Actin Directly Interacts with Phospholipase D, Inhibiting Its Activity
2001; Elsevier BV; Volume: 276; Issue: 30 Linguagem: Inglês
10.1074/jbc.m008521200
ISSN1083-351X
AutoresSukmook Lee, Jong Bae Park, Jong Hyun Kim, Yong Kim, Jung Hwan Kim, Kum-Joo Shin, Jun Sung Lee, Sang Hoon Ha, Pann‐Ghill Suh, Sung Ho Ryu,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoMammalian phospholipase D (PLD) plays a key role in several signal transduction pathways and is involved in many diverse functions. To elucidate the complex molecular regulation of PLD, we investigated PLD-binding proteins obtained from rat brain extract. Here we report that a 43-kDa protein in the rat brain, β-actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. We also determined that the region between amino acids 613 and 723 of PLD2 is required for the direct binding of β-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified β-actin potently inhibited both phosphatidylinositol-4,5-bisphosphate- and oleate-dependent PLD2 activities in a concentration-dependent manner (IC50 = 5 nm). In a previous paper, we reported that α-actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 (ARF1)-reversible manner (Park, J. B., Kim, J. H., Kim, Y., Ha, S. H., Kim, J. H., Yoo, J.-S., Du, G., Frohman, M. A., Suh, P.-G., and Ryu, S. H. (2000) J. Biol. Chem. 275, 21295–21301). In vitro binding analyses showed that β-actin could displace α-actinin binding to PLD2, demonstrating independent interaction between cytoskeletal proteins and PLD2. Furthermore, ARF1 could steer the PLD2 activity in a positive direction regardless of the inhibitory effect of β-actin on PLD2. We also observed that β-actin regulates PLD1 and PLD2 with similar binding and inhibitory potencies. Immunocytochemical and co-immunoprecipitation studies demonstrated the in vivointeraction between the two PLD isozymes and actin in cells. Taken together, these results suggest that the regulation of PLD by cytoskeletal proteins, β-actin and α-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions. Mammalian phospholipase D (PLD) plays a key role in several signal transduction pathways and is involved in many diverse functions. To elucidate the complex molecular regulation of PLD, we investigated PLD-binding proteins obtained from rat brain extract. Here we report that a 43-kDa protein in the rat brain, β-actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. We also determined that the region between amino acids 613 and 723 of PLD2 is required for the direct binding of β-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified β-actin potently inhibited both phosphatidylinositol-4,5-bisphosphate- and oleate-dependent PLD2 activities in a concentration-dependent manner (IC50 = 5 nm). In a previous paper, we reported that α-actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 (ARF1)-reversible manner (Park, J. B., Kim, J. H., Kim, Y., Ha, S. H., Kim, J. H., Yoo, J.-S., Du, G., Frohman, M. A., Suh, P.-G., and Ryu, S. H. (2000) J. Biol. Chem. 275, 21295–21301). In vitro binding analyses showed that β-actin could displace α-actinin binding to PLD2, demonstrating independent interaction between cytoskeletal proteins and PLD2. Furthermore, ARF1 could steer the PLD2 activity in a positive direction regardless of the inhibitory effect of β-actin on PLD2. We also observed that β-actin regulates PLD1 and PLD2 with similar binding and inhibitory potencies. Immunocytochemical and co-immunoprecipitation studies demonstrated the in vivointeraction between the two PLD isozymes and actin in cells. Taken together, these results suggest that the regulation of PLD by cytoskeletal proteins, β-actin and α-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions. phospholipase D phosphatidylcholine phosphatidylinositol-4,5-bisphosphate ADP-ribosylation factor guanosine 5′-3′-O-(thio) triphosphate Triton X-100 polyacrylamide gel electrophoresis glutathione S-transferase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid pheochromocytoma green fluorescent protein Mammalian phospholipase D (PLD)1 hydrolyzes phosphatidylcholine (PC) to generate phosphatidic acid and choline in response to a variety of signals, which can include hormones, neurotransmitters, and growth factors (1Exton J.H. Biochim. Biophys. Acta. 1997; 1439: 121-133Crossref Scopus (327) Google Scholar). phosphatidic acid itself has been shown to be an intracellular lipid second messenger and to be involved in multiple physiological events such as the promotion of mitogenesis, stimulation of respiratory bursts, secretory processes, actin cytoskeletal reorganization, and the activation of Raf-1 kinase and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinase isoforms in a large number of cells. These relationships 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. 