Ectodomain Cleavage of ErbB-4
2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês
10.1074/jbc.m302111200
ISSN1083-351X
AutoresQiu-chen Cheng, Oleg Tikhomirov, Wenli Zhou, Graham Carpenter,
Tópico(s)Peptidase Inhibition and Analysis
ResumoEctodomain cleavage of the ErbB-4 receptor tyrosine kinase generates a membrane-associated fragment of 80 kDa (m80) that has been subjected to N-terminal sequencing. The sequence obtained shows that the N terminus of this fragment begins with Ser-652 of ErbB-4. When a 12-residue peptide corresponding to ErbB-4 residues 646–657 was incubated with recombinant tumor necrosis factor-α-converting enzyme, fragments representing residues 646–651 and 652–657 were obtained. These data indicate that ectodomain cleavage of ErbB-4 occurs between His-651 and Ser-652, placing the cleavage site within the ectodomain stalk region approximately 8 residues prior to the transmembrane domain. Several experiments have characterized other aspects of the m80 ErbB-4 fragment. Inhibition of ErbB-4 tyrosine kinase activity with pan-ErbB tyrosine kinase inhibitors indicates that kinase activity is stringently required for heregulin-dependent, but not 12-O-tetradecanoylphorbol-13-acetate-induced, ErbB-4 ectodomain cleavage and formation of the m80 fragment. When the m80 ErbB-4 fragment is generated by cell treatment with heregulin or 12-O-tetradecanoylphorbol-13-acetate, the fragment associates with intact ErbB-2. However, this fragment does not associate with the intact ErbB-4 molecule. Ectodomain cleavage of the ErbB-4 receptor tyrosine kinase generates a membrane-associated fragment of 80 kDa (m80) that has been subjected to N-terminal sequencing. The sequence obtained shows that the N terminus of this fragment begins with Ser-652 of ErbB-4. When a 12-residue peptide corresponding to ErbB-4 residues 646–657 was incubated with recombinant tumor necrosis factor-α-converting enzyme, fragments representing residues 646–651 and 652–657 were obtained. These data indicate that ectodomain cleavage of ErbB-4 occurs between His-651 and Ser-652, placing the cleavage site within the ectodomain stalk region approximately 8 residues prior to the transmembrane domain. Several experiments have characterized other aspects of the m80 ErbB-4 fragment. Inhibition of ErbB-4 tyrosine kinase activity with pan-ErbB tyrosine kinase inhibitors indicates that kinase activity is stringently required for heregulin-dependent, but not 12-O-tetradecanoylphorbol-13-acetate-induced, ErbB-4 ectodomain cleavage and formation of the m80 fragment. When the m80 ErbB-4 fragment is generated by cell treatment with heregulin or 12-O-tetradecanoylphorbol-13-acetate, the fragment associates with intact ErbB-2. However, this fragment does not associate with the intact ErbB-4 molecule. ErbB-4 is a member of the ErbB receptor tyrosine kinase family, which also includes the epidermal growth factor receptor (ErbB-1), ErbB-2, and ErbB-3 (1Carpenter G. Exp. Cell Res. 2003; 284: 66-77Crossref PubMed Scopus (206) Google Scholar). ErbB-4 and ErbB-3 bind the neuregulin (heregulin) family of growth factors, and ErbB-4 also recognizes certain growth factors in the epidermal growth factor family of ErbB-1 agonists, such as betacellulin, epiregulin, and heparin-binding epidermal growth factor. ErbB-4 heterodimerizes with ErbB-2 as do ErbB-1 and ErbB-3; however, ErbB-4 can also signal through the formation of ErbB-4 homodimers. Within the receptor tyrosine kinase family, ErbB-4 is uniquely processed (2Ni C.-Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar) by a proteolytic pathway that is known to occur with certain other transmembrane proteins, such as Notch (3Fontini M.D. Nat. Rev. Mol. Cell Biol. 2002; 3: 673-684Crossref PubMed Scopus (345) Google Scholar), the low density lipoprotein receptor-related protein (4My P. Reddy Y.K. Herz J. J. Biol. Chem. 2002; 277: 18736-18743Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), the amyloid precursor protein (APP) 1The abbreviations used are: APP, amyloid precursor protein; ALLN, N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal; TPA, 12-O-tetradecanoylphorbol-13-acetate; DMEM, Dulbecco's modified Eagle's medium; PVDF, polyvinylidene difluoride; ADAM, a disintegrin and metalloprotease; TNFα, tumor necrosis factor-α; TACE, TNFα-converting enzyme. (5Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), and the adhesion molecules CD44 (6Okamoto I. Kawano Y. Murakami D. Sasayama T. Araki N. Miki T. Wong A.J. Saya H. J. Cell Biol. 2001; 155: 755-762Crossref PubMed Scopus (310) Google Scholar, 7Lammich S. Okochi M. Takeda M. Kaether C. Capell A. Zimmer A.