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Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts,1 Department of Pathology and Laboratory Medicine, Institute for Medicine and Engineering, The Abramson Family Cancer Research Institute, University of Pennsylvania Medical School, Philadelphia, Pennsylvania2
Received 7 May 2004/ Returned for modification 15 June 2004/ Accepted 30 July 2004
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAddendum in proofReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAddendum in proofReferences |
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Human NOTCH1 (hN1) has a crucial influence upon several stages of lymphocyte development (reviewed in reference 31). Expression of constitutively active hN1 in hematopoietic stem cells induces the development of CD4+ CD8+ double-positive (DP) immature T cells in the bone marrow, inhibits normal marrow B-cell development, and eventually leads to the appearance of fatal T-cell leukemias (32). Conversely, inducible notch1 knockout mice fail to develop mature T cells due to a requirement for notch1 during early stages of intrathymic T-cell development (33). Thus, although Notch receptors are essential for the development of multicellular organisms, their activation must be precisely regulated.
Notch receptors are first synthesized as 300- to 350-kDa type I single-pass transmembrane glycoproteins. During maturation, Notch precursor polypeptides are proteolytically processed by a furin-like convertase at a site called S1 (Fig. 1A), yielding two noncovalently associated subunits (26). The resulting two associated subunits, here termed extracellular Notch (NEC) and transmembrane Notch (NTM), constitute the mature heterodimeric form of the protein present at the cell surface (2, 7).
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Notch ligands are transmembrane proteins that are members of the DSL (Delta, Serrate, and LAG-2) family. These proteins contain a cysteine-rich DSL domain essential for binding to the EGF-like domain in the NEC subunit of Notch receptors (16). In the current model for Notch activation, ligand binding to repeats within the EGF-like domain induces two sequential proteolytic events termed S2 and S3 cleavages. S2 cleavage is catalyzed by disintegrin-metalloproteases like tumor necrosis factor alpha (TNF-
)-converting enzyme (TACE) at a site lying 13 residues external to the transmembrane segment of the NTM subunit (8, 27). Following cleavage at S2, 
-secretase cleavage within the transmembrane domain at site S3 releases active ICN (39, 46). Once released from the plasma membrane, ICN translocates to the nucleus, where it interacts with members of the CSL (for CBF-1/RBPJ
, Suppressor of Hairless, and Lag-1) family of transcription factors, resulting in transcriptional activation of target genes (4, 11, 18, 20, 24, 39, 40, 43, 47, 48). The RAM and ANK domains of ICN participate in CSL binding, while the ANK domain is also essential for recruitment of coactivators and transactivation (21, 28, 38).
Enforced expression of forms of Notch lacking the NEC subunit results in phenotypes characteristic of constitutively active Notch signaling in a variety of model organisms (10, 13, 25, 30, 35, 42). Additionally, disruption of the mature heterodimeric form of the receptor leads to nuclear localization of a polypeptide of the size of ICN and increased transcriptional activation of a CSL-specific reporter gene (34). In contrast, forms of Notch lacking the EGF-like repeats, but retaining the NRR, are restrained in an inactive state and are unable to respond to ligand (20, 25, 35, 42). Furthermore, certain deletions and point mutations within the NRR lead to gain-of-function phenotypes in Drosophila (25, 42) and Caenorhabditis elegans (6, 15). Taken together, these studies suggest that a major role of the NRR region of the NEC subunit is to restrain activation of Notch receptors in the absence of ligands, yet permit activation in response to ligand binding.