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Lee S.D. Han J.M. Lee T.G. Kim Y. Park J.B. Lambeth J.D. Suh P.G. Ryu S.H. FEBS Lett. 1998; 430: 231-235Crossref PubMed Scopus (89) Google Scholar). PLD2 also depends on PIP2 but has a higher basal activity than PLD1 (16Lopez I. Arnold R.S. Lambeth J.D. J. Biol. Chem. 1998; 273: 12846-12852Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), and it has been proposed that PLD2 may be closely associated with different cellular inhibitors. Although many studies continue to focus on the functional relationships and the isozyme specificities of the PLD isozymes, the molecular mechanism of the regulation of the PLDs has not been fully elucidated. In this regard, the identification of PLD-binding partners may provide clues toward the understanding of the complex regulatory mechanism of PLD in different cells. It has been observed in many studies that PLD is crucially implicated in the actin-based cytoskeleton of cells. More recently, PLD activity has been found in the detergent-insoluble fraction of various cell types that contain a wide range of cytoskeletal proteins (17Hodgkin M.N. Clark J.M. Rose S. Saqib K. Wakelam M.J.O. Biochem. J. 1999; 339: 87-93Crossref PubMed Scopus (46) Google Scholar, 18Iyer S.S. Kunser D.J. J. Biol. Chem. 1999; 274: 2350-2359Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Several cytoskeletal proteins such as fodrin and gelsolin have been found to act as PLD-specific inhibitors in vitro (19Lukowski S. Mira J.P. Jachowski A. Geny B. Biochem. Biophys. Res. Commun. 1998; 248: 278-284Crossref PubMed Scopus (33) Google Scholar, 20Lukowski S. Lecomte M.C. Mira J.P. Marin P. Gautero H. Russo-Marie F. Geny B. J. Biol. Chem. 1996; 271: 24164-24171Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21Steed P.M. Nagar S. Wennogle L.P. Biochemistry. 1996; 35: 5229-5237Crossref PubMed Scopus (62) Google Scholar), and agonist-induced PLD stimulation can provoke changes in cell morphology through cytoskeletal rearrangement (5Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar, 22Ha K.S. Exton J.H. J. Cell Biol. 1993; 123: 1789-1796Crossref PubMed Scopus (154) Google Scholar, 23Ha K.S. Yeo E.J. Exton J.H. Biochem. J. 1994; 303: 55-59Crossref PubMed Scopus (56) Google Scholar, 24Colley W.C. Sung T.C. Roll R. Jenco J. Hammond S.M. Altshuller Y. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar). Furthermore, we reported previously that α-actinin, an F-actin cross-linking protein, also binds to PLD2 to inhibit its activity (25Park J.B. Kim J.H. Kim Y Ha S.H. Kim J.H. Yoo J. 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). Thus, there is a strong body of evidence supporting a possibly close regulatory association between PLD and the actin cytoskeleton. In our present study, we found for the first time that β-actin, a major cytoskeletal protein, negatively regulates PLD by direct binding. We also looked at the relationships and modes of action of ARF1 and other cytoskeletal proteins on PLD using PLD2 as a model enzyme, and the results obtained suggest possible mechanisms for the regulation of PLD by these cellular components. The enhanced chemiluminescence kit (ECL system), dipalmitoylphosphatidyl-[methyl-3H]choline, chelating-Sepharose, DEAE-Sepharose, and Sephadex-150 resin were purchased from Amersham Pharmacia Biotech.Dipalmitoyl-phosphatidylcholine, PIP2, dioleoyl-phosphatidylethanolamine, paraformaldehyde, and sodium oleate were purchased from Sigma. Anti-actin antibody was purchased from ICN Pharmaceuticals. GTPγS was obtained from Roche Molecular Biochemicals. Horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgA, IgM, and IgG were from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Dulbecco's modified Eagle's medium was purchased from Life Technologies, Inc. Immobilized protein A and fluorescein isothiocyanate-conjugated goat anti-rabbit antibody were purchased from Pierce. β-octylglucopyranoside was obtained from Calbiochem. Rhodamine-phalloidin was obtained from Molecular Probes. A polyclonal antibody that recognizes both PLD1 and PLD2 was generated as described previously (25Park J.B. Kim J.H. Kim Y Ha S.H. Kim J.H. Yoo J. 