-K. Edbauer D. Walter J. Steiner H. Haass C. J. Biol. Chem. 2002; 277: 44754-44759Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar) and E-cadherin (9Vecchi M. Baulida J. Carpenter G. J. Biol. Chem. 1996; 271: 18989-18995Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The first step in this pathway involves the release of the ErbB-4 ectodomain by a cleavage that produces two fragments as follows: a 120-kDa ectodomain fragment and an 80-kDa membrane-associated fragment, designated m80 (9Vecchi M. Baulida J. Carpenter G. J. Biol. Chem. 1996; 271: 18989-18995Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The latter fragment contains the ErbB-4 transmembrane domain and the entire cytoplasmic region, including the tyrosine kinase domain. Ectodomain cleavage of ErbB-4 in cells occurs at a low constitutive or basal level (10Vecchi M. Carpenter G. J. Cell Biol. 1997; 139: 995-1003Crossref PubMed Scopus (114) Google Scholar) that can be increased by TPA (9Vecchi M. Baulida J. Carpenter G. J. Biol. Chem. 1996; 271: 18989-18995Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) or by heregulin or other growth factors that bind ErbB-4 (11Zhou W. Carpenter G. J. Biol. Chem. 2000; 275: 34737-34743Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Also, cleavage is potentiated by the addition to cells of pervanadate, a tyrosine phosphatase inhibitor that provokes ErbB-4 tyrosine phosphorylation (12Vecchi M. Rudolph-Owen L.A. Brown C.L. Dempsey P.J. Carpenter G. J. Biol. Chem. 1998; 273: 20589-20595Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The ectodomain cleavage of ErbB-4 is sensitive to metalloprotease inhibitors (10Vecchi M. Carpenter G. J. Cell Biol. 1997; 139: 995-1003Crossref PubMed Scopus (114) Google Scholar) and does not occur in cells genetically deficient in tumor necrosis factor-α-converting enzyme (TACE), a transmembrane metalloprotease, also known as adisintegrin and metalloprotease or ADAM17 (13Rio C. Buxbaum J.D. Peschon J.J. Corfas G. J. Biol. Chem. 2000; 275: 10379-10387Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). The mechanism by which TACE participates in this cleavage is not understood. Interestingly, a splice form of ErbB-4 known as Jm-b is present in human and mouse tissues and is resistant to ectodomain cleavage (14Elenius K. Corfas G. Paul S. Choi C.J. Rio C. Plowman G.D. Klagsbrun M. J. Biol. Chem. 1997; 272: 26762-26768Abstract Full Text Full Text PDF Scopus (188) Google Scholar). Compared with the cleavable Jm-a isoform of ErbB-4, the Jm-b isoform contains a unique sequence within the ectodomain stalk region. The ErbB-4 m80 fragment that is generated by ectodomain cleavage is further processed by two competing protease activities. The fragment can be degraded by proteosome activity following polyubiquitination (10Vecchi M. Carpenter G. J. Cell Biol. 1997; 139: 995-1003Crossref PubMed Scopus (114) Google Scholar). More importantly, the m80 fragment is also a substrate for γ-secretase activity (2Ni C.-Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar, 15Lee H.-J. Jung K.-M. Huang Y.A. Bennett L.B. Lee J.S. Mei L. Kim T.-W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). γ-Secretase is a multicomponent proteolytic activity that cleaves proteins, such as Notch and APP, within their transmembrane domains and releases the cytoplasmic domains from the plasma membrane allowing them to relocalize within the cell (6Okamoto I. Kawano Y. Murakami D. Sasayama T. Araki N. Miki T. Wong A.J. Saya H. J. Cell Biol. 2001; 155: 755-762Crossref PubMed Scopus (310) Google Scholar). In the case of ErbB-4, as well as Notch and APP, the cytoplasmic domain is rapidly found in the nucleus (2Ni C.-Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar, 15Lee H.-J. Jung K.-M. Huang Y.A. Bennett L.B. Lee J.S. Mei L. Kim T.-W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Although the function of the ErbB-4 cytoplasmic domain in the nucleus is not known, evidence indicates that heregulin-dependent cell death in T47D cells requires γ-secretase activity (2Ni C.-Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar). The capacity of ErbB-4 to be processed by γ-secretase activity requires ectodomain cleavage as a prerequisite (2Ni C.-Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar). This is analogous to the processing of Notch and APP (5Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) and is likely the point at which ErbB-4 processing is regulated by TPA or heregulin. Therefore, we have continued to investigate the ectodomain cleavage event and report herein several relevant findings. Materials—Heregulin β1 was obtained from R & D Systems, Inc. TPA and protease inhibitor mixture were products of Sigma and Aldrich. N-Acetyl-l-leucinyl-l-leucinyl-l-norleucinal (ALLN) and PD153035 were purchased from Calbiochem-Novabiochem. CI-1003 was a gift from David Fry, Pfizer. Horseradish peroxidase-conjugated Protein A, Protein A-Sepharose, and Protein G-Sepharose were from Zymed Laboratories Inc. Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum were from Invitrogen. Polyclonal antibody to residues 1291–1308 of the ErbB-4 C-terminal domain was obtained from Santa Cruz Biotechnology and was used for immunoprecipitation. Another polyclonal antibody to residues 1108–1264 in the ErbB-4 C-terminal domain was a gift of Matthias Kraus, University of Alabama, Birmingham, and was used for Western blotting. A monoclonal antibody to the ectodomain of ErbB-4 was purchased from NeoMarkers, Inc. Anti-phosphotyrosine (monoclonal) was a product of Transduction Laboratories. Recombinant TACE catalytic domain (16Plowman G.D. Culouscou J.-M. Whitney G.S. Green J.M. Carlton G.W. Foy L. Neubauer M.D. Shoyab M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1746-1750Crossref PubMed Scopus (689) Google Scholar) and the pro-TNFα peptide (Dnp-SPLAQAVRSSSR) were gifts of David Becherer, Glaxo-SmithKline. The ErbB-4 peptide (Ac-YPWTGHSTLPQH) was a gift of James Tam, Vanderbilt University. Polyvinylidene difluoride (PVDF) membranes were from Bio-Rad. Anti-TACE, a purified monoclonal antibody to human TACE (M220), was generously provided by Roy Black, Immunex. N-terminal Sequence Analysis—T47-14 cells were grown to confluence in 10 15-cm culture dishes and were then treated with TPA (100 ng/ml) for 45 min at 37 °C. Subsequently, the cells were lysed in cold buffer (1% Nonidet P-40, 10 mm Tris, pH 7.4, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, aprotinin (10 μg/ml), leupeptin (10 μg/ml)), and the lysate was clarified by centrifugation (14,000 × g, 10 min). Polyclonal antibody (50 μg) to the ErbB-4 C-terminal domain was then added and incubated for 2 h at 4 °C before the addition of Protein A-Sepharose. After 2 h the resulting precipitate was washed in cold lysis buffer three times. The precipitate was then treated with a buffer containing 10 mm Tris-Cl, pH 7.4, 150 mm NaCl, and 1.5% SDS for 10 min at 100 °C to elute precipitated proteins. The eluate was then diluted 10-fold into lysis buffer. The diluted eluate was used for a second immunoprecipitation that employed a 2-h incubation with a monoclonal antibody to the ErbB-4 C-terminal domain and Protein G-Sepharose followed by washing with lysis buffer. This precipitate was placed on an SDS-PAGE, and after electrophoresis, the proteins were transferred to a PVDF membrane. The membrane was stained with Coomassie Blue, and a band at 80 kDa was cut out and eluted. Edman microsequencing using an Applied Biosystems 492 sequencer was performed by the Vanderbilt Peptide Sequencing and Amino Acid Analysis Shared Resource. Analysis of Peptide Cleavage—Two synthetic 12-residue peptides were used as substrates: a pro-TNFα peptide that contains a dinitrophenyl group at the N terminus and that contains the known cleavage site present in the TNFα precursor, and an ErbB-4 peptide that is acetylated at the N terminus. Each peptide was incubated at 37 °C for the indicated period of time in 20 mm Hepes buffer, pH 7.4, with or without the indicated concentration of recombinant human TACE catalytic domain (16Plowman G.D. Culouscou J.