Munc-18-1 Inhibits Phospholipase D Activity by Direct Interaction in an Epidermal Growth Factor-reversible Manner
2004; Elsevier BV; Volume: 279; Issue: 16 Linguagem: Inglês
10.1074/jbc.m310976200
ISSN1083-351X
AutoresHye Young Lee, Jong Bae Park, Il Ho Jang, Young Chan Chae, Jong Hyun Kim, Il Shin Kim, Pann‐Ghill Suh, Sung Ho Ryu,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoMammalian phospholipase D (PLD) has been reported to be a key enzyme for epidermal growth factor (EGF)-induced cellular signaling, however, the regulatory mechanism of PLD is still unclear. In this report, we found that Munc-18-1 is a potent negative regulator of PLD in the basal state and that its inhibition is abolished by EGF stimulation. We investigated PLD-binding proteins obtained from rat brain extract, and identified a 67-kDa protein as Munc-18-1 by peptide-mass finger-printing. The direct association between PLD and Munc-18-1 was confirmed by in vitro binding analysis using the purified proteins, and their binding sites were identified as the phox homology domain of PLD and multiple sites of Munc-18-1. PLD activity was potently inhibited by Munc-18-1 in vitro (IC50 = 2–5 nm), and the cotransfection of COS-7 cells with Munc-18-1 and PLD inhibited basal PLD activity in vivo. In the basal state, Munc-18-1 coprecipitated with PLD and colocalized with PLD2 at the plasma membrane of COS-7 cells. EGF treatment triggered the dissociation of Munc-18-1 from PLD when PLD was activated by EGF. The dissociation of the endogenous interaction between Munc-18-1 and PLD, and the activation of PLD by EGF were also observed in primary cultured chromaffin cells. These results suggest that Munc-18-1 is a potent negative regulator of basal PLD activity and that EGF stimulation abolishes this interaction. Mammalian phospholipase D (PLD) has been reported to be a key enzyme for epidermal growth factor (EGF)-induced cellular signaling, however, the regulatory mechanism of PLD is still unclear. In this report, we found that Munc-18-1 is a potent negative regulator of PLD in the basal state and that its inhibition is abolished by EGF stimulation. We investigated PLD-binding proteins obtained from rat brain extract, and identified a 67-kDa protein as Munc-18-1 by peptide-mass finger-printing. The direct association between PLD and Munc-18-1 was confirmed by in vitro binding analysis using the purified proteins, and their binding sites were identified as the phox homology domain of PLD and multiple sites of Munc-18-1. PLD activity was potently inhibited by Munc-18-1 in vitro (IC50 = 2–5 nm), and the cotransfection of COS-7 cells with Munc-18-1 and PLD inhibited basal PLD activity in vivo. In the basal state, Munc-18-1 coprecipitated with PLD and colocalized with PLD2 at the plasma membrane of COS-7 cells. EGF treatment triggered the dissociation of Munc-18-1 from PLD when PLD was activated by EGF. The dissociation of the endogenous interaction between Munc-18-1 and PLD, and the activation of PLD by EGF were also observed in primary cultured chromaffin cells. These results suggest that Munc-18-1 is a potent negative regulator of basal PLD activity and that EGF stimulation abolishes this interaction. Mammalian phospholipase D (PLD) 1The abbreviations used are: PLD, phospholipase D; EGF, epidermal growth factor; ARF, ADP-ribosylation factor; SNX, sorting nexin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; PX, phox homology; SH, Src homology; PA, phosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; PBt, phosphatidylbutanol; Sf9, Spodoptera frugiperda 9; GST, glutathione