Transmembrane Neuregulins Interact with LIM Kinase 1, a Cytoplasmic Protein Kinase Implicated in Development of Visuospatial Cognition
1998; Elsevier BV; Volume: 273; Issue: 32 Linguagem: Inglês
10.1074/jbc.273.32.20525
ISSN1083-351X
AutoresJay Y. Wang, Kristen Frenzel, Duanzhi Wen, Douglas L. Falls,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe neuregulins are receptor tyrosine kinase ligands that play a critical role in the development of the heart, nervous system, and breast. Unlike many extracellular signaling molecules, such as the neurotrophins, most neuregulins are synthesized as transmembrane proteins. To determine the functions of the highly conserved neuregulin cytoplasmic tail, a yeast two-hybrid screen was performed to identify proteins that interact with the 157-amino acid sequence common to the cytoplasmic tails of all transmembrane neuregulin isoforms.This screen revealed that the neuregulin cytoplasmic tail interacts with the LIM domain region of the nonreceptor protein kinase LIM kinase 1 (LIMK1). Interaction between the neuregulin cytoplasmic tail and full-length LIMK1 was demonstrated by in vitro binding and co-immunoprecipitation assays. Transmembrane neuregulins with each of the three known neuregulin cytoplasmic tail isoforms interacted with LIMK1. In contrast, the cytoplasmic tail of TGF-α did not interact with LIMK1. In vivo, neuregulin and LIMK1 are co-localized at the neuromuscular synapse, suggesting that LIMK1, like neuregulin, may play a role in synapse formation and maintenance. To our knowledge, LIMK1 is the first identified protein shown to interact with the cytoplasmic tail of a receptor tyrosine kinase ligand. The neuregulins are receptor tyrosine kinase ligands that play a critical role in the development of the heart, nervous system, and breast. Unlike many extracellular signaling molecules, such as the neurotrophins, most neuregulins are synthesized as transmembrane proteins. To determine the functions of the highly conserved neuregulin cytoplasmic tail, a yeast two-hybrid screen was performed to identify proteins that interact with the 157-amino acid sequence common to the cytoplasmic tails of all transmembrane neuregulin isoforms. This screen revealed that the neuregulin cytoplasmic tail interacts with the LIM domain region of the nonreceptor protein kinase LIM kinase 1 (LIMK1). Interaction between the neuregulin cytoplasmic tail and full-length LIMK1 was demonstrated by in vitro binding and co-immunoprecipitation assays. Transmembrane neuregulins with each of the three known neuregulin cytoplasmic tail isoforms interacted with LIMK1. In contrast, the cytoplasmic tail of TGF-α did not interact with LIMK1. In vivo, neuregulin and LIMK1 are co-localized at the neuromuscular synapse, suggesting that LIMK1, like neuregulin, may play a role in synapse formation and maintenance. To our knowledge, LIMK1 is the first identified protein shown to interact with the cytoplasmic tail of a receptor tyrosine kinase ligand. The neuregulins (NRGs) 1The abbreviations used are: NRGneuregulinCAPS3-(cyclohexylamino)-1-propanesulfonic acidGSTglutathioneS-transferaseLIMK1LIM kinase 1LIMK1-ldrLIMK1 LIM domain regionPBSphosphate-buffered salinePCRpolymerase chain reactionPVDFpolyvinylidene difluorideRTKreceptor tyrosine kinaseTGF-αtransforming growth factor αTMtransmembraneAbantibody. were originally identified in searches for ligands of the receptor tyrosine kinase erbB2 (1Peles E. Bacus S.S. Koski R.A. Lu H.S. Wen D. Ogden S.G. Levy R.B. Yarden Y. Cell. 1992; 69: 205-216Abstract Full Text PDF PubMed Scopus (478) Google Scholar, 2Wen D. Peles E. Cupples R. Suggs S.V. Bacus S.S. Luo Y. Trail G. Hu S. Silbiger S.M. Levy R.B. Koski R.A. Lu H.S. Yarden Y. Cell. 1992; 69: 559-572Abstract Full Text PDF PubMed Scopus (526) Google Scholar, 3Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N. Raab H. Lewis G.D. Shepard H.M. Kuang W.