-M. Whitney G.S. Green J.M. Carlton G.W. Foy L. Neubauer M.D. Shoyab M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1746-1750Crossref PubMed Scopus (689) Google Scholar) in a final volume of 50 μl. To assess cleavage of the peptide substrates by mass spectrometry, an aliquot (∼1 μl) was mixed with 1 μl of α-cyano-4-hydroxycinnamic acid (CHCA) matrix and spotted on a sample plate for mass spectrometry. This matrix was made using a saturated solution of α-cyano-4-hydroxycinnamic acid in 50:50 (v/v) mixture of acetonitrile and 0.1% trifluoroacetic acid in water. The samples were then assayed on a PerSeptive Biosystems Elite matrix-assisted laser desorption/ionization-time of flight-mass spectrometer utilizing the reflectron detector. Standardization of peptide masses was performed by utilizing a peptide mixture supplied by the Vanderbilt Mass Spectrometry Facility. Analysis was performed by the Vanderbilt Peptide Sequencing and Amino Acid Analysis Shared Resource. Cell Culture—T47D cells are a human mammary carcinoma cell line obtained from the American Tissue Culture Collection. T47-14 cells are NIH/3T3 cells that have been transfected to express human ErbB-4 and were provided by Matthias Kraus, University of Alabama-Birmingham. Both have been described previously (9Vecchi M. Baulida J. Carpenter G. J. Biol. Chem. 1996; 271: 18989-18995Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11Zhou W. Carpenter G. J. Biol. Chem. 2000; 275: 34737-34743Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The cells were grown in DMEM containing 10% fetal bovine serum and used for experiments when nearly confluent. Prior to each experiment the cells were incubated for about 24 h in DMEM plus 0.5% fetal bovine serum. Immediately prior to the addition of growth factors or TPA at 37 °C, the cultures were washed with phosphate-buffered saline and DMEM containing 20 mm Hepes, pH 7.2, and 0.1% bovine serum albumin was added. Cell Lysis and Immunologic Analysis—After each experimental treatment, the cells were washed with ice-cold phosphate-buffered saline and immediately lysed by the addition of cold TGH lysis buffer (1% Triton X-100, 10% glycerol, 20 mm Hepes, pH 7.2, 100 mm NaCl), supplemented with protease inhibitor mixture P8340 (Sigma) and 50 mm NaF, for 30 min at 4 °C. The lysate was then centrifuged (14,000 × g, 10 min) and the supernatant used for immunologic measurement and determination of protein concentration (Bio-Rad Protein Assay Reagent). For subsequent immunoprecipitations ∼1 mg of lysate was incubated with the indicated antibody for 30 min at 4 °C with rocking followed by the addition of Protein A- or Protein G-Sepharose as appropriate, and the incubation was continued for3hat4 °C. The precipitates were then washed with TGH lysis buffer three times. Laemmli sample buffer (1.5×) was then added, and the samples were heated (100 °C) for 5 min. Subsequently the samples were vortexed and centrifuged (8,000 rpm, 2 min) to remove the Sepharose beads. The samples were then loaded on SDS-PAGE (7.5%). After electrophoresis the separated proteins were transferred to Protran nitrocellulose membranes (Schleicher & Schuell). The membranes were blocked with 5% dry milk in TBST buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 0.05% Tween 20) for 1 h at room temperature with rocking. The membranes were then washed twice with TBST buffer and incubated overnight at 4 °C with the indicated antibody in TBST buffer containing 1.0% bovine serum albumin. The membranes were then washed four times with TBST, and the horseradish peroxidase-conjugated Protein A was added for 1 h at room temperature with rocking. The samples were washed four times with TBST, and bound horseradish peroxidase was detected by enhanced chemiluminescence. Where indicated, blots were stripped and reprobed with a second antibody. The