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). Anti-ARF monoclonal antibody was provided kindly by Dr. Richard A. Kahn (Emory University, Atlanta, GA). Full-length cDNAs of murine PLD2 and its N-terminal deletion mutant were provided generously by Dr. Michael A. Frohman (State University of New York, NY). Hexa-histidine (His6)-tagged PLD1 and PLD2 were purified from detergent extracts of baculovirus-infected sf9 cells by chelating-Sepharose affinity column chromatography as described previously (26Kim 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). Rat brains (3 g) were homogenized in homogenation buffer (20 mm Tris/HCl, pH 7.5, 1 mm MgCl2, 1 mm EDTA, 1 mm EGTA, and 150 mm NaCl) using a polytron homogenizer. After centrifugation at 100,000 × g for 1 h at 4 °C, the resulting supernatant was used to investigate potential PLD2-binding partners. Protein concentrations in the brain extract were determined using the methods developed by Bradford (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar). Affinity-purified anti-PLD antibodies immobilized on protein A resin (PLD antibody complex) were first incubated with purified recombinant PLD2 (3 µg) for 2 h. After a brief centrifugation, the immune complexes were washed three times with radioimmune precipitation buffer (50 mm Tris/HCl, pH 8.5, 0.1% SDS, 150 mm NaCl, 1% TX-100, and 1% deoxycholate). The prepared brain extract (3 mg of protein) was then incubated with the complexes for 2 h at 4 °C. Finally, the co-precipitated proteins were washed again three times with radioimmune precipitation buffer, loaded onto a gel, and visualized by Coomassie Brilliant Blue staining. The technique used was performed as described previously (25Park J.B. Kim J.H. Kim Y Ha S.H. Kim J.H. Yoo J. 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). In brief, the fraction containing 43-kDa protein (p43) after co-immunoprecipitation from rat brain extract was separated by 8% SDS-PAGE, and the band corresponding to p43 was excised and digested with trypsin (Roche Molecular Biochemicals) for 6 h at 37 °C. The masses of the tryptic peptides obtained were determined with a Voyager DE time-of-flight mass spectrometer (Perceptive Biosystems, Inc., Framingham, MA) in the Korea Basic Science Institute. Delayed ion extraction resulted in peptide masses with better than 50 ppm mass accuracy on average. Using the amino acid sequences and the mass numbers of the tryptic peptides of p43, the Swiss-Prot data base was searched for a protein match. β-actin was purified from rat brain as described previously (28Bray D. Thomas C. J. Mol. Biol. 1976; 105: 527-544Crossref PubMed Scopus (124) Google Scholar) and assessed to be >90% pure by Coomassie staining. Myristoylated recombinant ARF1 was expressed in Escherichia coli and purified (29Lambeth J.D. Kwak J.-Y. Bowman E.P. Perry D. Uhlinger D.J. Lopez I. J. Biol. Chem. 1995; 270: 2431-2434Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The full-length cDNA of human PLD2 was digested into fragments containing specific domains. These individual PLD2 fragments were then ligated into the EcoRI or SmaI site of the pGEX4T3 vector. Human β-actin cDNA, kindly provided by Dr. Jungchul Kim (Kyungpook National University, Korea), was then ligated into the EcoRI site of the pGEX4T1 vector (Amersham Pharmacia Biotech). Subcloning and the polymerase chain reaction were used to produce the expression vectors encoding the respective GST fusion proteins (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). E. coli BL21 cells were transformed with individual expression vectors encoding the GST fusion proteins, and after harvesting the cells the GST fusion proteins expressed were purified by standard methods (31Lee 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) using glutathione-Sepharose 4B (Amersham Pharmacia Biotech). All operations were performed at 4 °C