-J. Wood W.I. Goeddel D.V. Vandlen R.L. 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The extracellular epidermal growth factor-like domain of TM-NRGs activates the receptor tyrosine kinases erbB2, erbB3, and erbB4. Most prior NRG studies have focused on the interaction of the NRG extracellular domain with these receptor tyrosine kinases (RTKs) and the biological consequences of erbB2/erbB3/erbB4 activation by NRG. In contrast, this study focused on the long intracellular region of TM-NRG isoforms (see Fig. 1). The high degree of amino acid sequence conservation of this intracellular region (4Falls D.L. Rosen K.M. Corfas G. Lane W.S. Fischbach G.D. Cell. 1993; 72: 801-815Abstract Full Text PDF PubMed Scopus (554) Google Scholar) suggests that it has important biological functions. Grimm and Leder (22Grimm S. Leder P. J. Exp. Med. 1997; 185: 1137-1142Crossref PubMed Scopus (34) Google Scholar) recently reported that one form of the NRG cytoplasmic tail (the b-tail) can activate apoptosis in TM-NRG-transfected HEK 293 cells. Two other potential biological functions of the NRG cytoplasmic tail are regulation of NRG protein trafficking and of proteolytic release of the NRG ectodomain into the extracellular space (see Refs. 13Wen D.Z. Suggs S.V. Karunagaran D. Liu N.L. Cupples R.L. Luo Y. Janssen A.M. Benbaruch N. Trollinger D.B. Jacobsen V.L. Meng S.Y. Lu H.S. Hu S. Chang D. Yang W.N. Yanigahara D. Koski R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (234) Google Scholar, 17Burgess T.L. Ross S.L. Qian Y. Brankow D. Hu S. J. Biol. Chem. 1995; 270: 19188-19196Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 23Bosenberg M.W. Pandiella A. Massagué J. Cell. 1992; 71: 1157-1165Abstract Full Text PDF PubMed Scopus (109) Google Scholar, and 24Briley G.P. Hissong M.A. Chiu M.L. Lee D.C. Mol. Biol. Cell. 1997; 8: 1619-1631Crossref PubMed Scopus (69) Google Scholar). Another intriguing possibility is that transmembrane NRG may function not only as a receptor ligand but also as a receptor and that the NRG cytoplasmic tail mediates outside-in signal transduction. If NRG acts as a "receptor" and the RTKs erbB2, erbB3, and erbB4 are its "ligand," bi-directional signaling could occur between cells expressing TM-NRG and cells expressing the RTKs erbB2, erbB3, and erbB4. The idea of bi-directional signaling between cells expressing an RTK TM ligand and cells expressing the cognate RTK was first suggested by Pfeffer and Ullrich (25Pfeffer S. Ullrich A. Nature. 1985; 313: 184Crossref PubMed Scopus (50) Google Scholar), and recent in vivo and in vitro studies of the interaction between the TM ligand LERK-2 and the RTK Nuk (26Henkemeyer M. Orioli D. Henderson J.T. Saxton T.M. Roder J. Pawson T. Klein R. Cell. 1996; 86: 35-46Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 27Holland S.J. Gale N.W. Mbamalu G. Yancopoulos G.D. Henkemeyer M. 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Koski R.A. Lu H.S. Yarden Y. Cell. 1992; 69: 559-572Abstract Full Text PDF PubMed Scopus (526) Google Scholar, 3Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N. Raab H. Lewis G.D. Shepard H.M. Kuang W.-J. Wood W.I. Goeddel D.V. Vandlen R.L. Science. 