stripping solution contained 62.5 mm Tris-Cl, pH 6.7, 100 mm β-mercaptoethanol, and 2% SDS. Ectodomain Cleavage Site—To obtain N-terminal sequence information regarding the m80 ErbB-4 fragment, a large number of cultures of T47-14 cells were treated with TPA for 30 min, and the m80 fragment was immunopurified by two rounds of antibody precipitation. A single Coomassie Blue-stained 80-kDa band was then eluted and subjected to automated Edman microsequencing. The first seven cycles yielded the sequence STLPQ(R/H)A. The alignment of this sequence with the known sequence of ErbB-4 is presented in Fig. 1. The sequence obtained for the N terminus of the m80 fragment aligns correctly with the ErbB-4 Jm-a isoform sequence (16Plowman G.D. Culouscou J.-M. Whitney G.S. Green J.M. Carlton G.W. Foy L. Neubauer M.D. Shoyab M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1746-1750Crossref PubMed Scopus (689) Google Scholar) beginning with Ser-652 and is within the receptor stalk region between the second cysteine-rich domain and the transmembrane domain. Also presented in Fig. 1 are sequences for the stalk regions of the Jm-b ErbB-4 isoform, ErbB-1, ErbB-2, and ErbB-3. The above data indicate that ectodomain cleavage of ErbB-4 probably occurs between His-651 and Ser-652. However, it is possible that cleavage occurs prior to this point in the sequence and that the isolated m80 fragment involves a subsequent loss of N-terminal residues due to secondary proteolytic activity. To test the capacity of TACE to cleave ErbB-4 within the stalk region, a 12-residue peptide (YPWTGHSTLPQH) corresponding to residues 646–657 of ErbB-4 was incubated with recombinant TACE. Cleavage of the peptide was assessed by mass spectrometry. This approach has been used before with other cell surface molecules that are subject to TACE-mediated ectodomain cleavage. Since the TACE-dependent cleavage site of pro-TNFα has been well characterized by this approach (17Moss M.L. Jin S.-L.C. Milla M.E. Burkhart W. Carter H.L. Chen W.-J. Clay W.C. Didsbury J.R. Hassler D. Hofman C.R. Kost T.A. Lambert M.H. Leesnitzer M.A. McCauley P. McGehan G. Mitchell J. Moyer M. Pahel G. Rocque W. Overton L.K. Schoenen F. Seaton T. Su J.-L. Warner J. Willard D. Becherer J.D. Nature. 1997; 385: 733-736Crossref PubMed Scopus (1490) Google Scholar, 18Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2728) Google Scholar), a 12-residue peptide (SPLAQAVRSSSR) including the known cleavage site within stalk region of pro-TNFα was used as a control. The initial experiments utilized reaction conditions that have been reported previously to support the TACE-dependent cleavage of the pro-TNFα peptide substrate. The results, shown in Fig. 2A, demonstrate that under these conditions the pro-TNFα substrate peptide was cleaved following incubation with TACE and that two major products were detected, peptides a and b. These two peptides correspond to those expected for cleavage of the substrate between Ala-76 and Val-77, the known pro-TNFα cleavage site (17Moss M.L. Jin S.-L.C. Milla M.E. Burkhart W. Carter H.L. Chen W.-J. Clay W.C. Didsbury J.R. Hassler D. Hofman C.R. Kost T.A. Lambert M.H. Leesnitzer M.A. McCauley P. McGehan G. Mitchell J. Moyer M. Pahel G. Rocque W. Overton L.K. Schoenen F. Seaton T. Su J.-L. Warner J. Willard D. Becherer J.D. Nature. 1997; 385: 733-736Crossref PubMed Scopus (1490) Google Scholar, 18Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2728) Google Scholar). Under these same reaction conditions, however, the ErbB-4 peptide was not cleaved (data not shown). In view of the above result, the reaction conditions were readjusted to include the addition of more enzyme and a prolonged incubation time to test for less efficient TACE cleavage of the ErbB-4 peptide compared with the pro-TNFα substrate. Specifically, the amount of TACE was increased from 8 to 250 nm and the incubation time from 3 to 16 h. The results are shown in Fig. 2B. In the absence of added TACE no degradation products from the ErbB-4 peptide were detected. Under these conditions the presence of TACE did induce the cleavage of the ErbB-4 substrate peptide with