S-transferase; His6, hexahistidine; PLC, phospholipase C; FISH, five SH3 domains. is a membrane-bound enzyme that hydrolyzes phosphatidylcholine to generate a multifunctional lipid, phosphatidic acid (PA), in response to a variety of signals, including hormones, neurotransmitters, and growth factors (1Exton J.H. Biochim. Biophys. Acta. 1999; 1439: 121-133Crossref PubMed Scopus (337) Google Scholar). PA itself has been shown to be an intracellular lipid second messenger and to be involved in multiple physiological events, such as membrane vesicle trafficking, cytoskeletal dynamics, mitogenesis, and respiratory bursts in a large number of cells. These findings suggest that agonist-induced PLD activation may play roles in multiple signaling events (2Jones D. Morgan C. Cockcroft S. Biochim. Biophys. Acta. 1999; 1439: 229-244Crossref PubMed Scopus (168) Google Scholar, 3Danniel L.W. Sciorra V.A. Ghosh S. Biochim. Biophys. Acta. 1999; 1439: 265-276Crossref PubMed Scopus (36) Google Scholar, 4Olson S.C. Lambeth J.D. Chem. Phys. Lipids. 1996; 80: 3-19Crossref PubMed Scopus (54) Google Scholar, 5Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar, 6Rizzo M.A. Shome K. Vasudevan C. Stolz D.B. Sung T.C. Frohman M.A. Watkins S.C. Romero G. J. Biol. 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Many reports have suggested that PLD activity may be involved in various cellular physiologies (11Exton J.H. Ann. N. Y. Acad. Sci. 2000; 905: 61-68Crossref PubMed Scopus (79) Google Scholar). However, the regulatory mechanisms of PLD activity in its various functions have not been well characterized. Therefore, the identification of novel proteins that interact with PLD may provide clues about its function, not only in terms of the mechanisms that regulate PLD, but also in terms of the functional association between these regulators and specific cellular events. Many reports have suggested that PLD plays a role in the transduction of the intracellular signals initiated by various agonists (1Exton J.H. Biochim. Biophys. Acta. 1999; 1439: 121-133Crossref PubMed Scopus (337) Google Scholar, 12Exton J.H. FEBS Lett. 2002; 531: 58-61Crossref PubMed Scopus (234) Google Scholar). Because the product of PLD, PA, is involved in many cellular signaling events, agonist-induced PLD activation must be tightly controlled. Moreover, PLD is known to be positively regulated by various proteins and signals, for example, by ADP-ribosylation factor (ARF), protein kinase C, and EGF (12Exton J.H. FEBS Lett. 2002; 531: 58-61Crossref PubMed Scopus (234) Google Scholar, 13Cook S.J. Wakelam M.J. Biochem. J. 1992; 285: 247-253Crossref PubMed Scopus (76) Google Scholar). In particular, EGF stimulation increases PLD1 activity and this activation is downstream of PIP2 hydrolyzing phospholipase C and subsequent protein kinase Cα activation (14Han J.M. Kim Y. Lee J.S. Lee C.S. Lee B.D. Ohba M. Kuroki T. Suh P.G. Ryu S.H. Mol. Biol. Cell. 2002; 13: 3976-3988Crossref PubMed Scopus (49) Google Scholar). However, the molecular mechanism of PLD2 activation by EGF is at present unclear, even though a recent report suggested that ARF4 is a potential mediator (15Kim S.W. Hayashi M. Lo J.F. Yang Y. Yoo J.S. Lee J.D. J. Biol. Chem. 2003; 278: 2661-2668Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Moreover, α-/β-synuclein (16Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (383) Google Scholar), α-actinin (17Park J.B. Kim J.H. Kim Y. Ha S.H. Yoo J.S. Du G. Frohman M.A. Suh P.G. Ryu S.H. J. Biol. Chem. 2000; 275: 21295-21301Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), actin (18Lee