Although it is clear that the NRR participates in the regulation of Notch activity, there is limited information about the mechanism by which this region restrains signaling. Here, we establish that the NECC region within the NRR is sufficient to form a heterodimer with the extracellular region of NTM (NTMX) and show that the LNR domain is necessary for preventing metalloprotease cleavage at the S2 site in the absence of ligand, even though it is not required for heterodimerization. In addition, Notch polypeptides lacking both the ligand-binding and the LNR domains activate CSL reporter genes in cultured cells and drive the appearance of CD4+ CD8+ DP T cells in the peripheral blood when expressed in hematopoetic stem cells of mice, confirming that the removal of the LNR domain results in a ligand-independent gain-of-function phenotype. Together, these findings identify two distinguishable activities residing within the NRR: heterodimerization, mediated through the NECC region; and prevention of ligand-independent proteolysis, which also requires the LNR domain.
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MATERIALS AND METHODS |
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E (residues Gly-1674 to Lys-2556) were engineered in frame at the 3' end of the hN1 signal peptide in the pcDNA3 plasmid vector. For metalloprotease sensitivity experiments, three myc epitope tags were added in series at the C-terminal end of the NTM subunit of each construct. The bacterial hN2 NECC-NTMX-H6 expression construct (residues Asp-1537 to Gln-1677) was engineered in the plasmid vector pET-21a(+) (Novagen). The construct was designed such that only a single additional methionine was added at the N terminus of the encoded hN2 polypeptides and a hexahistidine tag was added to the C terminus. To permit stable expression in bone marrow cells, cDNAs encoding hN1 NRR-NTM and NECC-NTM were cloned into the backbone of the retroviral shuttle vector MigRI (32). Expression of Notch proteins in cultured cells. U2OS and 293T cells (American Type Culture Collection) were transiently transfected with Lipofectamine Plus reagent (Invitrogen). U2OS and 293T cells were maintained in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (Sigma), 2 mM L-glutamine (Gibco), 100 U of penicillin/ml (Gibco), and 100 µg of streptomycin/ml (Gibco). Cells were grown at 37°C under 5% CO2.
NRR-NTMX association studies. 293T cells transfected with control vector or with vectors encoding different soluble forms of Notch heterodimers were grown on six-well plates for 72 h. Three days after transfection, the cells were washed twice with ice-cold Hanks balanced salt solution and collected by centrifugation at 500 x g for 5 min. Cell pellets were lysed by heating (100°C) for 10 min in sodium dodecyl sulfate (SDS) loading buffer in the presence of reducing agent and analyzed by Western blot after SDS-polyacrylamide gel electrophoresis (PAGE), using an antibody directed against the HA epitope tag (HA.11; Covance). Immunoprecipitates from conditioned media were prepared by mixing 1 ml of conditioned medium with anti-HA or anti-FLAG (Sigma) beads at 4°C for 2 h. Following incubation, the beads were washed three times with 1 ml of ice-cold buffer A (50 mM Tris, pH 7.5, 100 mM NaCl, 1% NP-40) and heated (100°C) for 10 min in SDS loading buffer in the presence of reducing agent. The recovered proteins were resolved by SDS-PAGE and analyzed by Western blotting with antibodies against the epitope tags.
Recombinant protein expression and purification. The hN2 NECC-NTMX-H6 polypeptide (residues Asp-1537 to Gln-1677) was subcloned into the plasmid vector pET-21a(+) (Novagen) with a hexahistidine tag at the C-terminal end for affinity purification. The hN2 expression plasmid was then transformed into Escherichia coli strain BL21(DE3)pLysS. Protein expression was induced by addition of 0.4 mM isopropyl-1-thio-ß-D-galactopyranoside to log-phase cell cultures grown in Luria-Bertani broth. After induction for 4 h, cells were recovered by centrifugation and resuspended in ice-cold 50 mM Tris buffer, pH 8.0, containing 300 mM NaCl, 20% (wt/vol) sucrose, 10 mM ß-mercaptoethanol, 0.5 mM EDTA, 10 mM imidazole, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 2-µg/ml pepstatin, 5-µg/ml aprotinin, and 2-µg/ml leupeptin. Cells were lysed by freezing and thawing, followed by three cycles of sonication for 30 s on ice. Insoluble material was removed from the lysate by centrifugation, and the supernatant was adsorbed to nickel-nitrilotriacetic acid (NTA)-agarose resin (QIAGEN) at 4°C for 2 h. After extensive washing with buffer B (50 mM Tris buffer [pH 8.0], 300 mM NaCl, 10 mM imidazole), bound NECC-NTMX-H6 was eluted from the Ni-NTA agarose resin with buffer C (buffer B containing 250 mM imidazole). After dialysis at 4°C for 12 to 16 h against buffer D (50 mM Tris buffer [pH 8.0], 300 mM NaCl), NECC-NTMX-H6 was further purified by size exclusion chromatography on a Superdex 75 column (Amersham Biosciences) in buffer D.