1992; 256: 1205-1210Crossref PubMed Scopus (925) Google Scholar, 4Falls D.L. Rosen K.M. Corfas G. Lane W.S. Fischbach G.D. Cell. 1993; 72: 801-815Abstract Full Text PDF PubMed Scopus (554) Google Scholar, 5Goodearl A. Davis J.B. Mistry K. Minghetti L. Otsu M. Waterfield M.D. Stroobant P. J. Biol. Chem. 1993; 268: 18095-18102Abstract Full Text PDF PubMed Google Scholar, 6Marchionni M.A. Goodearl A.D.J. Chen M.S. Bermingham-McDonogh O. Kirk C. Hendricks M. Danehy F. Misumi D. Sudhalter J. Kobayashi K. Wroblewski D. Lynch C. Baldassare M. Hiles I. Davis J.B. Hsuan J.J. Totty N.F. Otsu M. McBurney R.N. Waterfield M.D. Stroobant P. Gwynne D. Nature. 1993; 362: 312-318Crossref PubMed Scopus (681) Google Scholar). These proteins might now be considered forms of NRG1 in light of the recent discovery of related proteins encoded by two other NRG family genes. These NRG1-related proteins have been dubbed NRG2 (or Don-1) (36Carraway K.L. Weber J.L. Unger M.J. Ledesma J. Yu N. Gassmann M. Lai C. Nature. 1997; 387: 512-516Crossref PubMed Scopus (342) Google Scholar, 37Chang H. Riese D.J. Gilbert W. Stern D.F. McMahan U.J. Nature. 1997; 387: 509-512Crossref PubMed Scopus (256) Google Scholar, 38Busfield S.J. Michnick D.A. Chickering T.W. Revett T.L. Ma J.Y. Woolf E.A. Comrack C.A. Dussault B.J. Woolf J. Goodearl A.D.J. Gearing D.P. Mol. Cell. Biol. 1997; 17: 4007-4014Crossref PubMed Scopus (106) Google Scholar) and NRG3 (39Zhang D. Sliwkowski M.X. Mark M. Frantz G. Akita R. Sun Y. Hillan K. Crowley C. Brush J. Godowski P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9562-9567Crossref PubMed Scopus (328) Google Scholar). All of the protein isoforms that are the subject of this study are produced from transcripts of the NRG1 gene. The cDNA clones encoding rat NRG isoforms are described in Ref. 13Wen D.Z. Suggs S.V. Karunagaran D. Liu N.L. Cupples R.L. Luo Y. Janssen A.M. Benbaruch N. Trollinger D.B. Jacobsen V.L. Meng S.Y. Lu H.S. Hu S. Chang D. Yang W.N. Yanigahara D. Koski R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (234) Google Scholar. The cDNA clone that encodes the full-length murine LIMK1 was a gift from E. Robertson (Department of Molecular and Cellular Biology, Harvard University) (31Cheng A.K. Robertson E.J. Mech. Dev. 1995; 52: 187-197Crossref PubMed Scopus (47) Google Scholar). The yeast two-hybrid bait vector pBTM116 and prey vector pVP16 were provided by S. Hollenberg (Fred Hutchinson Cancer Research Center, Seattle, WA) (40Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar). The mouse brain library in pVP16, the PER bait, and the PER prey were provided by C. Weitz and N. Gekakis (Harvard Medical School). Polyclonal antibody 1310, raised against the common region of the NRG cytoplasmic tail, and the immunizing peptide were a gift from T. Burgess (Amgen, Inc.) (immunizing peptide, CNSFLRHARETPDSYRDS) (17Burgess T.L. Ross S.L. Qian Y. Brankow D. Hu S. J. Biol. Chem. 1995; 270: 19188-19196Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Antibody sc-537, also recognizing the common region of the NRG cytoplasmic tail (immunizing peptide, FLRHARETPDSYRDSPHSER) and anti-Myc mouse monoclonal antibody 9e10 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody sc-537 was used for the immunohistochemical experiments because at the time these experiments were conducted, little Ab 1310 remained. Antibodies sc-537 and 1310 have given similar results in our experiments. Mouse monoclonal antibody 7D5, directed against the NRG ectodomain, was purchased from NeoMarkers (Fremont, CA). Anti-SV2 hybridoma supernatant was a gift of Dr. Kathy Buckley (Harvard Medical School) (41Buckley K. Kelly R.B. J. Cell Biol. 1985; 100: 1284-1294Crossref PubMed Scopus (576) Google Scholar). The anti-FLAG mouse monoclonal antibody M2 was purchased from International Biotechnologies, Inc. (New Haven, CT). All secondary antibodies for Western blot and immunofluorescence experiments were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Culture