in a refrigerated room or on ice. The two recombinant PLD isozymes prepared or COS-7 cell extracts overexpressing wild type and the N-terminal deletion mutant of murine PLD2 (Δ1–185) were first bound to PLD antibody complexes. These were then respectively incubated with the indicated amounts of purified β-actin or ARF1 for 15 min at 37 °C. At this stage, all of the PLD-binding partners were present in the PLD assay buffer (50 mm HEPES/NaOH, pH 7.3, 3 mm EGTA, 3 mm CaCl2, 3 mm MgCl2, and 80 mm KC1) containing 0.5% β-octylglucopyranoside. After brief centrifugation, the co-precipitated complexes were washed three times in the same buffer before being loaded onto a polyacrylamide gel. The in vitrobinding of the GST-PLD2 fragments with β-actin was also performed in the same buffer containing 1% TX-100. All procedures using α-actinin binding were similar to those used for β-actin binding. In brief, PLD2 immune complexes were incubated with α-actinin and β-actin at 37 °C for 15 min in the PLD assay buffer, and the resulting co-precipitates were washed with the same buffer containing 0.25% CHAPS as described previously (25Park J.B. Kim J.H. Kim Y Ha S.H. Kim J.H. Yoo J. 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). PIP2-dependent PLD activity was assayed by measuring choline release from phosphatidylcholine (12Brown 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) with minor modifications. In brief, the reaction was carried out at 37 °C for 15 min in a 125-µl assay mixture containing the PLD assay buffer, the PLD preparation, and 25 µl of phospholipid vesicles composed of dioleoyl-phosphatidylethanolamine, PIP2, dipalmitoyl-phosphatidylcholine, and dipalmitoyl[methyl-3H]choline (a total of 150,000 cpm/assay) in a molar ratio of 16:1.4:1. Oleate-dependent PLD activity was assayed as described earlier (26Kim 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). In brief, PC vesicles (25 µl) containing 5 nmol of dipalmitoyl-phosphatidylcholine and 200,000 dpm of dipalmitoylphosphatidyl-[methyl-3H]choline were added to a 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. The final concentration of PC in the reaction mixture was 25 µm. The assay mixture was then incubated at 30 °C for 1 h, and the reaction was terminated by the addition of 0.3 ml of 1 n HCl/5 mm EGTA and 1 ml of chloroform/methanol/HCl (50:50:0.3). After a brief centrifugation, the amount of [methyl-3H]choline in 0.5 ml of the aqueous phase was quantified by liquid scintillation counting. Proteins were denatured by boiling for 5 min at 95 °C in a Laemmli sample buffer (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar), separated by SDS-PAGE, and transferred to nitrocellulose membranes by electroblotting using the Bio-Rad wet transfer system. After blocking in TTBS buffer (10 mm Tris/HCl, pH 7.5, 150 mmNaCl, and 0.05% Tween 20) containing 5% skim milk powder, the membranes were incubated with individual monoclonal or polyclonal antibodies, which was subsequently followed by another incubation with anti-mouse or anti-rabbit IgG, as required, coupled with horseradish peroxidase. Detection was performed using an enhanced chemiluminescence kit according to manufacturer instructions. The tetracycline-regulated (Tet-off) expression system (Life Technologies, Inc.) was used to induce the expression of PLD2 in PC12 cells (33Lee S.D. Lee B.D. Han J.M. Kim J.H. Kim Y. Suh P.G. Ryu S.H. J. Neurochem. 2000; 75: 1053-1059Crossref PubMed Scopus (52) Google Scholar). Clonal cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 0.5 µg/ml tetracycline, 10% (v/v) equine serum, and 5% fetal calf serum. To induce PLD2, the cells were grown in the same medium without tetracycline. COS-7 cells were maintained in a growth medium composed of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a humidified CO2-controlled (5%) incubator. For transfection and the transient expression of PLD isoforms, COS-7 cells were plated at a density of 1 × 106 cells/well in 100-mm dishes and transfected using LipofectAMINE (Life Technologies, Inc.) as described previously (34Kim J.H. Lee B.D. Kim Y. Lee S.D. Suh P.G. Ryu S.H. J. Immunol. 