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J. Biol. Chem., Vol. 277, Issue 8, 6318-6323, February 22, 2002

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Presenilin-dependent -Secretase-like Intramembrane Cleavage of ErbB4^*

Hahn-Jun Lee^§, Kwang-Mook Jung^§, Yang Z. Huang^¶, Lori B. Bennett, Joanne S. Lee, Lin Mei^¶, and Tae-Wan Kim

From the  Department of Pathology, Taub Institute for Research on Alzheimer's Disease and the Aging Brain, College of Physicians and Surgeons, Columbia University, New York, New York 10032 and the ^¶ Department of Neurobiology, Pathology, and Physical Medicine and Rehabilitation, University of Alabama School of Medicine, Birmingham, Alabama 35294

Received for publication, October 17, 2001, and in revised form, December 5, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

An unusual protease -secretase requires functional presenilins and cleaves substrates (e.g. amyloid -protein precursor and Notch) with very loose amino acid sequence specificity within the transmembrane region. Here we report that ErbB4, a tyrosine kinase receptor for neuregulins, is a substrate for presenilin-dependent -secretase. Our studies show that constitutive ectodomain shedding of full-length ErbB4 yields the ~80-kDa membrane-associated C-terminal fragment (B4-CTF). Subsequent intramembrane cleavage of the B4-CTF was inhibited in the cells devoid of functional presenilins or by treatment of cells with a -secretase inhibitor, leading to enhanced accumulation of B4-CTF. Furthermore, an in vitro -secretase assay demonstrated that the intracellular domain of ErbB4 (B4-ICD) was produced and subsequently released into the soluble fraction in a presenilin-dependent manner. We have also shown that ectopically expressed B4-ICD is localized to the nucleus, suggesting that the presenilin-dependent cleavage of ErbB4 generates the soluble B4-ICD that functions in the nucleus presumably at transcriptional level. Our study indicates that ErbB4 represents a first receptor tyrosine kinase that undergoes intramembrane proteolysis and may mediate a novel signaling function independent of its canonical role as a receptor tyrosine kinase. Our studies also support the idea that presenilins play a generic role in intramembrane cleavage of selected type I membrane proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

ErbB4 is a type I membrane receptor tyrosine kinase, which belongs to the epidermal growth receptor family and mediates response to multiple growth factors, including neuregulins (reviewed in Refs. 1-3). ErbB4 has been implicated in many important biological and pathological processes, such as cardiovascular, mammary gland, and neural development, as well as malignancy and heart disease (1-3).

Presenilins (PS1 and PS2),¹ gene products of the major early-onset familial Alzheimer's disease genes (reviewed in Refs. 4-6), are required for the activity of -secretase, an unusual aspartyl protease that cleaves substrates within the predicted transmembrane region (7; reviewed in Refs. 8-10). Two of the characteristics of -secretase include a lack of requirement for specific amino acid target sequences within the transmembrane domain and a requirement for ectodomain shedding to produce membrane-anchored truncated C-terminal derivatives (10, 11). These observations imply that the presenilins may also be involved in the intramembrane cleavage of other type I membrane proteins. The -secretase cleavage of amyloid -protein precursor (APP) is a critical rate-limiting step toward the production of amyloid -peptide (A) in Alzheimer's disease (6). In addition to APP, transmembrane cleavage of Notch, which releases the Lin-12/Notch intracellular domain, plays a pivotal role in cell fate determination (12, 13). Ectopically expressed intracellular domains of APP (AICD) and Notch (NICD) appear to be localized in the nucleus and participate in gene transcription (12-20).

Here, we report that ErbB4, a tyrosine kinase receptor for neuregulins, represents a novel substrate that undergoes presenilin-dependent -secretase-like proteolysis and the resulting soluble intracellular domain of ErbB4 localizes to the nucleus.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Plasmids-- The expression construct encoding full-length ErbB4 with C-terminal HA epitope tag has been described elsewhere (21) and used as a template for PCR amplification using High Fidelity PCR Master (Roche Molecular Biochemicals, Indianapolis, IN). An ErbB-E2 construct was generated by adding N-terminal signal peptide and HA tag and C-terminal 6xHis and V5 tags by two overlapping PCRs using the following primers: 5'-SP common ErbB4, aaaatgaagccggcgacaggactttgggtctgggtgagccttctcgtggcggcggggaccgtccagcccagcgattctTATCATATGATGTGCCAGAT; 5'-JMaH1, TATCCATATGATGTGCCAGATTATGCAAGCCTCgggtgtaacggtcccactagtcatgactgcattta; 5'-JMaH2, TATCCATATGATGTGCCAGATTATGCAAGCCTCcatggaccgggtcctgacaactgtacaaagtgctctc; and 3'-ErbB4a, caccacagtattccggtgtctgtagggtggaggcgg. Capital letters denotes the incorporated HA tag sequences. PCR products were subcloned into pEF6/V5-His TOPO vector according to the manufacturer's instructions (Invitrogen, Carlsbad, CA), and all constructs were fully verified by DNA sequencing. GFP and GAL4-BD constructs were generated as previously described (21).