In vitro furin processing of recombinant hN2 NECC-NTMX. Purified NECC-NTMX-H6 was incubated in buffer E (50 mM Tris [pH 7.5], 1 mM CaCl2) with soluble furin (New England BioLabs) at 30°C for 4 h, using 1 U of furin per 50 µg of protein. After cleavage, the furin was removed by adsorbing the NECC-NTMX-H6 heterodimer to Ni-NTA-agarose at 4°C for 2 h after cleavage and then eluting it in buffer C. The processed NECC-NTMX-H6 heterodimer was purified and analyzed by size exclusion chromatography on a Superdex 75 column in buffer D. For analysis and identification of the cleaved products by reversed-phase high-performance liquid chromatography (HPLC), aliquots from the peak were acidified by addition of 5% (vol/vol) acetic acid, applied to an analytical-scale C18 column, and resolved with a water-acetonitrile gradient. The two eluted peptides (monitored by A229) were collected, lyophilized, resuspended in water, and analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.
Transcriptional activation assays. Plasmids were introduced into U2OS cells by transfection with Lipofectamine (Invitrogen). To assay activation of CSL-dependent transcription, cells were cotransfected in triplicate with various Notch1 expression constructs in pcDNA3, a CSL firefly luciferase reporter plasmid (17), and an internal control sea pansy Renilla luciferase plasmid (Promega). Normalized firefly luciferase activities were measured in whole-cell extracts prepared 44 to 48 h after transfection with the Promega dual luciferase kit.
Metalloprotease sensitivity assay. U2OS cells transiently transfected with myc-tagged forms of hN1 (NRR-NTM-myc and NECC-NTM-myc) were treated with the presenilin inhibitor compound E (CalBiochem) at a concentration of 1 µM or with carrier alone (0.01% dimethyl sulfoxide) as described previously (45). Cells were washed once with ice-cold Hanks buffered saline and lysed in buffer F (50 mM Tris [pH 7.5], 100 mM NaCl, 10 mM phenylmethylsulfonyl fluoride, 10-µg/ml aprotonin, 10-µg/ml leupeptin, 1% NP-40) for 15 min on ice. Lysates were cleared by centrifugation at 16,000 x g for 15 min at 4°C. hN1 polypeptides were immunoprecipitated from whole-cell lysates (2 x 106 cells) by incubation for 12 to 16 h at 4°C with anti-myc antibody (9E10) cross-linked to protein G-Sepharose beads (Sigma). After incubation, beads were washed twice with buffer F and once with a mixture of 50 mM Tris, pH 7.5, and 100 mM NaCl. Anti-myc immunoprecipitates were treated with lambda protein phosphatase (New England BioLabs) as recommended by the manufacturer prior to Western blot analysis.
Murine bone marrow transplantation assays.