1999; 163: 5462-5470PubMed Google Scholar). PLD2-inducible PC12 cells cultured in the presence or absence of tetracycline or COS-7 cells overexpressing PLD1 were lysed with PLD assay buffer containing 1% cholate, 1% TX-100, 1 mm phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 5 µg/ml aprotinin. After brief sonication, the cell lysates were incubated for 2 h with constant agitation and centrifuged at 100,000 × g for 1 h. The cell extracts (1 mg of protein) recovered were incubated with anti-PLD antibody-immobilized on protein A resin for 2 h. After brief centrifugation, the co-immunoprecipitated complexes were washed three times with ice-cold radioimmune precipitation buffer before being loaded onto a polyacrylamide gel for immunoblot analysis. Cells transfected with the wild type and the N-terminal deletion mutant of murine PLD2 (Δ1–185) were disrupted by sonication in ice-cold PLD assay buffer. The lysates were then centrifuged at 100,000 × g for 1 h at 4 °C, and the pellet was resuspended in the same buffer and referred to as membranes. Immunocytochemistry was performed as described previously (35Kim Y. Han J.M. Han B.R. Lee K.A. Kim J.H. Lee B.D. Jang I.H. Suh P.G. Ryu S.H. J. Biol. Chem. 2000; 275: 13621-13627Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In brief, PC12 cells grown on coverslips in the presence or absence of tetracyclin were rinsed with PBS four times and fixed with 3.7% (w/v) paraformaldehyde for 10 min at 37 °C. After rinsing with PBS and blocking with PBS containing 1% goat serum and 0.1% TX-100 for 4 h at 4 °C, the cells were incubated with 2 µg/ml primary polyclonal antibody specific to PLD overnight at 4 °C. The cells were washed six times with PBS containing 0.05% TX-100 and then incubated in this washing medium with fluorescein isothiocyanate-labeled goat anti-rabbit secondary antibody and rhodamine-phalloidin for 1 h to visualize PLD2 and filamentous actin (F-actin), respectively. To visualize F-actin in COS-7 cells overexpressing GFP, GFP-PLD1, or GFP-PLD2 (36Kim Y. Kim J.E. Lee S.D. Lee T.G. Kim J.H. Park J.B. Han J.M. Jang S.K. Suh P.G. Ryu S.H. Biochim. Biophys. Acta. 1999; 1436: 319-330Crossref PubMed Scopus (38) Google Scholar), they were incubated with rhodamine-phalloidin as described above. Slides were then examined under a fluorescence microscope (Nikon, Melville, NY). Because the regulation of PLD could possibly occur through direct interaction between PLD and other binding partners, we started our investigation by looking for cellular PLD2-binding proteins from rat brain extract using purified PLD2 complexed with anti-PLD antibody. After the precipitation (PLD2 precipitate) and protein analysis by SDS-PAGE, we found that the co-precipitate contained major PLD2-binding proteins with relative molecular masses of 48 (p48), 43 (p43), and 35 kDa (p35) and some minor proteins. As shown in Fig. 1, these bands appeared in distinctive patterns only in PLD2 immunoprecipitates. A major band corresponding to p43 in the PLD2 precipitates was excised from the gel for identification by peptide mass fingerprinting. A trypsinized peptide mixture of p43 was then subjected to matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Fig. 2Ashows the matrix-assisted laser desorption ionization mass spectrum of the digested peptides of p43. The masses obtained, marked as P1–P7, were compared with proteins in the Swiss-Prot data base using the MS-Fit peptide mass search program. As shown in TableI, the peptides exhibited molecular masses that were almost identical to the calculated masses of the corresponding theoretically predicted tryptic peptides of β-actin. The accuracy of this peptide search result was obtained with 50 ppm, and the analyzed peptides covered 21% of the β-actin sequence (Table. I). To substantiate the identity of this protein further, the presence of actin in the PLD2 precipitate was confirmed using a monoclonal antibody to actin. As shown in Fig. 2 B, actin was strongly detected in the PLD2 precipitate but not in a control immune complex. On the basis of these results, we concluded that the 43-kDa protein in the PLD2 precipitate from the rat brain extract was β-actin.Figure 2Identification of p43 as β-actin. A, p43 isolated from proteins that co-precipitated with PLD2 was digested with trypsin, and the resulting peptide mixture was analyzed by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Thearrows indicate matched peaks among