the appearance of three peptide products. The peptide products denoted B and A correspond, respectively, to peptides having the sequence YPWTGH and STLPQH. These are the products expected by cleavage of the substrate between His and Ser residues corresponding to residues 651 and 652 of ErbB-4. Also, the sequence of peptide A corresponds to the N-terminal sequence obtained for the isolated m80 ErbB-4 fragment. Under these conditions in vitro a third peptide, denoted C, was also produced and corresponds to the sequence YPWTGHSTLP of the ErbB-4 peptide. This most likely arises from a second cleavage site in the substrate between residues Pro and Gln and would predict the presence of a fourth product, a dipeptide Gln-His, that was not detected in the chromatogram. Most likely this represents an in vitro cleavage that does not occur in vivo. Role of Tyrosine Kinase Activity in ErbB-4 Ectodomain Cleavage—A significant question in the ligand-induced proteolytic processing of ErbB-4 is whether its growth factor-dependent tyrosine kinase activity is required. One approach would be to transfect a kinase-negative ErbB-4 mutant into recipient cells and then assay heregulin-induced cleavage. However, we have reported previously that exogenously expressed ErbB-4, in several recipient cell lines, does not exhibit heregulin-induced proteolytic processing (11Zhou W. Carpenter G. J. Biol. Chem. 2000; 275: 34737-34743Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The reasons for this are not clear but preclude a mutagenesis approach. Several highly selective tyrosine kinase chemical inhibitors have been developed to ErbB-1 and can be used at higher concentrations to inhibit all ErbB tyrosine kinase activities. In the experiment shown in Fig. 3, PD153035 (19Fry D.W. Kraker A.J. McMichael A. Ambroso L.A. Nelson J.M. Leopold W.R. Connors R.W. Bridges A.J. Science. 1994; 265: 1093-1095Crossref PubMed Scopus (815) Google Scholar) was used at a concentration experimentally determined to completely prevent heregulin-dependent ErbB-4 tyrosine kinase activity. When T47D cells were treated with heregulin or TPA, formation of the m80 ErbB-4 fragment was increased. When the cells were exposed to PD153035 and heregulin or TPA, the amount of m80 fragment formed was significantly decreased, particularly in the case of heregulin stimulation. Blotting with anti-phosphotyrosine shows that heregulin provoked autophosphorylation of ErbB-4, but TPA did not. Similar results also were obtained with a different ErbB kinase inhibitor CI-1003 (20Slichenmyer W.J. Elliott W.L. Fry D.W. Semin. Oncol. 2001; 28: 80-85Crossref PubMed Google Scholar). The data show, somewhat surprisingly, that the tyrosine kinase inhibitors abrogate TPA induction of ErbB-4 ectodomain cleavage, although TPA does not induce a detectable level of ErbB-4 tyrosine phosphorylation. In this experiment ErbB-4 cleavage was measured in T47D cells, a breast cancer line in which TPA also induces the shedding of endogenous heregulin from its cell surface precursor. 2G. Carpenter and Q.-C. Cheng, unpublished data. It seems possible, therefore, that this TPA-dependent autocrine circuit contributes to the TPA-induced cleavage of ErbB-4. Although TPA treatment does not produce a detectable level of ErbB-4 autophosphorylation, it is possible that autocrine stimulation occurs at a low level compared with the addition of a saturating quantity of exogenous heregulin. To test the T47-14 cells, an NIH/3T3 fibroblast cell line that expresses exogenous ErbB-4 was treated with TPA in the presence and absence of tyrosine kinase inhibitors. These cells do not express detectable levels of endogenous heregulin. The results, shown in Fig. 4, demonstrate that in this cell line TPA-induced cleavage was not sensitive to inhibitors of ErbB tyrosine kinase activity. In a previous study, we reported that ErbB-4 is rapidly translocated to a detergent-insoluble fraction following the addition of heregulin but not TPA (11Zhou W. Carpenter G. J. Biol. Chem. 2000; 275: 34737-34743Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). This event correlates with the sensitivity of ErbB-4-expressing