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LNX functions as a RING type E3 ubiquitin ligase that targets the cell fate determinant Numb for ubiquitin-dependent degradation

2002; Springer Nature; Volume: 21; Issue: 1 Linguagem: Inglês

10.1093/emboj/21.1.93

1460-2075

Jing Nie,

Cancer-related Molecular Pathways

Article15 January 2002free access LNX functions as a RING type E3 ubiquitin ligase that targets the cell fate determinant Numb for ubiquitin-dependent degradation Jing Nie Jing Nie The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author Melanie A. McGill Melanie A. McGill The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author Matt Dermer Matt Dermer The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author Sascha E. Dho Sascha E. Dho The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author Cheryl D. Wolting Cheryl D. Wolting The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author C. Jane McGlade Corresponding Author C. Jane McGlade The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author Jing Nie Jing Nie The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author Melanie A. McGill Melanie A. McGill The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author Matt Dermer Matt Dermer The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author Sascha E. Dho Sascha E. Dho The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author Cheryl D. Wolting Cheryl D. Wolting The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author C. Jane McGlade Corresponding Author C. Jane McGlade The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author Author Information Jing Nie1, Melanie A. McGill1, Matt Dermer1, Sascha E. Dho1, Cheryl D. Wolting1 and C. Jane McGlade 1 1The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:93-102https://doi.org/10.1093/emboj/21.1.93 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info LNX is a RING finger and PDZ domain containing protein that interacts with the cell fate determinant Numb. To investigate the function of LNX, we tested its RING finger domain for ubiquitin ligase activity. The isolated RING finger domain was able to function as an E2-dependent, E3 ubiquitin ligase in vitro and mutation of a conserved cysteine residue within the RING domain abolished its activity, indicating that LNX is the first described PDZ domain-containing member of the E3 ubiquitin ligase family. We have identified Numb as a substrate of LNX E3 activity in vitro and in vivo. In addition to the RING finger, a region of LNX, including the Numb PTB domain-binding site and the first PDZ domain, is required for Numb ubiquitylation. Expression of wild-type but not mutant LNX causes proteasome-dependent degradation of Numb and can enhance Notch signalling. These results suggest that the levels of mammalian Numb protein and therefore, by extension, the processes of asymmetric cell division and cell fate determination may be regulated by ubiquitin-dependent proteolysis. Introduction The numb gene in Drosophila affects binary cell fate decisions of cells in the peripheral and central nervous system, as well as muscle cells during development (Uemura et al., 1989; Brewster and Bodmer, 1995; Spana and Doe, 1996; Ruiz and Bate, 1997; Park et al., 1998). Drosophila Numb (dNumb) is a membrane- associated protein expressed in progenitor cells of these lineages. During cell division, dNumb asymmetrically localizes and subsequently segregates to one daughter cell, where it functions as an intrinsic determinant of cell fate (Rhyu et al., 1994; Knoblich et al., 1995; Spana et al. 1995; Vervoort et al., 1997). Evidence suggests that, in Drosophila, Numb proteins influence cell fate by inhibiting the action of Notch (Frise et al., 1996; Guo et al., 1996; Spana and Doe, 1996). Conserved Numb genes have been identified in mouse, rat, chicken and human (Verdi et al., 1996; Zhong et al., 1996; Wakamatsu et al., 1999). In addition, a related gene called Nbl (Numblike) has also been identified in mammals (Zhong et al., 1997; Dho et al., 1998). Heterologous expression of mammalian Numb (mNumb) in Drosophila produces a phenotype (transformation of hair cells to neurons) similar to ectopic expression of dNumb, suggesting that these proteins have conserved functional properties (Verdi et al., 1996; Zhong et al., 1996). In addition, there is evidence that the vertebrate Numb protein is asymmetrically distributed in dividing neuroepithelial cells that undergo asymmetric cell divisions and can inhibit the ability of Notch to suppress neuronal differentiation (Zhong et al., 1996; Wakamatsu et al., 1999). Homozygous deletion of the Numb gene in mice results in early embryonic lethality (Zhong et al., 2000; Zilian et al., 2001). Together, these results suggest that the levels of Numb protein are important in regulating developmental processes controlled by Notch. However, the mechanisms that regulate the asymmetric distribution and the levels of the vertebrate Numb protein are currently unknown. The structure of both Drosophila and vertebrate Numb proteins suggests that they function as adaptor proteins to mediate the formation of multiprotein complexes. mNumb has an N-terminal phosphotyrosine-binding (PTB) domain that interacts with ligands in a phosphotyrosine-independent manner (Li et al., 1997, 1998; Dho et al., 1998; Yaich et al., 1998). mNumb PTB binding partners include Notch, mdm2 and LNX, although the biological importance of these interactions has not yet been established (Zhong et al., 1996; Dho et al., 1998; Juven-Gershon et al., 1998). Within its C-terminal region, Numb also contains an Eps15 homology (EH) domain-binding motif (Salcini et al., 1997; Paoluzi et al., 1998) and two tripeptide sequences that fit the binding motif for α-adaptin, a component of the AP-2 complex. Mammalian Numb has recently been demonstrated to localize to clathrin-coated pits and early endosomes, and overexpression of fragments of Numb can block internalization of the EGFR and transferrin receptors, implying a role for Numb in intracellular vesicle trafficking events (Santolini et al., 2000). Previously, LNX was identified as a mNumb PTB domain-interacting protein (Dho et al., 1998). In addition to the Numb PTB domain-binding motif, LNX contains four PDZ domains, which also presumably mediate protein–protein interactions although their targets are not yet identified. LNX also contains an N-terminal RING finger domain. RING finger domains are conserved, cysteine-rich, zinc-binding domains found in a diverse group of proteins that until recently appeared to be functionally unrelated (Borden and Freemont, 1996; Saurin et al., 1996). However, an accumulating body of evidence suggests that many RING finger-containing proteins are involved in protein ubiquitylation (Tyers and Willems, 1999; Jackson et al., 2000; Joazeiro and Weissman, 2000). The ubiquitylation pathway involves a multiprotein cascade in which the first step is the attachment of free ubiquitin to a ubiquitin-activating enzyme (E1). Ubiquitin is then transferred to a ubiquitin-conjugating enzyme (E2), which along with a ubiquitin ligase (E3) protein, ligates the ubiquitin to a specific protein target (Hershko and Ciechanover, 1998). The specificity of this process is determined by the E3 component, which is proposed to function as an adaptor to bind substrates selectively (Tyers and Willems, 1999; Joazeiro and Weissman, 2000). Ubiquitylated proteins are then targeted to the proteasome or, in some cases (as with some plasma membrane proteins), to the endocytic pathway, resulting, in either case, in the degradation of the protein (Hershko and Ciechanover, 1998; Hicke, 1999; Rotin et al., 2000). RING finger domain proteins Rbx1 or Apc11 function as components of ubiquitin ligase complexes such as Skp1–Cdc53/CUL1-F-box protein (SCF), anaphase-promoting complex (APC) and VHL–elonginC/elonginB (VCB), and facilitate the transfer of ubiquitin from E2 to the substrate (Tyers and Willems, 1999). Another large family of RING finger-containing proteins, such as Cbl, Siah-1 and mdm2, which are otherwise unrelated to Apc11 or Rbx1, also have RING finger-dependent E3 ubiquitin ligase activity (Hu et al., 1997; Joazeiro et al., 1999; Lorick et al., 1999). In light of the mounting evidence for E3-like activity of proteins with RING finger domains, we tested whether LNX could promote ubiquitin-mediated protein degradation. Here we show that LNX has E3 ubiquitin ligase activity and this activity requires an intact RING finger domain. Furthermore, we identify Numb as a LNX substrate and find that expression of LNX results in the ubiquitylation and degradation of Numb proteins. Results The LNX RING finger domain mediates E2-dependent ubiquitylation activity To determine whether LNX can function in a manner similar to the recently characterized RING finger domain-containing E3 ubiquitin ligases, recombinant glutathione S-transferase (GST)–LNX was used in an in vitro ubiquitylation assay. GST–LNX fusion proteins were incubated with ubiquitin, recombinant E1 and E2 (UbcH5B), and ubiquitin ligase activity measured by detection of