S. Park J.B. Kim J.H. Kim Y. Kim J.H. Shin K.J. Lee J.S. Ha S.H. Suh P.G. Ryu S.H. J. Biol. Chem. 2001; 276: 28252-28260Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and collapsin response mediator protein-2 (19Lee S. Kim J.H. Lee C.S. Kim J.H. Kim Y. Heo K. Ihara Y. Goshima Y. Suh P.G. Ryu S.H. J. Biol. Chem. 2002; 277: 6542-6549Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) have been reported to inhibit basal PLD activity, but the nature of the signal-dependent modulation of PLD activity by its negative regulator after agonist stimulation remains unknown. Munc-18-1 is a 67-kDa protein, which was originally identified as a major brain protein, which binds to syntaxin, a synaptic vesicle fusion protein (20Hata Y. Slaughter C.A. Südhof T.C. Nature. 1993; 366: 347-351Crossref PubMed Scopus (591) Google Scholar, 21Pevsner J. Hsu S.C. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1445-1449Crossref PubMed Scopus (353) Google Scholar, 22Garcia E.P. Gatti E. Butler M. Burton J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2003-2007Crossref PubMed Scopus (224) Google Scholar). Mammals express three highly homologous isoforms of Munc-18 (23Tellam J.T. McIntosh S. James D.E. J. Biol. Chem. 1995; 270: 5857-5863Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Munc-18-1 is enriched in neurons, whereas Munc-18-2 and Munc-18-3 are expressed ubiquitously (23Tellam J.T. McIntosh S. James D.E. J. Biol. Chem. 1995; 270: 5857-5863Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Knockouts of Munc-18-1 have demonstrated that this protein is essential for synaptic vesicle exocytosis (24Harrison S.D. Broadie K. van de Goor J. Rubin G.M. Neuron. 1994; 13: 555-566Abstract Full Text PDF PubMed Scopus (208) Google Scholar, 25Wu M.N. Littleton J.T. Bhat M.A. Prokop A. Bellen H.J. EMBO J. 1998; 17: 127-139Crossref PubMed Scopus (157) Google Scholar), but the molecular mode of action of Munc-18-1 is not known in detail. Even though Munc-18-1 is an important protein in neuronal exocytosis and its protein-protein interactions may be critical in the fusion event, relatively few of its binding partners have been identified (26Verhage M. de Vries K.J. Roshol H. Burbach J.P. Gispen W.H. Sudhof T.C. Neuron. 1997; 18: 453-461Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 27Biederer T. Sudhof T.C. J. Biol. Chem. 2000; 275: 39803-39806Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 28Coppola T. Frantz C. Perret-Menoud V. Gattesco S. Hirling H. Regazzi R. Mol. Biol. Cell. 2002; 13: 1906-1915Crossref PubMed Scopus (114) Google Scholar). Furthermore, it has not been previously reported that Munc-18-1 can dynamically inhibit enzyme activity by direct interaction or that it is involved in cellular signaling events. In this study, we found for the first time that Munc-18-1 is a PLD-interacting molecule, and its inhibitory effect on PLD occurs by direct interaction with the PX domain of PLD. Furthermore, we found that this negative regulation of PLD activity by Munc-18-1 can be overcome by EGF stimulation, which thus offers a novel mechanism for PLD regulation. Materials—The enhanced chemiluminescence (ECL) kit, dipalmitoylphosphatidyl-[methyl-3H]choline, chelating Sepharose, and glutathione-Sepharose 4B were purchased from Amersham Biosciences; [3H]myristic acid from PerkinElmer Life Sciences; Silica Gel 60 thinlayer chromatography plates from MERCK (Darmstadt, Germany); Protein A-Sepharose from RepliGen (Needham, MA); Dulbecco's modified Eagle's medium and LipofectAMINE from Invitrogen (Carlsbad, CA); bovine calf serum from HyClone (Logan, UT); EGF