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Regulation of P53 Localization and Transcription by the HECT Domain E3 Ligase WWP1

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Regulation of P53 Localization and Transcription by the HECT Domain E3 Ligase WWP1 Oncogene (2007) 26, 1477–1483 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc SHORT COMMUNICATION Regulation of p53 localization and transcription by the HECT domain E3 ligase WWP1 A Laine and Z Ronai Signal Transduction Program, The Burnham Institute for Medical Research, La Jolla, CA, USA As a key cellular regulatory protein p53 is subject to tight 2003; Doran et al., 2004; Shmueli and Oren, 2005; regulation by several E3 ligases. Here, we demonstrate the Harris and Levine, 2005). Common to these ligases is role of HECT domain E3 ligase, WWP1, in regulating p53 their role in limiting p53 stability and activity. In localization and activity. WWP1 associates withp53 and searching for protein ligases with different effects on p53 induces p53 ubiquitylation. Unlike other E3 ligases, activity, we identified the HECT domain E3 ligase WWP1 increases p53 stability; inhibition of WWP1 WWP1, whose interaction with p53 increases its stability expression or expression of a ligase-mutant form results while reducing its transcriptional activity. in decreased p53 expression. WWP1-mediated stabilization WW domain-containing protein 1 (WWP1) was first of p53 is associated withincreased accumulation of p53 in identified as a novelprotein based on its WW modules– cytoplasm witha concomitant decrease in its transcrip- a 35–40 amino-acid (aa) region exhibiting high affinity tional activities. WWP1 effects are independent of Mdm2 towards the PY motif, a proline-rich sequence followed as they are seen in cells lacking Mdm2 expression. by a tyrosine residue (Verdecia et al., 2003). WWP1 Whereas WWP1 limits p53 activity, p53 reduces expres- shares a characteristic domain organization with the E3 sion of WWP1, pointing to a possible feedback loop ligases Nedd4 and Smurfs, which consists of a C2 mechanism. Taken together, these findings identify the first domain, 2–4 WW domains, and a HECT domain instance of a ubiquitin ligase that causes stabilization of (Kasanov et al., 2001). Although WWP1 has been p53 while inactivating its transcriptional activities. shown to function as an E3 ubiquitin ligase, only few Oncogene (2007) 26, 1477–1483. doi:10.1038/sj.onc.1209924; substrates have been identified. Among these are published online 21 August 2006 Kru¨ ppel-like factors (Conkright et al., 2001; Zhang et al., 2004; Chen et al., 2005) and Smad7 (Moren et al., Keywords: p53; WWP1; ubiquitin; HECHT; E3 ligases; 2005), which have been shown to be regulated by transcription WWP1. Additionally, it has been reported that WWP1 is essentialfor embryonic development in Caenorhabdi- tis elegans (Huang et al., 2000). WWP1 is a member of the Nedd4 family of E3 ligases, The tumor-suppressor protein p53 is a primary coordi- which includes Nedd4, Itch and WWP2 (Sudol and nator of cellular responses to a wide range of stresses Hunter, 2000; Ingham et al., 2004). Members of the (Oren, 2003). After genotoxic stress, p53 is rapidly Nedd4 family have been shown to be involved in activated and either promotes cell growth arrest or regulating cellular signaling and protein sorting (Galinier apoptosis, depending on the type, strength and duration et al., 2002). Nedd4 was initially found to regulate cell of the stimulus (reviewed by Oren (2003), Sengupta and surface stability of the epithelial sodium channel (ENaC) Harris (2005) and Poyurovsky and Prives (2006)). Under (Staub et al., 1996). Itch has been shown to be regulated normalgrowth conditions, however, the levelofp53 by JNK and to play a role in TNF signaling (Chang expression and activity is kept under tight control, et al., 2006). Additionally, Itch has been shown to preventing its activity under nonwarranted conditions. interact with, ubiquitinate, and degrade p73 (Rossi et al., Such tight regulation is for the most part mediated by 2005). WWP2 has been shown to regulate the stability of limiting p53 stability by one of several ubiquitin ligases, the Oct-4 transcription factor, a master regulator including Mdm2, ARF-BP1, Pirh1 and the component affecting the fate of pluripotent embryonic stem cells of the signalosome, Cop9, which efficiently targets p53 (Xu et al., 2004). WWP1, WWP2 and Itch have been to ubiquitylation and proteasome-dependent degrada- implicated in vascular protein sorting, which is exploited tion (Haupt et al., 1997; Kubbutat et al., 1997; Ashcroft by enveloped viruses (Martin-Serrano et al., 2005). and Vousden, 1999; Brooks and Gu, 2003; Leng et al., Earlier studies have implicated the role of the proline- rich domain of p53 in its activities (Walker and Levine, Correspondence: Professor Z Ronai, SignalTransduction Program, 1996; Zhu et al., 2000). For example, the phosphoryla- The Burnham