Cell Culture, Transfection, and Generation of Stable Cell Lines-- Stable PS12 cell lines were generated by transfecting 293 cells in a 100-mm dish with 5 µg of each plasmid: wild-type PS1and PS2 for dW cell lines or D385A PS1 and D366A PS2 for dAsp (pcDNA3.1/Zeo+) using SuperFect transfection reagent (Qiagen) (22). Individual Zeocin-resistant colonies were isolated and screened for PS1 and PS2 expression by Western blotting using PS1Loop and PS2Loop antibodies (23). Stable cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin in the presence of 250 µg/ml Zeocin (Invitrogen). For transient transfection, cells were transfected in 100-mm dishes with 10 µg of plasmid.

Antibodies and Western Blot Analyses-- At 48 h post-transfection, cells were lysed using buffer IP (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.25% Nonidet P-40, and 2 mM EDTA) supplemented with a protease inhibitor mixture tablet (Roche Molecular Biochemicals). Protein quantification, SDS-PAGE (4-20 or 8%), and Western blot analyses were carried out as described previously (24). Primary antibodies were used at the following dilutions: PS1Loop at 1:3000; PS2Loop at 1:2500; anti-APP R1 at 1:2000; anti-HA (HA 11, Covance) at 1:1000; anti-ErbB4 (Ab-2, Neomarker) at 1:200.

Preparation of Membrane Fractions and in Vitro Generation of the Intracellular Domains of ErbB4-- Active membrane preparation and in vitro generation of the intracellular domains of ErbB4 (B4-ICD) and Notch 1 (NICD) were performed as described in Ref. 17 with minor modifications. Briefly, cells were washed twice with ice-cold phosphate-buffered saline and homogenized with buffer HS (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 250 mM sucrose) plus protease inhibitor using a 22-gauge needle. All subsequent steps were carried out at 4 °C unless indicated otherwise. The samples were then centrifuged at 1500 × g for 10 min to remove nuclei and cell debris. Membranes were pelleted from the postnuclear supernatants by centrifugation for 20 min at 14,000 × g and resuspended in buffer H (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 5 mM EDTA) plus protease inhibitors. In vitro B4-ICD and NICD generation experiments were performed by incubating the membrane fractions at 37 °C for the indicated time in a volume of 50 µl as 2.5 mg/ml protein solution. After incubation, the soluble and membrane-associated ErbB4 or Notch fragments were separated by centrifugation of the reaction mixtures at 200,000 × g for 30 min.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

To identify novel substrates for presenilin-dependent -secretase, we conducted proteomic analysis by subcellular fractionation and two-dimensional electrophoresis. To detect the accumulation of novel substrates for -secretase, we first treated 293 cells with a -secretase inhibitor, Compound E (25). To achieve the enrichment of target transmembrane proteins, we performed subcellular fractionation to isolate membrane proteins using the 293 cells grown in the presence or absence of Compound E (Fig. 1A). Both heavy (P2) and light (P3) membrane fractions were extracted with carbonate (100 mM Na₂CO₃ at pH11) to remove peripherally attached membrane proteins (Fig. 1A). Under these conditions, C-terminal fragments of APP (C83/99), known -secretase substrates, substantially accumulated in all -secretase-treated cells (Fig. 1B). The remaining integral membrane proteins (CP2 and CP3) were further analyzed by two-dimensional electrophoresis (Fig. 1C) (data not shown for CP2). Less than 1% of total identified spots in two-dimensional gels accumulated in substantially higher levels in -secretase-treated cells as compared with control cells.