from the Daewoong Pharmaceutical Co. (Seoul, Republic of Korea); cholic acid from U. S. Biochemical Corp. (Cleveland, OH); horseradish peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG, IgM, and IgA from Kirkegaard and Perry Laboratories, Inc. (Gaithersburg, MD); and monoclonal anti-Munc-18 antibody from BD Transduction Laboratories (Franklin Lakes, NJ). Polyclonal antibody (mSTP4) recognizing both PLD1 and PLD2 was produced as described previously (29Kim J.H. Kim Y. Lee S.D. Lopez I. Arnold R.S. Lambeth J.D. Suh P.G. Ryu S.H. FEBS Lett. 1999; 454: 42-46Crossref PubMed Scopus (83) Google Scholar). All other chemicals were purchased from Sigma. Coimmunoprecipitation Analysis of PLD from Rat Brain Extract— Rat brains (3 g) were lysed by homogenization in buffer A (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm MgCl2, 1 mm EGTA, 1% Triton X-100, 1% cholic acid). Lysates were centrifuged at 100,000 × g for 30 min at 4 °C, and the supernatants were incubated with anti-PLD antibody immobilized to Protein A-Sepharose beads. The precipitates were washed four times and subjected to SDS-PAGE followed by immunoblot analysis. Identification of Protein by Peptide Mass Fingerprinting Using Matrix-assisted Laser Desorption Ionization (MALDI) Time-of-flight Mass Spectrometry—After silver staining, candidate bands were excised from the gel and digested with trypsin. A 1-μl aliquot of the total 30 μl of digest was used for peptide mass fingerprinting. Masses of the tryptic peptides were measured using a Bruker Reflex III mass spectrometer, as previously described (17Park J.B. Kim J.H. Kim Y. Ha S.H. Yoo J.S. Du G. Frohman M.A. Suh P.G. Ryu S.H. J. Biol. Chem. 2000; 275: 21295-21301Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 30Kim J.H. Lee S. Kim J.H. Lee T.G. Hirata M. Suh P.G. Ryu S.H. Biochemistry. 2002; 41: 3414-3421Crossref PubMed Scopus (69) Google Scholar). MALDI was performed using α-cyano-4-hydroxycinnamic acid as the matrix. Trypsin autolysis products were used for internal calibration. Delayed ion extraction resulted in peptide masses with a mass accuracy of better than 0.1 Da on average. Mass values were compared against the NCBI data base using Profound (31Jensen O.N. Podtelejnikov A.V. Mann M. Anal. Chem. 1997; 69: 4741-4750Crossref PubMed Scopus (244) Google Scholar). Purification of Recombinant PLDs from Baculovirus-transfected Sf9 Cells—His6-tagged rat PLD1 and human PLD2 were purified from detergent extracts of baculovirus-infected Sf9 cells by chelating-Sepharose affinity column chromatography, as described previously (29Kim J.H. Kim Y. Lee S.D. Lopez I. Arnold R.S. Lambeth J.D. Suh P.G. Ryu S.H. FEBS Lett. 1999; 454: 42-46Crossref PubMed Scopus (83) Google Scholar). Construction and Preparation of Glutathione S-Transferase (GST) Fusion, FLAG- or His6-tagged Proteins—The full-length cDNAs of rat PLD1 or human PLD2 were digested into fragments containing specific regions and ligated into pGEX4T3 vector, as previously reported (18Lee S. Park J.B. Kim J.H. Kim Y. Kim J.H. Shin K.J. Lee J.S. Ha S.H. Suh P.G. Ryu S.H. J. Biol. Chem. 2001; 276: 28252-28260Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The full-length cDNA of human PLD2 was deleted (167–206) and ligated into pcDNA 3.1 vector for transfecting into COS-7 cells. Murine Munc-18-1 plasmid in pGEX vector was generously provided by Dr. Thomas C. Südhof (Howard Hughes Medical Institute, University of Texas Southwestern Medical School, Dallas, TX) (20Hata Y. Slaughter C.A. Südhof T.C. Nature. 