conditions for COS-7 cells were as follows: 37 °C; 8% CO2; medium consisting of Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Plasmid DNA for transfections was purified using Qiagen Plasmid Maxi kits. COS-7 cells were transfected using DEAE-dextran. Briefly, 5 × 105 cells were plated in each 100-mm dish 24 h prior to transfection. The DNA transfection solution was prepared by adding 10 μg of plasmid DNA, 30 μl of 50 mg/ml DEAE-dextran (Sigma), and 6 μl of 100 mm chloroquine (Sigma) to 6 ml of Dulbecco's modified Eagle's medium containing 10% Nu-Serum (Collaborative Biomedical Products). Cells were rinsed with PBS (Life Technologies, Inc.) or medium without serum and then incubated with DNA transfection solution (6 ml/dish) for 4 h in the incubator. Cells were then shocked with 10% Me2SO in serum-free Dulbecco's modified Eagle's medium (6 ml/dish) for 3 min. The Me2SO-containing medium was replaced with normal growth medium, and the dishes were returned to the incubator. Details of plasmid construct structure are provided in the legend to Fig. 2. To make yeast bait constructs, the target sequences (NRG and TGF-α cytoplasmic tails) were amplified by polymerase chain reaction (PCR). The PCR products were gel purified and subcloned into pBTM116 using the EcoRI andBamHI sites. As illustrated in Fig. 2, the bait proteins are fusions of the LexA DNA binding domain (N-terminally fused) and the bait sequence (C-terminally fused). Myc/pcDNA3.1, a mammalian expression vector designed to express recombinant proteins with an N-terminal Myc tag, was prepared by ligating a Myc-tag encoding cassette into pcDNA3.1(+) (Invitrogen, San Diego, CA) using the BstXI and NotI sites. The cassette was made by annealing the two oligos, 5′-CTGGATC ATG GGG GAA CAG AAA CTC ATC TCT GAA GAG GAT CTG GC-3′ and 5′-GGCCGC CAG ATC CTC TTC AGA GAT GAG TTT CTG TTC CCC CAT GATCCAGCACA-3′. The same strategy was used to generate flag/pcDNA3.1, a mammalian expression vector designed to produce N-terminally FLAG-tagged recombinant proteins. The FLAG-encoding cassette was prepared by annealing the oligos 5′-GATCCATC ATG GAC TAC AAG GAC GAC GAT GAC AAG G-3′ and 5′-AATTC CTT GTC ATC GTC GTC CTT GTA GTC CAT GATG-3′. This cassette was ligated between theEcoRI and BamHI sites of pcDNA3.1(+). To create the Myc-tagged full-length LIMK1 construct, a NotI site was introduced into the LIMK1 cDNA immediately upstream of the initiator ATG using a multi-step strategy (details available upon request). This modified LIMK1 cDNA was then ligated into Myc/pcDNA3.1. As illustrated in Fig. 2, the resulting construct encodes a fusion protein with a Myc-epitope tag appended onto the N-terminal end of full-length LIMK1. To create FLAG-tagged NRG cytoplasmic tails, insert sequences from the appropriate yeast two-hybrid bait plasmid (NRGc bait, NRGb bait, or NRGa bait) were ligated into the flag/pcDNA3.1 vector. As illustrated in Fig. 2, the resulting construct encodes a fusion protein (flag-NRGc-tail, flag-NRGb-tail, and flag-NRGa-tail) with a FLAG epitope tag appended onto the N-terminal end of each cytoplasmic tail form. These FLAG-tagged NRG cytoplasmic tail proteins do not include the extracellular or TM domain of NRG and are therefore expected to be soluble cytoplasmic proteins. To make expression vectors for full-length rat NRGs, the cDNAs R22 (NDF β2a; GenBank accession number U02318), R19 (NDF α2b; accession number U02316), and R44 (NDF α2c; accession number U02324) were subcloned into pcDNA3.1(+) using the NotI andEcoRV sites. To generate GST fusion protein constructs, the yeast two-hybrid bait constructs NRGas-bait and NRGc-bait were digested with EcoRI and SalI, and the