View larger version (82K): [in this window] [in a new window]   Fig. 1.   Proteomic approach to identify novel -secretase substrates by subcellular fraction and two-dimensional electrophoresis. A, schematic illustrations of the subcellular fractionation procedure. B, membrane fractions of -secretase-treated or control cells were subjected to SDS-PAGE and Western blot analyses using anti-APP antibodies C7. 20 µg (S1-P3) or 10 µg (CP2 and CP3) of protein was loaded in each lane. C, CP3 fractions that were prepared from 293 cells grown in the absence (Control) or presence (+ -secretase inhibitor; compound E, 8 h, 50 nM) were further resolved in the first dimension isoelectric focusing by 7 cm non-linear IPGphor strip and in the second dimension by 4-20% SDS-PAGE. The spot corresponding to ErbB4 is indicated by the arrow.

Based on the molecular size of protein spots detected and selectively present in the inhibitor-treated cells, we selected several type I membrane candidate substrates. The following criteria were used to select the candidate substrates for further verification and analyses: (i) size of the predicted intracellular domain; (ii) phorbol ester-enhanced or metalloprotease-mediated ectodomain shedding; (iii) presence of extracellular dimerization motif. One of these candidates was ErbB4, a type I membrane receptor tyrosine kinase (26). ErbB4 was shown to undergo constitutive ectodomain shedding to produce membrane-anchored C-terminal fragment (27, 28) This cleavage appears to be mediated by tumor necrosis factor- converting enzyme or a related metalloprotease (27, 28) and is greatly potentiated by the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (29), similar to what has been reported for APP (30, 31). The reported size (~80 kDa) of metalloprotease-generated ErbB4 C-terminal fragment corresponded to one of the spots in the two-dimensional gel from Compound E-treated samples (Fig. 1C).

When we transiently expressed the constructs encoding full-length ErbB4 with C-terminal HA epitope tag (21), full-length ErbB4 (~180 kDa) was constitutively processed to generate a C-terminal fragment of ~80 kDa (Fig. 2A), which was tightly associated with the membrane.² To evaluate the involvement of -secretase in the ErbB4 proteolysis, we studied the effects of the deficiency in presenilin-dependent -secretase activity. For this purpose, the accumulation of the ~80-kDa membrane-associated ErbB4 C-terminal fragments (B4-CTF) was examined in the stable 293 transfectants harboring constructs encoding wild-type (dW) or loss-of-function (dAsp: D385A-PS1 and D366A-PS2) forms of PS1 and PS2 (14, 32). Similar to what has previously been reported using the Chinese hamster ovary cells, which overexpress both human APP and D257A-PS1/D366A-PS2 (14, 32, 33), dAsp 293 cells accumulated endogenous APP C-terminal fragments to a substantially higher level than either parental 293 or double wild-type cells (dW), and full-length PS1 and PS2 failed to undergo endoproteolytic processing (Fig. 2B). The accumulation of ErbB4 C-terminal fragments was dramatically elevated in dAsp cells whereas the levels of full-length form were virtually unaffected (Fig. 2B). As for the case in APP, the enhanced accumulation of B4-CTF was likely due to the increased stability. This may be attributed to the deficient -secretase activity rather than increased ectodomain shedding, because the secretion of the extracellular domain was not significantly affected in dAsp cells as compared with dW cells (data not shown).

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Accumulation of membrane-associated C-terminal derivatives of ErbB4 in cells lacking -secretase activity. A, HEK293 cells transfected with vector alone or full-length ErbB4 cDNA (with C-terminal HA tag) (ErbB4-HA) were lysed and analyzed by Western blot analyses (8% SDS-PAGE) using indicated antibodies. B, parental 293 cells (293) and stable 293 cells co-expressing either wild-type PS1 and PS2 (dW) or loss-of-function variants of PS1 and PS2 (D385A-PS1 and D366A-PS2, respectively; dAsp). Cell lysates were analyzed by Western blotting (4-20% SDS-PAGE) using the indicated antibodies. Locations of full-length presenilins and C-terminal fragments are indicated by single and double asterisks, respectively.

Next we investigated if a -secretase inhibitor blocks the putative transmembrane cleavage of ErbB4 to cause an accumulation of B4-CTF similar to that observed in dAsp cells. Treatment of the cells with a potent -secretase inhibitor Compound E (IC₅₀ = ~0.3 nM (25)) led to the accumulation of transgene-derived ErbB4 C-terminal fragments in 293 cells (Fig. 3A) as well as endogenous ErbB4 fragments (detected by anti-ErbB4 antibodies) in T47D cells (Fig. 3B). Our studies suggest that constitutive ectodomain shedding of full-length ErbB4 yields the ~80-kDa membrane-associated B4-CTF, and the subsequent intramembrane cleavage of the B4-CTF was inhibited in cells devoid of functional presenilins or by treatment of cells with a -secretase inhibitor, leading to enhanced accumulation of B4-CTF.