1993; 366: 347-351Crossref PubMed Scopus (591) Google Scholar). A 1.8-Kb Munc-18-1 insert was obtained by PCR and also digested into fragments containing specific domains. Domains 1, 2-1, and 3-a were digested with BamHI or EcoRI, domain 2-2 was digested with EcoRI or XhoI, and domain 3-b with BamHI or XhoI, as previously reported (32Misura K.M. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (612) Google Scholar). These fragments were ligated into pGEX4T1 vector. The 3.0-kb murine Munc-18-1 insert was ligated into the NcoI or HindIII site of the pRSETB vector for transformation into Escherichia coli to produce His6-tagged Munc-18-1, which was purified by chelating-Sepharose affinity column chromatography using 80 mm imidazole as eluant. The 1.8-kb Munc-18-1 insert was digested and ligated into the EcoRI or HindIII sites of the cytomegalovirus vector for transfecting into COS-7 cells, thus resulting in the FLAG tagging of Munc-18-1. GST fusion PX domains of p40phox, FISH, and sorting nexin 1 (SNX1) were kindly provided by Dr. Michael B. Yaffe (Massachusetts Institute of Technology, MA) (33Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Crossref PubMed Scopus (498) Google Scholar), Dr. Peter Lock (The Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Australia) (34Lock P. Abram C.L. Gibson T. Courtneidge S.A. EMBO J. 1998; 17: 4346-4357Crossref PubMed Scopus (150) Google Scholar), and Dr. Rohan D. Teasdale (Institute for Molecular Bioscience, The University of Queensland, Australia) (35Teasdale R.D. Loci D. Houghton F. Karlsson L. Gleeson P.A. Biochem. J. 2001; 358: 7-16Crossref PubMed Scopus (134) Google Scholar), respectively. Inserts of p40phox, FISH, and the SNX1-PX domain were obtained by PCR and digested into fragments containing specific domains by using EcoRI or BamHI, and XhoI or EcoRI. These fragments were then ligated into pGEX4T1 vector. E. coli BL21 cells were transformed with individual expression vectors encoding the GST fusion proteins and purified by standard methods (36Lee C. Kim S.R. Chung J.K. Frohman M.A. Kilimann M.W. Rhee S.G. J. Biol. Chem. 2000; 275: 18751-18758Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) that involved: lysing E. coli with a buffer containing 1% Triton X-100 and 1% cholic acid, centrifuging the lysate at 100,000 × g for 30 min at 4 °C, and a final purification with glutathione-Sepharose 4B. To obtain the soluble GST fusion Munc-18-1, the protein was eluted three times using 50 mm Tris/HCl, pH 7.0, 10 mm glutathione, 1 mm dithiothreitol as buffer for 1 h at 4 °C. Immunoblot Analysis—Proteins were denatured by boiling for 5 min at 95 °C in Laemmli sample buffer (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207159) Google Scholar), separated by SDS-PAGE, and immunoblotted as described previously (19Lee S. Kim J.H. Lee C.S. Kim J.H. Kim Y. Heo K. Ihara Y. Goshima Y. Suh P.G. Ryu S.H. J. Biol. Chem. 2002; 277: 6542-6549Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Binding Analysis in Vitro—In vitro binding was performed in 300 μl of PLD assay buffer (50 mm HEPES/NaOH, pH 7.3, 3 mm EGTA, 3 mm CaCl2, 3 mm MgCl2, and 80 mm KCl) containing 0.1% Triton X-100 and 0.1% cholic acid at 4 °C for 2 h. The resulting complexes were washed three times with 1 ml of PLD assay buffer containing 0.1% Triton X-100 and 0.1% cholic acid, and once with a buffer without detergent. Samples were then subjected to SDS-PAGE, and immunoblotting using anti-Munc-18 or anti-PLD antibody. PLD Activity Assay in Vitro—PIP2-dependent PLD activity was assayed by measuring choline release from phosphatidylcholine using dipalmitoylphosphatidyl [methyl-3H]choline with minor