ubiquitylated GST fusions proteins using anti-ubiquitin antibodies. E2-dependent ubiquitin ligase activity was detected in reactions containing pGST-LNXWT but not in reactions containing a form of LNX in which a conserved cysteine residue (C48) is mutated to alanine (LNXC48A; Figure 1A). The isolated RING finger domain also promoted E2-dependent E3 activity and mutation of C48 abolished this activity (Figure 1A). To further assess whether LNX has E3 activity in vivo, 293T cells were co-transfected with HA-tagged ubiquitin together with increasing amounts of FLAG-tagged LNXWT or LNXC48A. Anti-HA immunoblotting revealed a dose-dependent increase in the ubiquitylated proteins detected in LNXWT immunoprecipitates but not LNXC48A (Figure 1B). Figure 1.The LNX RING finger domain has E2-dependent ubiquitin ligase activity. (A) GS-bound GST fusions of LNXWT and the isolated RING finger domain or LNXC48A were incubated in an in vitro ubiquitylation reaction mixture in the absence (−) or presence (+) of E2 (UbcH5B) bacterial lysate for 90 min at room temperature. The reaction products were resolved by SDS–PAGE and ubiquitylated proteins detected by western blotting using anti-ubiquitin antibody. GS-bound fusion proteins or GST alone added to the reactions were quantitated by SDS–PAGE and stained with Coomassie Blue (data not shown). (B) In vivo E3 activity of LNX requires the RING finger. 293T cells were transiently co-transfected with HA-ubiquitin and either pFLAG-CMV-2 vector alone or the indicated amount of FLAG-tagged LNXWT or LNXC48A. Equivalent amounts of protein lysate were immunoprecipitated (IP) with anti-LNX antibody and immunoblotted (IB) with anti-HA monoclonal antibody to detect ubiquitylated proteins (top) or anti-LNX polyclonal antibody (bottom). Download figure Download PowerPoint LNX promotes the ubiquitylation of Numb LNX was initially identified as a Numb PTB domain-binding protein, therefore we tested whether Numb is a substrate for LNX-mediated ubiquitylation. The p72 isoform of Numb was transcribed and translated in vitro in the presence of [35S]methionine, and then incubated with recombinant E1, E2 (Ubc5B) and ubiquitin in the presence of purified GST or GST–wild-type or C48A LNXC48A fusion proteins. In the presence of wild-type GST–LNX, Numb was modified significantly, as indicated by the shift in migration of the input Numb protein into discrete bands and a high molecular weight smear (Figure 2A). The C48A mutant form of GST–LNX and GST alone did not cause a change in Numb mobility. To test whether LNX would promote Numb ubiquitylation in vivo, FLAG-tagged LNXWT or LNXC48A was co-transfected into 293T cells with HA-tagged ubiquitin. Endogenous Numb proteins were immunoprecipitated from cell lysates with anti-Numb antibody and immunoblotted with anti-HA antibody to detect ubiquitylated proteins. As shown in Figure 2B, a dose-dependent accumulation of ubiquitylated proteins was detected in Numb immunoprecipitates from LNXWT transfected cells but not LNXC48A transfected cells. To determine whether Numb itself is ubiquitylated, rather than Numb-associated proteins, the cell lysates were boiled in the presence of SDS to disrupt protein–protein interactions and then immunoprecipitated with anti-HA. Numb-reactive bands were detected in anti-HA immunoprecipitates from boiled lysates, indicating that Numb was directly modified by HA-ubiquitin in the presence of wild-type but not mutant LNX (Figure 2C). Furthermore, in the presence of a proteasome inhibitor, MG132, Numb reactivity could be detected as a high molecular weight smear, suggesting it had been modified by polyubiquitylation. Figure 2.Numb is a substrate for LNX-mediated ubiquitylation. (A) Numb is a LNX substrate in vitro. Numb was transcribed and translated in vitro in the presence of [35S]methionine using wheat germ extract. Radiolabelled Numb was added to an in vitro ubiquitylation reaction containing E1, E2 (UbcH5B) and ubiquitin in the presence of GST alone, GST–LNXWT or GST–LNXC48A. Reactions were separated by SDS–PAGE and the Numb proteins visualized by autoradiography. The position of unmodified Numb is indicated by an arrow and the ubiquitylated Numb by a bracket. (B) LNX promotes the ubiquitylation of Numb in mammalian cells. Numb protein was immunoprecipitated from half of the cell lysates from the 293T cells transiently co-transfected with pMT HA-ubiquitin and either pFLAG-LNXWT or LNXC48A used in Figure 1B and immunoblotted with anti-HA monoclonal antibody (top). The membrane was then stripped and reprobed with anti-Numb