX as PPXY and show that the amino-terminal WW domain is responsible for this interaction. Using the WWOX WW domains as a probe, we screened high-density protein arrays and identified five candidate-binding partners. The binding to one of these candidates, small membrane protein of the lysosome/late endosome (SIMPLE), was further analysed, and we observed that a specific PPSY motif in the SIMPLE amino-acid sequence was required to interact with the amino-terminal WW domain of WWOX. In addition, immunofluorescence staining demonstrated that endogenous WWOX and SIMPLE co-localize to perinuclear compartments of MCF-7 human breast cancer cells. These studies demonstrate that WWOX contains a Group I WW domain that binds known cellular proteins containing the specific ligand PPXY. Identification and characterization of WWOX interacting proteins will lead to an understanding of the biological functions of WWOX in normal and tumor cells.

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Section: Discussionmentioning

confidence: 59%

Section: Discussionmentioning

confidence: 84%

WWOX binds the specific proline-rich ligand PPXY: identification of candidate interacting proteins

et al. 2004

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“…3). Unlike known Nedd4-1 substrates, including LAPTM5, which typically contain PY motifs (27)(28)(29)(30)32), PTEN does not possess this motif, possibly explaining the observed lack of its interaction with Nedd4-1 ( Fig. 3 C and D) and unaffected PTEN ubiquitination in Nedd4-1-deficient cells (Fig.…”

Section: Discussionmentioning

confidence: 97%

“…The WW domain of Nedd4 proteins recognizes and binds a short amino acid sequence in substrate proteins, called the PY motif (L/PPxY) (26)(27)(28)(29)(30). By using two independent approaches, we recently generated distinct strains of mice with disrupted Nedd4-1 gene, leading to a complete loss of Nedd4-1 protein and embryonic lethality at mid to late gestation (F.F.…”

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confidence: 99%

The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization

Fouladkou

Landry²,

Kawabe

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

165

140

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PTEN is a tumor suppressor frequently mutated in cancer. Recent reports implicated Nedd4-1 as the E3 ubiquitin ligase for PTEN that regulates its stability and nuclear localization. We tested the physiological role of Nedd4-1 as a PTEN regulator by using cells and tissues derived from two independently generated strains of mice with their Nedd4-1 gene disrupted. PTEN stability and ubiquitination were indistinguishable between the wild-type and Nedd4-1-deficient cells, and an interaction between the two proteins could not be detected. Moreover, PTEN subcellular distribution, showing prominent cytoplasmic and nuclear staining, was independent of Nedd4-1 presence. Finally, activation of PKB/Akt, a major downstream target of cytoplasmic PTEN activity, and the ability of PTEN to transactivate the Rad51 promoter, a measure of its nuclear function, were unaffected by the loss of Nedd4-1. Taken together, our results fail to support a role for Nedd4-1 as the E3 ligase regulating PTEN stability and subcellular localization.E3 ligase ͉ tumor suppressor ͉ P13K signal ͉ WW domain P TEN is one of the most frequently mutated genes in human cancer (1-3). In the cytoplasm, PTEN functions as a lipid phosphatase by dephosphorylating the D3 position of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ], and directly antagonizing PI3K (4-6). Consistent with a negative regulatory role for PTEN in regulation of PI3 signaling, PTEN-deficient cells and tissues exhibit defects in cell proliferation, growth, survival, death, protein translation, metabolism, migration, and structural organization (2,3,7,8).One of the most intriguing features of PTEN is its subcellular localization. Although PTEN was originally found to be a cytoplasmic protein (9), its nuclear localization in many cell types has been reported by using various independently developed monoclonal and polyclonal anti-PTEN antibodies (10)(11)(12)(13)(14). A number of attempts have been made to uncouple the cytoplasmic and nuclear roles of PTEN, yielding incongruous results. For example, differing accounts of the respective levels of cytoplasmic and nuclear PTEN throughout the stages of the cell cycle have been documented (15, 16). Functionally, nuclear PTEN has been shown to induce cell cycle arrest in certain cell types (17), whereas in others, nuclear accumulation of PTEN increased upon stimulation with proapoptotic factors and correlated with induction of apoptosis (18). Finally, nuclear-targeted PTEN was shown to impede the growth of U251MG glioblastoma cells and down-regulate p70S6K in a PKB/Akt-independent manner but was unable to inhibit cell invasion (19).Recently, published work revealed a phosphatase activityindependent nuclear role for PTEN in the regulation of chromosomal stability and repair of DNA damage (20). Wang et al. (21) and Trotman et al. (22) reported that PTEN stability and nuclear translocation were regulated by ubiquitination mediated by the ubiquitin ligase Nedd4-1. Although the Nedd4-1-mediated polyubiquitination of PTEN in the cytosol caused ...

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“…WWP1 shares a characteristic domain organization with the E3 ligases Nedd4 and Smurfs, which consists of a C2 domain, 2-4 WW domains, and a HECT domain (Kasanov et al, 2001). Although WWP1 has been shown to function as an E3 ubiquitin ligase, only few substrates have been identified.…”

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confidence: 99%

Regulation of p53 localization and transcription by the HECT domain E3 ligase WWP1

2006

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As a key cellular regulatory protein p53 is subject to tight regulation by several E3 ligases. Here, we demonstrate the role of HECT domain E3 ligase, WWP1, in regulating p53 localization and activity. WWP1 associates with p53 and induces p53 ubiquitylation. Unlike other E3 ligases, WWP1 increases p53 stability; inhibition of WWP1 expression or expression of a ligase-mutant form results in decreased p53 expression. WWP1-mediated stabilization of p53 is associated with increased accumulation of p53 in cytoplasm with a concomitant decrease in its transcriptional activities. WWP1 effects are independent of Mdm2 as they are seen in cells lacking Mdm2 expression. Whereas WWP1 limits p53 activity, p53 reduces expression of WWP1, pointing to a possible feedback loop mechanism. Taken together, these findings identify the first instance of a ubiquitin ligase that causes stabilization of p53 while inactivating its transcriptional activities.

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Characterizing Class I WW domains defines key specificity determinants and generates mutant domains with novel specificities

Abstract: Class I WW domains contain a highly conserved set of residues that are important in selecting Pro-Xxx-Tyr containing peptide ligands. The presence of these residues within an uncharacterized WW domain can be used to predict its ability to bind Pro-Xxx-Tyr containing peptide ligands.

Cited by 97 publications

References 65 publications

WWOX binds the specific proline-rich ligand PPXY: identification of candidate interacting proteins

WWOX binds the specific proline-rich ligand PPXY: identification of candidate interacting proteins

The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization

Regulation of p53 localization and transcription by the HECT domain E3 ligase WWP1

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