modifications of a previously described method (38Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (821) Google Scholar). The reaction was carried out for 20 min at 37 °C in 125 μl of assay mixture containing PLD assay buffer (50 mm HEPES/NaOH, pH 7.3, 3 mm EGTA, 3 mm CaCl2, 3 mm MgCl2, and 80 mm KCl). Oleate-dependent PLD activity was assayed as previously described (29Kim J.H. Kim Y. Lee S.D. Lopez I. Arnold R.S. Lambeth J.D. Suh P.G. Ryu S.H. FEBS Lett. 1999; 454: 42-46Crossref PubMed Scopus (83) Google Scholar). The reaction mixture (175 μl) containing 50 mm HEPES/NaOH, pH 7.0, 2 mm EGTA, 1.7 mm CaCl2, 20 μm sodium oleate, and 0.1 m KCl was incubated at 30 °C for 1 h. Cell Culture—COS-7 cells were maintained in growth medium composed of Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum at 37 °C in a humidified CO2-controlled (5%) incubator. COS-7 cells were transfected using LipofectAMINE, as described previously (19Lee S. Kim J.H. Lee C.S. Kim J.H. Kim Y. Heo K. Ihara Y. Goshima Y. Suh P.G. Ryu S.H. J. Biol. Chem. 2002; 277: 6542-6549Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 39Kim J.H. Lee B.D. Kim Y. Lee S.D. Suh P.G. Ryu S.H. J. Immunol. 1999; 163: 5462-5470PubMed Google Scholar). Bovine adrenal chromaffin cells were prepared as described previously (40Kilpatrick D.L. Ledbetter F.H. Carson K.A. Kirshner A.G. Slepetis R. Kirshner N. J. Neurochem. 1980; 35: 679-692Crossref PubMed Scopus (203) Google Scholar) and primary cultured in a growth medium composed of Dulbecco's modified Eagle's medium/F-12 supplemented with 10% bovine calf serum at 37 °C in a humidified CO2-controlled (5%) incubator. PLD Activity Assay in Cells—PLD activity was assayed by measuring the formation of PBt (39Kim J.H. Lee B.D. Kim Y. Lee S.D. Suh P.G. Ryu S.H. J. Immunol. 1999; 163: 5462-5470PubMed Google Scholar). The intensities of PBt spots in the presence of 1-butanol were measured, and PLD activity was obtained by subtracting the corresponding intensities of spots obtained in the absence of 1-butanol. Immunoprecipitation—Immunoprecipitation was performed as described previously (14Han J.M. Kim Y. Lee J.S. Lee C.S. Lee B.D. Ohba M. Kuroki T. Suh P.G. Ryu S.H. Mol. Biol. Cell. 2002; 13: 3976-3988Crossref PubMed Scopus (49) Google Scholar). Briefly, cells were lysed with PLD assay buffer (50 mm HEPES/NaOH, pH 7.3, 3 mm EGTA, 3 mm CaCl2, 3 mm MgCl2, and 80 mm KCl) containing 1% Triton X-100, 1% cholic acid, and 1 mm phenylmethylsulfonyl fluoride. After brief sonication, the cell lysates were centrifuged at 100,000 × g for 30 min at 4 °C. The supernatants (1 mg of protein) were then incubated with anti-PLD antibody immobilized on protein A-Sepharose for 6 h at 4 °C. The resulting immune complexes were washed three times with 1 ml of PLD assay buffer containing 1% Triton X-100 and 1% cholic acid and once with a buffer without detergent. Samples were then subjected to SDS-PAGE, and immunoblotting using anti-Munc-18 or anti-PLD antibody. Immunocytochemical Analysis—COS-7 cells were grown on coverslips and transfected. After stimulation with 100 nm EGF for 0, 0.5, 1, 2, or 5 min, immunocytochemical analysis was performed as described previously (14Han J.M. Kim Y. Lee J.S. Lee C.S. Lee B.D. Ohba M. Kuroki T. Suh P.G. Ryu S.H. Mol. Biol. Cell. 2002; 13: 3976-3988Crossref PubMed Scopus (49) Google Scholar). Cells were washed with phosphate-buffered saline and fixed in 4% (w/v) paraformaldehyde, and then incubated in blocking buffer (1% horse serum in phosphate-buffered saline containing 0.2% Triton X-100). Subsequently, the cells were incubated with primary