View larger version (46K): [in this window] [in a new window]   Fig. 3.   The accumulation of C-terminal proteolytic derivatives of ErbB4 by -secretase inhibitor. A, ErbB4-HA-transfected 293 cells were incubated in the presence of the indicated concentrations of -secretase inhibitor compound E (Cpd. E) for 16 h. Detergent lysates were analyzed by Western blot (4-20% SDS-PAGE). B, T47D cells that express high levels of endogenous ErbB4 were treated with the medium containing the indicated concentrations of Compound E. Total lysates were analyzed by Western blot analyses using anti-ErbB4 (8% SDS-PAGE).

To directly assess the intramembrane cleavage of ErbB4 without stimulation with ligand, we expressed constructs encoding the truncated ErbB4 lacking the majority of entire extracellular regions (Fig. 4A). To trace the precursor and product upon cleavage, the truncated construct (E2) encoded a polypeptide containing an HA epitope tag located immediately after the signal peptide in addition to the C-terminal V5 and 6xHis tags (Fig. 4A). ErbB4 constructs encoding extracellular 79 amino acids (E2, Fig. 4A) were found to undergo proteolytic processing in the absence of the majority of the extracellular domain, yielding C-terminal fragments that are similar to the fragment generated from full-length ErbB4. Increased accumulation of these fragments (indicated by the asterisk in Fig. 4B) in dAsp indicates that these fragments represent a direct substrate for -secretase cleavage.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   In vitro generation of the ErbB4 intracellular domain (B4-ICD). A, schematic representation of full-length (FL) and truncated (E2) ErbB4 constructs and comparison of amino acid sequences of transmembrane regions of ErbB4 with mNotch 1 and APP. B, processing of ErbB4 variants lacking extracellular domains (E2) in wild-type (dW) and double aspartate (dAsp) 293 cells. dW and dAsp cell lines were transfected with the indicated constructs and analyzed by Western blot analyses using the antibodies at the bottom. Note that full-length ErbB4 contains C-terminal HA tag, whereas E2 constructs harbor N-terminal HA tag and C-terminal V5/6xHis tag. Asterisks denote the metalloprotease-derived C-terminal fragments (*) and degradation products (**). C, in vitro production of B4-ICD. Membrane fractions from E2-transfected dW or dAsp cells were incubated at 37 °C for the indicated hours with or without a -secretase inhibitor (1 µM Compound E (Cpd. E)). After the reaction, both supernatant (S100) and the pellet (P100) were analyzed by Western blot analyses using anti-V5 antibody. D, membrane fractions prepared from cells transfected with Notch-E constructs were subjected to the in vitro NICD generation assay as described in C. S100 fractions were analyzed by anti-myc antibody.

Alignment of transmembrane amino acid sequences revealed that ErbB4 has a conserved valine residue, which is topologically similar with the -secretase cleavage site in Notch (Fig. 4A (15)). Recent studies demonstrated that APP also undergoes a similar cleavage, which is reminiscent of the S3 cleavage of Notch at the conserved valine residue (at site 49 of the A), in addition to the expected -secretase cleavage (at sites 40 and 42) (17-19). To directly demonstrate the -secretase-like cleavage of ErbB4 and the subsequent release of soluble intracellular domains of ErbB4 (B4-ICD), we performed in vitro -CTF (e.g. B4-ICD) generation experiments (17-19). Incubation of the cytosol-free membrane fractions prepared from dW cells transfected with ErbB4-E2 constructs (Fig. 3A) led to the release of V5-epitope-tagged B4-ICD into the soluble fraction (Fig. 4C). The release of B4-ICD was increased in a time-dependent manner in dW cells, whereas inhibition occurred in both membrane fractions from dAsp cells and dW cells incubated with Compound E (Fig. 4C), indicating that presenilin-dependent -secretase cleavage is responsible for the release of B4-ICD. In our system, Notch intracellular domain (NICD) was also generated in a -secretase inhibitor-sensitive manner when the membrane fractions were prepared from dW cells transfected with the Notch-E construct (Fig. 4D (15)).