antibody (middle). Total cell lysates were blotted with anti-LNX to monitor LNX expression levels (bottom, as in Figure 1B). (C) 293T cells were transiently co-transfected with pMT HA-ubiquitin and either FLAG-CMV-2 vector alone, FLAG-tagged LNXWT or LNXC48A, which disrupts the RING finger domain. Some transfected cells were incubated overnight with 20 μM MG132 before harvest. Cells were lysed in 1 ml of modified HNTG-ZE lysis buffer containing 1% SDS and then boiled at 95°C for 5 min. The lysates were then diluted with 10 ml of HNTG-ZE lysis buffer and subjected to immunoprecipitation with anti-HA antibody, separated by SDS–PAGE and immunoblotted with anti-Numb antibody. Download figure Download PowerPoint LNX substrate recognition requires the PTB domain-binding motif and the first PDZ domain Previously, we have shown that the interaction between LNX and Numb requires the NPAY(188) sequence motif between the RING finger domain and the first PDZ domain (Figure 3A), which serves as a phosphorylation-independent binding site for the Numb PTB domain (Dho et al., 1998; Li et al., 1998). To identify regions of LNX important for substrate recognition, we generated a series of truncation mutants, as depicted in Figure 3A. All of the LNX truncation mutants retained E2-dependent ubiquitin ligase activity in vitro, confirming that the RING finger domain is sufficient for E2 activation (Figure 3B). We then tested the ability of the LNX truncation mutants to ubiquitylate Numb in vitro. Wild-type LNX and LNX mutants lacking PDZ domains 2, 3 and 4 efficiently ubiquitylated in vitro translated Numb (Figure 3C). However, removal of the first PDZ domain resulted in a complete loss of substrate recognition, even though LNXΔPDZ retains the Numb PTB domain-binding site. Figure 3.LNX substrate recognition in vitro requires the PTB domain-binding motif and the first PDZ domain of LNX. (A) Schematic representation of LNX and LNX mutants constructed and expressed as either GST fusions or with a FLAG tag at the N-terminus. (B) Truncated LNX mutants retain E3 activity. GS-bound fusion proteins of wild-type, C48A and truncated LNX mutants depicted in (A) or GST alone were incubated in an in vitro ubiquitylation reaction mixture in the presence of E2 (UbcH5B) bacterial lysate for 90 min at room temperature. The reaction products were resolved by SDS–PAGE and ubiquitylated proteins detected by western blotting using anti-ubiquitin antibody. (C) LNX substrate recognition in vitro requires the PTB binding site and PDZ1. Numb was in vitro transcribed and translated in the presence of [35S]methionine using wheat germ extract. Radiolabelled Numb was added to an in vitro ubiquitylation reaction containing E1, E2 (UbcH5B) and ubiquitin in the presence or absence of GST–LNXWT or GST–LNXC48A or truncated LNX mutants. Reactions were separated by SDS–PAGE and the Numb proteins visualized by autoradiography. The position of unmodified Numb is indicated by an arrow and the ubiquitylated Numb by the square bracket. GS-bound fusion proteins or GST alone added to the reactions were quantitated by SDS–PAGE and stained with Coomassie Blue (bottom gel). LNX GST fusion proteins in each lane are indicated by arrowheads. Download figure Download PowerPoint The regions of LNX required for ubiquitylation of endogenous Numb were determined in cells co-transfected with mammalian expression vectors encoding FLAG epitope-tagged LNX or the LNX mutants depicted in Figure 3A and HA epitope-tagged ubiquitin. Anti-Numb immunoprecipitates were immunoblotted with anti-HA to detect ubiquitylated proteins (Figure 4A) or anti-FLAG to detect co-immunoprecipitation of LNX with Numb (Figure 4C). We have previously shown that mutation of the Numb PTB-binding site (Y188A) in full-length LNX abrogates binding of the Numb PTB domain (Dho et al., 1998). This mutation also attenuated, but did not eliminate, co-immunoprecipitation of full-length Numb and LNX (Figure 4C), and dramatically reduced ubiquitylation of Numb (Figure 4A). These results suggest that the interaction between Numb and LNX may involve regions in addition to the Numb PTB domain binding to Y188. Figure 4.LNX substrate recognition and co-immunoprecipitation with Numb in vivo requires the PTB binding motif and the first PDZ domain. 