All experiments were performed as described previously (4) and were conducted in accordance with National Institutes of Health guidelines for the care and use of animals and with an approved animal protocol from the University of Pennsylvania Animal Care and Use Committee. Briefly, cDNAs cloned into the MigRI vector were packaged into retroviruses by transient transfection of Bosc23 cells. After titering of viruses on NIH 3T3 cells, green fluorescent protein (GFP)-normalized retroviral supernatants were used to "spinoculate" bone marrow cells from female BALB/c mice (Taconic Farms). The retrovirally transduced bone marrow cells were then injected into lethally irradiated (900 rads) 4- to 8-week-old female syngeneic recipients. Three weeks posttransplantation, peripheral blood sampled from retro-orbital sinuses was assessed for the presence of GFP+ immature T cells by flow cytometric analysis (Becton Dickinson; FACSCalibur). Cells were incubated with biotinylated CD8 (53-6.7) and phycoerythrin-conjugated CD4 (RM4-5) (Pharmingen). Biotinylated antibodies were revealed with streptavidin-CyChrome. Dead cells, identified by forward scatter (FSC) and side scatter (SSC), were excluded from the analysis. Flow cytometry results were analyzed with CellQuest software.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAddendum in proofReferences |
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To determine whether the precursor is processed at the S1 site during maturation and whether the NRR and NTMX fragments remain associated with each other after cleavage at the S1 site during maturation, we expressed the NRR-NTMX precursor polypeptide of hN1 with FLAG and HA epitopes at the N and C termini, respectively, to facilitate detection of the individual subunits of the complex in immunoprecipitation assays (Fig. 1B). Following transient transfection into 293T cells, the single-chain precursor (NRR-NTMX) was detected in whole-cell extracts (Fig. 2A), whereas immunoprecipitates prepared from conditioned media using either FLAG or HA-coupled beads contained both epitope-tagged subunits, NRR and NTMX (Fig. 2B), but little or no uncleaved precursor. Thus, the two subunits remain stably associated as an NRR-NTMX heterodimer after furin cleavage and secretion into the conditioned media. The interaction between the NRR and NTMX subunits was disrupted by 0.1% SDS (Fig. 2C), demonstrating that association between the subunits is noncovalent, as seen with full-length hN1 (34).
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E). In Western blots, we consistently observe that NECC-NTM expresses at lower levels than does E (as in Fig. 6B) (data not shown), possibly explaining the observed difference in activity.
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Normally the product of S2 cleavage (NTM*, Fig. 6A) is rapidly cleaved at the S3 site by gamma-secretase to release ICN1 (i.e., the active form of Notch), a species that has a very short half-life. However, in the presence of gamma-secretase inhibitors, NTM* is stabilized, allowing it to accumulate and be detected (45). Cells transiently expressing NECC-NTM accumulate readily detectable amounts of NTM* in the presence of a gamma-secretase inhibitor, whereas cells expressing NRR-NTM, a molecule that still has the LNR modules but is quiescent in the reporter assay, do not (Fig. 6B). Together, these results show that although the LNR domain is not essential for association between the two Notch subunits, it does play a critical role in the regulation of receptor activity by preventing premature proteolytic activation in the absence of ligand.
Loss of the LNR modules causes Notch gain of function in a mouse model. To assess the biological activity of NECC-NTM in vivo, we scored its activity in a murine bone marrow transplantation assay. When used to reconstitute irradiated mice, bone marrow stem cells transduced with gain-of-function forms of Notch1 rapidly give rise to an abnormal CD4+ CD8+ DP T-cell population in the bone marrow that spills into the peripheral blood by 3 weeks posttransplantation (4, 31). In these experiments, we used the retroviral vector MigRI, which also drives the expression of GFP from a bicistronic mRNA with an internal ribosomal entry sequence, in order to gate on transduced cells. The GFP+ cells in the peripheral blood of a mouse reconstituted with hematopoetic stem cells transduced with NRR-NTM virus on day 54 are mainly CD4– CD8– (Fig. 7); most of these cells are B220+ B cells and Gr1+ granulocytes (not shown), a result similar to that seen with empty MigRI virus (4, 32). In contrast, 70% of the GFP+ cells in the peripheral blood of a mouse reconstituted with NECC-NTM-transduced marrow cells are CD4+ CD8+ immature DP T cells (Fig. 7). Similar results were obtained with nine mice in each group (not shown). Thus, the LNR region is necessary to prevent ligand-independent receptor activation in vivo.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAddendum in proofReferences |
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Prior studies had shown that mutations involving the LNR domain result in inappropriate increases in Notch signals, implying an important regulatory role for these domains. We noted previously that the folding and structural integrity of individual LNR modules require calcium (3, 44); that Notch heterodimers immunoprecipitated from whole-cell lysates dissociate in the presence of EDTA (a metal ion chelator) (34); and that EDTA treatment of intact cells results in release of NTM, nuclear translocation of ICN, and activation of CSL reporter genes (34). While the simplest explanation for these observations was for the LNR domain to participate directly in binding to the NTM subunit, observations described here argue against such a role, as the region lying C terminal of the LNR domain, NECC, is sufficient to form stable heterodimers with NX in secreted and recombinant Notch "minireceptors." This NECC region ranges in size from 70 to 103 residues among the mammalian Notch receptors and includes a number of highly conserved hydrophobic residues that might participate in the association with NTMX (Fig. 3C). Most striking among the conserved sequences is the cluster of aliphatic amino acids that lies near the NECC N-terminal end (Fig. 3C).