the measured tryptic peaks of p43 with calculated molecular masses of β-actin within 50 ppm. P1–P7, the molecular masses of the peptides obtained. B, equal aliquots of the co-immunoprecipitates used in Fig. 1 were separated by SDS-PAGE and analyzed by immunoblot analysis using antibodies directed against PLD or actin, respectively. The lane order is the same as that described in the Fig. 1 legend.View Large Image Figure ViewerDownload (PPT)Table IPeptide sequences and masses from p43 by matrix-assisted laser desorption ionization/time-of-flight mass spectrometryPeptideSequence1-aThe matched peptides cover 21% (81 of 375 amino acids) of the proteins.M + H+ObservedCalculated1-bMonoisotopic mass.DaP1LDLAGR (178–183)644.36644.37P2ILAPPER (329–335)795.49795.47P3GYSFTTTAER (197–206)1132.541132.52P4HQGVMVGMGQK (40–50)1187.571187.56P5QEYDESGPSIVHR (360–372)1516.721516.70P6SYELPDGQVITIGNER (239–254)1790.901790.89P7VAPEEHPVLLTEAPLNPK (96–113)1954.031954.061-a The matched peptides cover 21% (81 of 375 amino acids) of the proteins.1-b Monoisotopic mass. Open table in a new tab To determine whether β-actin associates directly with PLD2, β-actin from rat brain was purified to over 90% (data not shown) using the methods of Bray and Thomas (28Bray D. Thomas C. J. Mol. Biol. 1976; 105: 527-544Crossref PubMed Scopus (124) Google Scholar) and incubated with the PLD2-bound immune complexes. As shown in Fig.3A, the resulting co-precipitation demonstrated that β-actin interacts directly with PLD2. To identify the PLD2 sequence involved in the β-actin binding, we constructed the GST fusion proteins shown in Fig. 3 B and tested them for their ability to bind to purified β-actin. GST-PLD2 (amino acids 613–723) was found to be the region that most potently bound to β-actin (Fig. 3 C). It seems therefore that the region of the protein encoded between amino acids 613 and 723 may be important for the direct interaction with β-actin. We monitored PLD activity to determine the effect of β-actin on PLD2. As shown in Fig. 4A, the PIP2-dependent activity of PLD2 was inhibited specifically in a β-actin concentration-dependent manner. Using β-actin purified from rat brain, the concentration required for half-maximal inhibition was about 5 nm. To further confirm the inhibitory effect of β-actin on PLD2 activity, we constructed and purified a GST-β-actin for reconstitution assays of PLD activity. As expected, we observed that this GST-β-actin had an inhibitory effect that was similar to that of the β-actin purified from rat brain. To exclude the possibility that the β-actin-inhibited PLD2 activity might be caused by PIP2 sequestration or masking in the substrate phospholipid vesicles, we also performed a PLD2 activity assay in the absence of PIP2. We previously reported that PLD2 could be activated specifically by oleate (18:1) in the absence of PIP2 (26Kim 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). As shown in Fig. 4 B, PLD2 activated by oleate was inhibited progressively by increasing either the concentration of β-actin purified from rat brain or GST-β-actin with similar inhibitory potency. The inhibitory efficacy of β-actin, under the condition of the oleate assay, was close to that observed under the condition that included PIP2 (Fig.4 A). Taken together, these results suggest that the inhibitory effect of β-actin on PLD2 might be mediated by direct interaction. In a previous study, we found that α-actinin binds directly to the N-terminal region (amino acids 1–185) of PLD2 and inhibits activity of the enzyme (25Park J.B. Kim J.H. Kim Y Ha S.H. Kim J.H. Yoo J. 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). To clarify the relationship between the two cytoskeletal proteins, β-actin and α-actinin, in terms of PLD2 binding, in vitro binding assays were performed. Fig. 5Ademonstrates that an increase in β-actin reduced α-actinin binding to PLD2 in a competitive and concentration-dependent manner. In other words, the binding of β-actin to PLD2 induced the release of α-actinin already bound to PLD2. To exclude the possibility that this mode of competition occurs through the same binding site on the two proteins, we used an N-terminal α-actinin binding region deletion mutant of PLD2 (PLD2Δ (1)). In
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