cells to ectodomain cleavage initiated by heregulin. Therefore, we tested whether tyrosine kinase activity is necessary for heregulin-dependent translocation of ErbB-4 to the detergent-insoluble fraction in T47D cells. The results of this experiment, shown in Fig. 5A, demonstrate that tyrosine kinase activity is required for this translocation. To pursue the correlation between ectodomain cleavage of ErbB-4 and translocation to the detergent-insoluble fraction, we have measured the level of TACE found in the detergent-soluble and -insoluble fractions. The data, presented in Fig. 5B, indicate that significantly higher levels of TACE are present in the detergent-insoluble fraction compared with the soluble fraction. Although heregulin is required for ErbB-4 translocation to the insoluble fraction, the growth factor has only a small influence on the distribution of TACE. The addition of heregulin slightly increased (43%) the level of TACE in the detergent-soluble fraction and slightly decreased (30%) the amount of TACE in the insoluble fraction. Association of m80 and ErbB-2—A characteristic of ErbB receptors is their propensity to homo- and heterodimerize following the addition of ligand. ErbB-4 can homodimerize or heterodimerize with ErbB-2 (1Carpenter G. Exp. Cell Res. 2003; 284: 66-77Crossref PubMed Scopus (206) Google Scholar). The mechanism of ErbB-4 association with itself or ErbB-2 is not known. To test whether the m80 ErbB-4 fragment associates with the full-length ErbB-4 molecule, T47D cells were treated with heregulin or TPA, and lysates were prepared. One-half of each lysate was then incubated with an ErbB-4 antibody that recognizes an ectodomain epitope not present in the m80 fragment to selectively precipitate the full-length ErbB-4 molecule. The immunoprecipitates were then tested by Western blotting for ErbB-4-reactive material using an antibody to a C-terminal epitope present in both the full-length and m80 forms of ErbB-4. The results, shown in Fig. 6 (lanes 1–3), indicate that no m80 fragment could be detected. Although the level of full-length molecule is decreased by TPA or heregulin treatment, there is still a reasonable level available to potentially form an association complex with the m80 fragment. The remaining one-half of each lysate was used as a positive control. ErbB-4 was immunoprecipitated with an antibody to the ErbB-4 C terminus and then blotted with the same antibody. The results (Fig. 6, lanes 4–6) show that in this experiment the m80 fragment was, in fact, generated by heregulin or TPA. To assess the possible association of the ErbB-4 m80 fragment with ErbB-2, the experiment shown in Fig. 7 was conducted. T47D cells were treated with either heregulin (Fig. 7A) or TPA (Fig. 7B) in the presence or absence of ALLN, a protease inhibitor, which we have shown previously prevents the metabolic degradation of the m80 ErbB-4 fragment (10Vecchi M. Carpenter G. J. Cell Biol. 1997; 139: 995-1003Crossref PubMed Scopus (114) Google Scholar). After treating the cells, lysates were prepared and precipitated with an antibody to ErbB-2. The precipitates were subsequently analyzed by Western blotting with antibody to the C terminus of ErbB-4. The results show that following the addition of either heregulin or TPA to induce ErbB-4 ectodomain cleavage, the m80 fragment is found associated with ErbB-2, which is not cleaved. The presence of ALLN increases both the basal and agonist-stimulated levels of the m80 fragment associated with ErbB-2. In these experiments, the data from untreated cells (lane 1 in Fig. 7, A and B) show that ErbB-2 coprecipitates with ErbB-4 and implies the presence of preformed dimers or oligomers. Others have shown that the ErbB-1 receptor can self-associate in the absence of added ligand (21Gadella Jr., T.W. Jovin T.M. J. Cell Biol. 1995; 129: 1543-1558Crossref PubMed Scopus (371) Google Scholar, 22Sako Y. Minoguchi S. Yanagida T. Nat. Cell Biol. 2000; 2: 168-172Crossref PubMed Scopus (756) Google Scholar, 23Yi X. Sharma K.D. Takahashi T. Iwamoto R. Mekada E. Mol. Biol. Cell. 2002; 13: 2547-2557Cr
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