293T cells were co-transfected with pMT HA-ubiquitin and FLAG-tagged mutants of LNX as depicted in Figure 3A. Cell lysates were prepared 24 h after transfection and Numb immunoprecipitated from 1 mg of protein. Immunoprecipitates were resolved by SDS–PAGE and transferred to PVDF membrane. Replicate membranes were blotted with anti-HA to detect ubiquitylated proteins (A), anti-Numb (B) to detect precipitated Numb proteins and anti-LNX (C) to detect LNX co-immunoprecipitating with Numb. Whole-cell lysates (D) were blotted with anti-LNX antibody to confirm expression of the mutant LNX proteins. Download figure Download PowerPoint The second, third and fourth PDZ domains of LNX were dispensable for substrate recognition and binding to Numb (Figure 4A and C). In contrast, the LNX mutant lacking the first PDZ domain (LNXΔPDZ) did not interact with or ubiquitylate Numb, indicating that the Numb–LNX interaction also requires the first PDZ domain of LNX. Notably, the LNX construct lacking the second, third and fourth PDZ domains (LNXΔ234) appeared to bind to Numb and ubiquitylate substrates more efficiently than wild-type LNX (Figure 4A and C), suggesting that PDZ domains 2, 3 and 4 may have a regulatory function. In addition, some of the ubiquitylated proteins detected in LNXΔ234 transfected cells are likely to represent ubiquitylated forms of LNX, since these high molecular weight bands were also detected by anti-FLAG antibodies (Figure 4C and D). LNX expression results in Numb protein degradation Ubiquitylation of cellular proteins often leads to their degradation by the proteasome. To determine whether LNX-mediated ubiquitylation of Numb promotes its degradation, 293T cells were co-transfected with wild-type, C48A or Y188A mutant forms of LNX and p72 Numb. Protein lysates of transfected cells were separated and immunoblotted with anti-Numb. Transfected Numb was significantly degraded when co-transfected with wild-type LNX but not when transfected with the LNX mutants that were unable to ubiquitylate Numb efficiently (C48A and Y188A mutants; Figure 5A). Figure 5.LNX-mediated ubiquitylation results in degradation of Numb. (A) 293T cells were transiently co-transfected with pcDNA-p72 Numb and either pFLAG-CMV-2 vector alone, FLAG-tagged LNXWT or LNX mutants that disrupt either the RING finger domain (LNXC48A) or the Numb PTB domain binding site (LNXY188A). Twenty four hours after transfection, whole-cell lysates were prepared, quantitated and equivalent amounts of protein resolved by SDS–PAGE and immunoblotted with anti-Numb antiserum (top). The membrane was then stripped and immunoblotted with anti-LNX to verify equivalent loading (bottom). (B) To determine whether overexpression of another RING domain containing E3 could promote degradation of Numb, cells were co-transfected with pcDNA-p72 Numb and either empty pFLAG-CMV-2 vector, wild-type FLAG-tagged LNX or HA-Cbl. Total cell lysates were quantitated and equivalent amounts of protein separated by SDS–PAGE and immunoblotted with anti-Numb (top), anti-LNX (middle) or anti-c-Cbl (bottom) to detect expression. (C) LNX-induced degradation of Numb is proteasome dependent. Twenty four hours after co-transfection of 293T cells with plasmids encoding Numb, LNX or empty vector, cells were incubated with dimethyl sulfoxide (DMSO) or the concentrations of MG132 indicated dissolved in DMSO for the times indicated. Whole-cell lysates were prepared and the equivalent amount of total protein was separated by SDS–PAGE. The levels of Numb expression were detected by immunoblotting with anti-Numb (top). Membranes were then stripped and blotted with anti-LNX to verify loading and LNX expression (bottom). Download figure Download PowerPoint E3 enzymes are largely responsible for target specificity and therefore we tested whether Numb was a specific target of LNX or whether overexpression of c-Cbl, another RING finger domain containing E3, could cause degradation of co-transfected p72 Numb. Numb was co-transfected with LNX or c-Cbl in 293T cells and total protein lysates from transfected cells were immunoblotted with anti-Numb antiserum. In contrast to LNX transfected cells, in which most of the transfected Numb was degraded, transfection with c-Cbl had no detectable effect on p72 Numb protein levels (Figure 5B). To determine whether Numb degradation in the presence of LNX is proteasome dependent, 293T cells were co-transfected with p72 Numb and wild-type LNX and treated with either 10 or 25 μM MG132 for 6–22 h post-transfection. Numb protein levels were examined by immunoblotting of total cell lysates with anti-Numb antiserum. Treatment of transfected cells with either 10 or 25 μM MG132 for 22 h effectively blocked the degradation of Numb (Figure 5C). LNX expression causes degradation of endogenous Numb To further assess the effect of LNX expression on endogenous