NECC is not as well conserved in Drosophila Notch, consistent with the possibility that fly Notch may not require S1 cleavage for activation. However, significant sequence similarity does exist in this region, both within the hydrophobic stretch near the N-terminal end and at a series of invariant residues scattered throughout the region. Efforts to determine a high-resolution structure of a mammalian Notch heterodimer, which will reveal the nature of the interface in detail, are now under way.
Mutations in NECC and in its heterodimerization partner, the extracellular stub of NTM (NTMX), have been previously associated with gain-of-function phenotypes in both flies and worms. In worms, the S642N mutation in NECC of the glp-1 notch homolog causes a gain-of-function phenotype (6). In flies, simultaneous replacement of the two conserved cysteines in NTMX with serine residues leads to gain of function, as assessed by the development of an antineurogenic phenotype (25), whereas point mutations elsewhere within NTMX are associated with gain of function in worms (15). The biochemical studies reported here provide a rationalization for the observed gain of function in the genetic studies; various mutations in either NECC or NTMX can destabilize the heterodimer, causing premature subunit dissociation after S1 cleavage and subsequent Notch activation by the normal proteolytic cascade of S2 and S3 cleavages.
LNR domains are a unique feature of Notch receptors, existing in all members of the family as three tandemly repeated modules. Although the NECC region is sufficient to form a stable heterodimer with NTMX, it is not sufficient to prevent proteolytic activation in the absence of the adjacent LNR modules (Fig. 5 and 6). The implication of these studies is that LNR mutations cause gain-of-function phenotypes because they allow proteolysis at the S2 site even in the absence of ligand, although without promoting premature dissociation of the subunits. The effects we observed previously showing that EDTA dissociates and activates Notch heterodimers may be explained by loss of this "anti-protease" activity, as these experiments were performed (whole-cell lysates or intact cells at 25 to 37°C) under conditions in which proteases may have been active.
More generally, our studies show that stable heterodimer association and prevention of ligand-independent activating cleavages are separable functions that can be attributed to two different regions of the extracellular domain of Notch and raise further mechanistic questions. Most notably, does the LNR domain prevent ligand-independent proteolysis merely by sterically preventing access of metalloproteases to the S2 site, or do more complex mechanisms come into play? It will also be important to resolve the puzzle of how physiologic ligand binding overcomes the intrinsic restraint on signaling imposed by the LNR domain.
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ADDENDUM IN PROOF |
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ACKNOWLEDGMENTS |
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This research was supported by NIH grants CA92433 (to S.C.B.), CA82308 (to J.C.A.), AI47833 (to W.S.P.), and 1 F31 CA97547-01 (to C.S.I.). S.C.B. is a Pew Scholar in the Biomedical Sciences and an Established Investigator of the American Heart Association.
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FOOTNOTES |
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