antibodies for 2 h at room temperature and then incubated with the secondary antibodies, rhodamine-conjugated anti-rabbit antibody or fluorescein isothiocyanate-conjugated anti-mouse antibody, for 1 h at room temperature. Slides were then mounted and examined under a confocal microscope (Carl Zeiss, Germany). Munc-18-1 Was Identified as a PLD-interacting Protein in Rat Brain Extract—Because the regulation of PLD might occur via direct interaction with other binding partners, the enrichment of PLD in rat brain extract led us perform a coimmunoprecipitation assay to isolate proteins that bind directly with PLD. After the precipitation with anti-PLD antibody from rat brain extract and protein analysis by SDS-PAGE and silver staining, the coprecipitate was found to contain a PLD-binding protein with a relative molecular weight of 67,000 (Fig. 1A). A 67-kDa band in the PLD precipitate was excised from a gel for identification by peptide mass fingerprinting. A peptide mixture of the trypsin-digested p67 was eluted and analyzed by MALDI-TOF mass spectrometry. Fig. 1B shows the peptide mass map of the protein. A search for these masses in a comprehensive sequence data base showed that 6 masses matched the calculated tryptic peptide masses of Munc-18-1 with an accuracy of <0.1 Da (Fig. 1C). These peptides covered 12% of the sequence of Munc-18-1. To further verify the identity of this protein, PLDs were immunoprecipitated from rat brain extract and the presence of Munc-18-1 was confirmed by immunoblotting with anti-Munc-18 antibody. As shown in Fig. 1D, Munc-18-1 was specifically precipitated by anti-PLD antibody but not by preimmune serum. Munc-18-1 Interacts Directly with PLD2—To determine whether Munc-18-1 binds directly to PLD, an in vitro binding analysis was performed with the purified proteins. Because PLD2 was precipitated more than PLD1 in the rat brain extract immunoprecipitate, we used purified PLD2 for the in vitro binding analysis. As shown in Fig. 1E, the binding of purified Munc-18-1 increased on increasing the amount of PLD2. This result suggests that the coprecipitated Munc-18-1 by PLD antibody from rat brain extract may have been because of direct interaction with PLD2. PLD1 was also found to interact with Munc-18-1 in a similar manner (data not shown). The PLD2-PX Domain C-terminal Region Contains the Munc-18-1 Interacting Site—To determine the regions of PLD2 that interact with Munc-18-1, fragments of PLD2 were generated as GST fusion proteins (Fig. 2A). The ability of the six GST fusion proteins to bind Munc-18-1 was examined by using an in vitro binding assay with purified His6-Munc-18-1. GST-F1-(1–314) was found to bind to Munc-18-1 most strongly (Fig. 2B). To more precisely map the F1 region required for Munc-18-1 binding, GST fusion proteins of the F1 region serial deletion constructs, and of the PX, the pleckstrin homology, or the F1 region without the PX domain construct, were generated (Fig. 2A). As shown in Fig. 2C, F1-2-(1–206) interacted with Munc-18-1, but the shorter fragments showed little interaction. The PX domain (66Liscovitch M. J. Lipid Mediat. Cell Signal. 1996; 14: 215-221Crossref PubMed Scopus (55) Google Scholar–195) itself also interacted with Munc-18-1 but the pleckstrin homology domain or the F1 region, which do not contain the PX domain, had little bindings with Munc-18-1 (Fig. 2D). These results led us to conclude that the C-terminal region of the PLD2-PX domain (167–195) makes a major contribution to Munc-18-1 binding. Multiple Sites of Munc-18-1 Are Involved in the Interaction with PLD2—To determine the regions of Munc-18-1 that interact with PLD2, fragments of Munc-18-1 were generated as GST fusion proteins (Fig. 3A) considering the crystal structure of Munc-18-1 (32Misura K.M. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (612) Google Scholar). As shown in Fig. 3B, domain 1 (D1; 4–134), domain 2-2 (D2-2; 480–592), and domain 3-a (D3-a; 246–361) bound to PLD2 but not as well as whole Munc-18-1 bound to PLD2. This result suggests that multiple sites of Munc-18-1 cooperate to produce high affinity binding between Munc-18-1 and PLD2. Munc-18-1 Specifically Interacts with the PLD-PX Domain—To check whether Munc-18-1 binds to the PX domains of other proteins, we tested its interactions with the PX domains of PLD1, PLD2, p40phox, FISH, and SNX1. The sequence alignment of the PLD1-, PLD2-, p40phox-, FISH-, and SNX1-PX domains was performed using the Clustal W software (Fig. 4A). Using a GST pull-down assay, we found that Munc-18-1 does not bind to the PX domains of p40phox, FISH, or SNX1, whereas it clearly interacted with both PX domains of PLD1 and PLD2 (Fig. 4B). This result suggests that Munc-18-1 specifically interacts with the PLD-PX domain. According to the sequence alignment result, the homology between the PLD1-PX domain and the PLD2-PX domain is high (46%) and the homology between PLD-PX domains and other PX domains is relatively low (less than 20%). Especially, the homology between the C-terminal 29 amino acids of PLD-PX domains, which correspond to 167–195 of PLD2, is very high (68%), which supports that Munc-18-1 binds to the C-terminal of the PLD-PX domain. To further confirm the importance of the C-terminal of the PLD-PX domain, we constructed a PLD2 deletion mutant (Δ167–206) according to the result of Fig. 2C and tested the interaction with Munc-18-1 by in vitro binding analysis (Fig. 4C) and in vivo coimmunoprecipitation analysis (Fig. 4D). As shown in Fig. 4, C and D, the PLD2 deletion mutant (Δ167–206) did not interact with Munc-18 either in vitro or in vivo, whereas PLD2 wild type showed a normal interaction. Taken together these results and the result from Fig. 2D strongly support that the C-terminal region (167–195) of the PLD2-PX domain is critically important for Munc-18-1 interaction. Munc-18-1 Inhibits PLD Activity—As Munc-18-1 was found to bind to the PX domain of PLD, and the N-terminal region of PLD has been reported to be important for PLD activity regulation (10Frohman M.A. Sung T.C. Morris A.J. Biochim. Biophys. Acta. 1999; 1439: 175-186Crossref PubMed Scopus (277) Google Scholar), we tested the effect of Munc-18-1 on PLD activity in vitro and in vivo. As shown in Fig. 5, the in vitro activity of PLD was inhibited specifically by His6-Munc-18-1 in a concentration-dependent manner. The concentration required for half-maximal inhibition was determined to be ∼5 nm in a PIP2-dependent PLD activity assay. To exclude the possibility that the inhibition might have resulted from the masking of PIP2 by Munc-18-1, we examined the effect of Munc-18-1 on PLD2 activity in an oleate-dependent activity assay. The inhibition of PLD2 activity by Munc-18-1 was found to be unchanged; ∼2 nm Munc-18-1 was required to inhibit PLD2 activity by 50% (Fig. 5). To test the effect of Munc-18-1 on in vivo PLD activity, PLD1 or PLD2 and Munc-18-1 were cotransfected into COS-7 cells and the effect of Munc-18-1 on basal PLD activity was determined. As shown in Fig. 6, the transfection of increasing amounts of Munc-18-1 DNA resulted in the inhibition of endogenous and overexpressed PLD2 a
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