To gain insight into the biological functions of the B4-ICD produced by presenilin-dependent intramembrane cleavage, we then examine its subcellular localization. We determined the subcellular localization of B4-ICD by expressing either GFP alone or an N-terminal GFP-tagged version of ErbB4 containing the entire intracellular domain (GFP-B4ICD; Fig. 5). As shown in Fig. 5, GFP was distributed both in the nucleus and in the cytoplasm. In contrast, the GFP-B4ICD was located predominantly in the nucleus (Fig. 5). Deletion of the entire tyrosine kinase domain did not affect the nuclear localization.³ These results suggest that the B4-ICD may play an unique role in cell signaling in addition to the canonical pathway mediated by the tyrosine kinase activation. This observation is consistent with what has been reported for APP and Notch intracellular domains (AICD and NICD, respectively), because ectopically expressed AICD and NICD localize to the nucleus (13, 14) and appear to participate in gene transcription (15, 16).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Nuclear localization of the ErbB4 intracellular domain (B4-ICD). Confocal microscopy was performed to demonstrate the co-localization of GFP-B4ICD (B4-ICD fused with N-terminal GFP) and the DAPI nuclear staining (bottom panel). GFP controls are shown in the top panel.

With exception of the intracellular domain of Notch 1 (NICD) (12-15), the specific biological function mediated by B4-ICD and other ICD of known -secretase substrates (e.g. APP family) are currently unknown. The signaling specificity may be determined by a set of genes that are selectively activated (or inactivated) by each ICD. Although further experiments are necessary to clearly define the role of the nuclear targeting of B4-ICD, it is interesting to note that nuclear localization of ErbB4 and other ErbB family proteins have previously been described in vivo and in vitro (34-36). For instance, the immunoreactivity of EGF receptor cytoplasmic domain was found in the nucleus, and the occurrence of the nuclear EGFR in highly invasive cancer cell lines was increased (34). The nuclear ErbB4 immunoreactivity has also been observed in many cancer cell lines (35) as well as in cultured primary neurons.³ Our preliminary studies showed that overexpression of truncated ErbB4 inhibited cell growth, raising the possibility that nuclear localization of ErbB4 ICDs may play a role in cell growth and proliferation. These observations further imply that the proteolytic generation of B4-ICD or other ICD, such as B1-, B2-, and B3-ICD, might modulate cell growth and proliferation of normal and cancer cells.

These studies demonstrated that the ectodomain shedding of ErbB4 yields the ~80-kDa membrane-tethered B4-CTF, which serves as a substrate for the presenilin-dependent -secretase-like transmembrane cleavage. The subsequent intramembrane cleavage of ErbB4 requires functional presenilins and can be blocked by -secretase inhibitor in vitro as well as in intact cells. In addition, ectopically expressed B4-ICD was localized to the nucleus. These results suggest that the presenilin-dependent intramembrane cleavage of ErbB4 may mediate a novel signaling function from the cell surface receptor to the nucleus independent of its canonical role as a receptor tyrosine kinase (1-3). Our studies also support the idea that presenilins play a generic role in intramembrane cleavage to produce transcriptionally active intracellular domains of selected type I membrane proteins.

	ACKNOWLEDGEMENTS

We thank G. Struhl and R. Tanzi for helpful comments; T. Golde, A. Fauq, and C. Ziani-Cherif for synthesis and providing compound E; G. Thinakaran, N. Robakis, and D. Selkoe for antibodies; R. Kopan for Notch-E construct; M. Wolfe for the D385A-PS1 construct; and Elizabeth Maas for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants AG18026 (to T.-W. K.) and NS40480 (to L. M.) and by the American Health Assistance Foundation (to T.-W. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Both authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Pathology, Taub Institute for Research on Alzheimer's Disease and the Aging Brain, College of Physicians and Surgeons, Columbia University, 630 W. 168th St., P&S 14-511, New York, NY 10032. Tel.: 212-305-5786; Fax: 212-342-1839; E-mail: twk16@columbia.edu.

Published, JBC Papers in Press, December 10, 2001, DOI 10.1074/jbc.M110371200

² K.-M. Jung and T.-W. Kim, unpublished data.

³ Y. Z. Huang and L. Mei, unpublished data.

	ABBREVIATIONS

The abbreviations used are: PS1, presenilin 1; PS2, presenilin 2; A, amyloid -peptide; APP, amyloid -protein precursor; AICD, APP intracellular domain; B4-CTF, ErbB4 C-terminal fragment; B4-ICD, ErbB4 intracellular domain; NICD, Notch intracellular domain; HA, hemagglutinin; GFP, green fluorescence protein.

	REFERENCES

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