cytoplasmic tail-encoding fragment was ligated in-frame into the vector pGEX-4T-1 (Amersham Pharmacia Biotech). All constructs were verified by restriction digestion and by automated dye terminator cycle sequencing (Applied Biosystems). Western blot analysis confirmed that proteins of the expected size were produced in COS-7 cells transfected with each of the mammalian expression constructs. Expressed TM-NRGs sometimes appeared as a doublet on Western blots, presumably due to heterogeneous glycosylation (cf. Fig. 6 A). The yeast two-hybrid screening reported here employed the bait plasmid pBTM116, the prey plasmid pVP16, and the yeast strain L40 (40Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar). The bait construct (NRGc bait) used for screening the library encodes a fusion between the LexA DNA binding domain and the portion of the NRG cytoplasmic tail common to all transmembrane NRGs. The two-hybrid expression library screened was prepared by N. Gekakis and C. Weitz (Harvard University). Each prey plasmid in the library encodes a fusion protein consisting of: 1) a nuclear localization sequence, 2) the VP16 transactivation domain, and 3) the protein encoded by a brain cDNA (see Fig. 2). This library was prepared from mRNA obtained from the brain of a 3-week-old mouse. The first strand cDNA synthesis was random primed to minimize bias toward C-terminal sequences and was size-selected for a length of 300–800 base pairs. This length is sufficient to encompass individual protein domains but, in many cases, may encode only a portion of a protein. The partial-length prey resulting from this size selection may be advantageous in allowing identification of interactions between the bait protein and proteins for which a full-length prey protein would not interact in a two-hybrid assay, either because the full-length protein is membrane-associated or because it contains a regulatory domain that blocks interaction with the bait. The library had 2 × 106 primary recombinants. For screening, library plasmids were transformed into L40 yeast that had previously been transformed with the NRGc bait plasmid. The version of the yeast two-hybrid system used employs two independent reporter genes, HIS3 and LacZ. Colonies that grow on medium lacking histidine and that produce β-galactosidase are considered primary screen positives. Prey plasmids isolated from colonies positive in the primary screen were further tested in a secondary screen: 1) to confirm that the prey plasmid isolated from the initial positive interacts with the NRGc bait, and 2) to test the specificity of the interaction with the NRGc bait. For the specificity control, the candidate prey plasmid was transformed into L40 yeast containing a bait plasmid that encodes the PAS domain of the Drosophila periodic protein PER. This protein sequence has no known similarity to NRG. Only prey showing no interaction with the PER bait were further evaluated. For the β-galactosidase filter assay, colonies of transformed yeast were picked and patched in triplicate on medium lacking tryptophan, leucine, uracil, and lysine. The yeast patches were transferred onto nitrocellulose filters, and the β-galactosidase assay was performed as described elsewhere (40Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar). Strength of interaction was scored as described in the legend of Fig. 3. The positive control for this assay was yeast transformed with bait and prey plasmids, which both had an insert encoding the PAS domain of PER, a domain known to strongly self-associate (42Huang Z.J. Edery I. Rosbash M. Nature. 