Numb proteins, we transfected MDCK cells with wild-type or C48A LNX and HA-ubiquitin. Similar to the effect in 293T cells, Numb was ubiquitylated in the presence of wild-type but not mutant LNX (Figure 6A). Western analysis of total cell lysates with anti-Numb antibodies revealed that Numb protein levels were significantly reduced in the wild-type but not C48A transfected cells (Figure 6B). To visualize Numb protein levels in individual transfected cells, we stained MDCK cells transfected with either wild-type (Figure 6C, D and E) or C48A (Figure 6F, G and H) LNX with anti-Numb antiserum. LNX transfected cells were identified by anti-FLAG staining (Figure 6D and G) and Numb protein was detected by co-staining with anti-Numb (Figure 6C and F) and analysed by immunofluorescence and confocal microscopy. Merged images are shown in Figure 6E and H. In both non-transfected cells and cells transfected with mutant LNX (Figure 6C and F, shown in red), endogenous Numb staining was observed in vesicular structures, which we and others have identified as early endo somes (Santolini et al., 2000; S.E.Dho, C.A.Smith and C.J.McGlade, in preparation). In cells transfected with wild-type LNX (green), Numb staining was selectively lost (Figure 6C and merged image in E). In order to assess whether LNX overexpression had a non-specific effect, we also stained LNX transfected cells with anti-SHC antisera (Figure 6I–K) to determine the effect of LNX overexpression on other endogenous proteins. SHC staining was unaltered in the wild-type LNX transfected cells, confirming the specificity of the effect of LNX on Numb protein levels. Figure 6.LNX expression in MDCK cells causes degradation of endogenous Numb. (A) MDCK cells were transiently co-transfected with pMT HA-ubiquitin and either empty, pFLAG-CMV-2 or FLAG-tagged LNXWT or LNXC48A. Numb protein was immunoprecipitated from ∼1 mg of transfected cell lysate with anti-Numb antiserum and immunoblotted with anti-HA monoclonal antibody. (B) MDCK cells transfected with either empty pFLAG-CMV-2, FLAG -LNXWT or LNXC48A. Whole-cell lysates were prepared, quantitated and equivalent amounts of protein were resolved by SDS–PAGE and immunoblotted with anti-Numb antiserum (top). The membrane was then stripped and immunoblotted with anti-LNX antibody to verify expression of LNX (middle) and with anti-tubulin to verify equivalent protein loading (bottom). (C–K) MDCK cells were transfected with either FLAG-tagged LNXWT (shown in C, D and E) or LNXC48A (shown in F, G and H). Cells were then co-stained with anti-FLAG and goat anti-mouse antibody conjugated to AlexaFluor488 to detect LNX transfected cells (green; D and G) and anti-Numb was detected by goat anti-rabbit antibody conjugated to CY3 (red; C and F). Merged images are shown in (E) and (H). To determine the effect of LNX expression on another endogenous PTB domain-containing molecule, MDCK cells transfected with pFLAG-LNXWT (I, J and K) were co-stained with anti-FLAG as described above (green; J) to detect LNX-transfected cells and with anti-SHC antisera (red; I). The merged image is shown in (K). Images were obtained by confocal microscopy. Download figure Download PowerPoint Numb functions as an antagonist of the Notch signalling pathway and levels of Numb protein have been shown to modulate Notch-mediated activation of downstream targets such as Hes-1 (Frise et al., 1996; Guo et al., 1996; Spana and Doe, 1996; Artavanis-Tsakonas et al., 1999; Wakamatsu et al., 1999). Therefore we tested whether LNX expression and the consequent decrease in Numb protein levels could alter Notch-mediated activation of a Hes-1 luciferase reporter. A constitutively active form of Notch1 (ΔEC-Notch) activated Hes-1 luciferase when transfected into CHO cells (Figure 7A). Co-transfection of ΔEC-Notch with LNX resulted in ∼30% increase in Notch nuclear activity, as measured by Hes-1 promoter-driven luciferase activity (Figure 7A). LNX expression in CHO cells also caused ubiquitylation of endogenous Numb (Figure 7B) and significant diminution of Numb staining (Figure 7C), suggesting that LNX reduces levels of Numb proteins resulting in augmentation of Notch signalling. We cannot formally exclude the possibility that LNX acts directly on Notch, although this seems unlikely since LNX expression does not promote Notch ubiquitylation or alter Notch protein expression levels (data not shown). Figure 7.LNX expression enhances Notch nuclear activity. (A) CHO cells were transfected with ΔEC-Notch, a Hes-1 luciferase reporter construct and either empty pEF or pEF-