Institute for Medical Research, La Jolla, CA, 92037, tion of Thr81 within this domain has been shown to be USA. E-mail: [email protected] important in recruitment of the prolyl isomerase Pin1 Received 15 March 2006; revised 13 July 2006; accepted 14 July 2006; (Zacchi et al., 2002), which contributes to p53 stability published online 21 August 2006 and activity following stress. In searching for E3 ligases Regulation of p53 localization and transcription A Laine and Z Ronai 1478 that may associate with the proline-rich domains domain (aa 97–116), p53Dp7, still efficiently bound to (Bedford et al., 2000; Verdecia et al., 2000), we have WWP1. This observation suggests that the conforma- compared different members of the four WW-domains tion of p53, generated in the presence of the proline-rich for their association with p53. Among those, WWP1 domain, is important for efficient association with exhibited the strongest association (see data below). To WWP1. As WWP1 is part of a family of HECT-E3 map the region on p53 required for interaction with ligases that contain a C-terminal HECT domain and WWP1, we performed a GST-pull down assay. Surpris- N-terminalWW domains, we next determined whether ingly, WWP1 associated, not with the proline-rich other members of the Nedd4 family can also associate region (1–100) but rather with the DNA-binding region with p53. GST-pull down assay using full-length p53 of p53 (100–295) (Figure 1a). However, the proline-rich and the four members of the Nedd4 family revealed that region was required for efficient interaction in vivo, WWP1 displayed the strongest association. WWP2 because a mutant of p53 that lacks this region (aa showed weaker association whereas Nedd4 and Itch 68–91), p53D6, showed a weaker association with WWP1 showed no association (Figure 1c). These data suggest in a co-immunoprecipitation assay (Figure 1b). Another that WWP1 is the primary Nedd4 family member that mutant which lacks a region downstream of the proline associates with p53 and that both the DNA-binding and ∆ a b p53 p7 ---+ ∆ p53 p6 - - + - p53wt ---+ Flag-WWP1 ++++ IB : Flag IP : his 10% InputGST-p53GST-p53 FL GST-p53 1-100GST-p53 100-295GST-p53 295-393 100-393 IB : his 35S-WWP1 Autorad kDa IB : Flag 100 IP : Flag 73 IB : his 54 Coomassie 48 IB : Flag WCE 35 IB : his 12 34 5 1234 c 35S-Nedd4 + ++ 35S-Itch +++ 35S-WWP2 + ++ + 35S-WWP1 +++ kDa 130 100 Autorad 72 10% Input Coomassie GST GST-p53 FL Figure 1 WWP1 associates with p53. (a) In vitro GST pull down between 35S-radiolabeled WWP1 and either full-length p53 or different fragments of p53. Top panelis autoradiograph of bound WWP1 and lowerpanelis coomassie stain of GST proteins. (b) In vivo association. In p53À/À/Mdm2À/À MEFs either his-tagged wild-type p53 or p53D6 (deleted of aa 68-91) or p53D7 (deleted of aa 97–116) was co-transfected with Flag-WWP1. Proteins were precipitated using either anti-his or anti-flag antibodies and samples separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Western blot was performed to detect the respective proteins. (c) In vitro GST pull down between GST-p53 and other HECT E3 ligases. GST or GST-p53 full-length was incubated with 35S-radiolabeled WWP1, WWP2, Itch or Nedd4. Top panel is autoradiograph of bound E3 ligases and lower panel is coomassie stain of GST proteins. Oncogene Regulation of p53 localization and transcription A Laine and Z Ronai 1479 the proline-rich domains of p53 are required for this possible role of Mdm2, p53À/À/Mdm2À/À mouse embryo association. fibroblasts (DKO MEFs) were used. Coexpression of As WWP1 is an E3 ligase, we next determined the WWP1 with p53 in the DKO MEFs increased p53 possible role of WWP1 in p53 stability. To exclude the steady-state levels (Figure 2a). A ligase-dead mutant of a b MG132 Flag-WWP1mut - --++ Flag-WWP1mut - - - - - 1.0 Flag-WWP1wt ++- -- Flag-WWP1wt - --0.1 1.0 2.0 p53wt - + +++ p53wt -+++++ IB : p53 IB : p53 IB : Flag IB : Flag IB : GFP IB : GFP 123456 12345 c d MG132 Time 0 1230123 Flag-WWP1mut - - 1.0 2.02.0 Flag-WWP1wt ----++++ Flag-WWP1wt ----1.0 p53wt ++++++++ IB : p53 Autorad IB : Flag IB : Flag IB : hdm2 IB : GFP IB : GFP 12345678 12345 e f NT pS-controlpS-WWP-99 IB : WWP1 pS-controlpS-WWP-98pS-WWP-2663 pS-controlpS-WWP-98pS-WWP-2663 p53wt +++ Flag-WWP1wt +++ IB : p53 IB : Flag IB : p53 IB : GFP IB : GFP 123 12 3 IB : p21 IB : β-actin 123 Figure 2 Effect of WWP1 on p53. (a) WWP1 increases p53 protein levels. In p53À/À/Mdm2À/À MEFs, p53 was co-transfected with either wild-type or mutant WWP1. At 24 h after transfection, some cells were treated with 40 mM of MG132 for 4 h before harvesting. Proteins were separated by SDS–PAGE and analysed by Western blot. The band corresponding to p53 is indicated by the arrow. (b) WWP1 acts in a dose-dependent manner. In p53À/À/Mdm2À/À MEFs, increasing amounts of WWP1 (mg, indicated in figure) were co-transfected with a constant amount of p53.
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