1993; 364: 259-262Crossref PubMed Scopus (417) Google Scholar). For the liquid β-galactosidase assay, yeast transformants were inoculated into 3 ml of medium lacking tryptophan, leucine, uracil, and lysine and grown until A600 = 1.0. The assay was performed as described elsewhere (43Bartel P.L. Chien C.-T. Sternglanz R. Fields S. Cellular Interactions in Development: A Practical Approach.in: Hartley D.A. Oxford University Press, New York1993Google Scholar). β-Galactosidase unit activity was calculated using the formula: activity = 1000 ×A420/[(time in min) × (volume of culture in ml) × A600]. The GST expression vector pGEX-4T-1 and GST fusion protein constructs GST-NRGas-tail and GST-NRGc-tail were transformed into the bacterial strain BL21. An overnight culture in 2×YT medium was diluted 1:50 into 50 ml of fresh 2×YT and incubated at 37 °C in a shaking incubator for 90 min. Isopropyl-1-thio-β-d-galactopyranoside was then added to the culture to a final concentration of 0.1 mm, the culture was incubated for an additional 4 h, and then the bacteria were pelleted at 2500 × g. The pellet was washed once with 7 ml of STE buffer (150 mm NaCl, 10 mmTris-HCl, pH 8.0, 1 mm EDTA). The bacteria were resuspended in 5 ml of cold STE containing 100 μg/ml lysozyme and incubated on ice for 15 min. Five hundred microliters of 100 mmdithiothreitol and 1 ml of 10% sarkosyl w/v were added, and the volume was brought to 10 ml with cold STE. The bacteria were lysed by freezing and thawing five times in a dry ice-ethanol bath. The lysate was cleared by centrifugation at 16,000 × g for 20 min at 4 °C. The supernatant was transferred to fresh tubes, and Triton X-100 was added to a final concentration of 2% v/v. The lysate (≈10 ml) was then incubated with 100 μl of glutathione-agarose beads (Amersham Pharmacia Biotech) for 1 h at 4 °C. The beads were settled by centrifugation at 700 × g, washed four times with PBS and twice with TENT buffer (1% Triton X-100, 5 mm EDTA, 150 mm NaCl, 10 mmTris-HCl, pH 7.5), and used for in vitro binding assays without further treatment. For the in vitro binding assay, COS-7 cells were transfected as described above with the Myc-LIMK1 expression construct. Cells were lysed 60 h after transfection with 400 μl of TENT buffer per 100-mm dish. The cell lysate was cleared by centrifugation at 700 × g for 15 min at 4 °C. One milliliter of the cell lysate was incubated overnight at 4 °C with GST fusion protein immobilized on glutathione-agarose beads (20 μl). The beads were washed three times with TENT buffer and then resuspended in 50 μl of 2× SDS sample buffer with dithiothreitol and heated at 95 °C for 5 min. For Western blot analysis, 15 μl of this sample was loaded on a 10% SDS-polyacrylamide gel. Myc-tagged LIMK1 was detected with antibody 9e10. Samples were heated to 95 °C for 5 min immediately prior to loading on SDS-polyacrylamide minigels. Resolved proteins were then transferred to polyvinylidene difluoride membranes (Millipore) using a CAPS transfer buffer (10 mm CAPS, 10% methyl alcohol, pH 11) (44). The membranes were blocked with 5% nonfat dry milk in TBS buffer (100 mm Tris, 0.9% NaCl, pH 7.5) for 1 h at room temperature. The membranes were incubated overnight at 4 °C with 0.5 μg/ml Ab 9e10 (for LIMK1), 0.3 μg/ml Ab 1310 (for NRGs), or 0.5 μg/ml 7D5 (for NRGs) in 5 ml of the blocking solution. The membranes were washed four times with TTBS buffer (100 mm Tris, 0.9% NaCl, 0.1% Tween 20, pH 7.5) at room temperature. Bound primary antibodies were visualized with a horseradish peroxidase-conjugated goat anti-mouse IgG (1:50,000 in TTBS) or goat anti-rabbit IgG (1:50,000 in TTBS) and SuperSignal chemiluminescent substrate system (Pierce). For stripping and reprobing, blots
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