Collapsin Response Mediator Protein-2 Inhibits Neuronal Phospholipase D2 Activity by Direct Interaction
2002; Elsevier BV; Volume: 277; Issue: 8 Linguagem: Inglês
10.1074/jbc.m108047200
ISSN1083-351X
AutoresSukmook Lee, Jung Hwan Kim, Chang Sup Lee, Jong Hyun Kim, Youndong Kim, Kyun Heo, Yasuo Ihara, Yoshio Goshima, Pann‐Ghill Suh, Sung Ho Ryu,
Tópico(s)Zebrafish Biomedical Research Applications
ResumoAlthough the functional significance of neuronal phospholipase D (PLD) is being recognized, little is known about its regulatory role in neuronal cells. To elucidate the regulatory mechanism of neuronal PLD, we investigated PLD2-binding neuronal protein from rat brain cytosol. During the fractionation of rat brain cytosol by four-column chromatography, a 62-kDa PLD2-interacting protein was detected by PLD2 overlay assay and identified as collapsin response mediator protein-2 (CRMP-2), which controls neuronal axon guidance and outgrowth. Using bacterially expressed glutathioneS-transferase fusion proteins, we found that two regions (amino acids 65–192 (the phagocytic oxidase domain) and 724–825) of PLD2 and a single region (amino acids 243–300) of CRMP-2 are required for the direct binding of both proteins. A co-immunoprecipitation study in COS-7 cells also showed anin vivo interaction between CRMP-2 and PLD2. Interestingly, CRMP-2 was found to potently inhibit PLD2activity in a concentration-dependent manner (IC50 = 30 nm). Overexpression studies also showed that CRMP-2 is an in vivo inhibitor of PLD2 in PC12 cells. Moreover, increasing the concentration of semaphorin 3A, one of the repulsive axon guidance cues, showed that PLD2 activity can be inhibited in PC12 cells. Immunocytochemistry further revealed that PLD2 is co-localized with CRMP-2 in the distal tips of neurites, its possible action site, in differentiated PC12 cells. Taken together, our results indicate that CRMP-2 may interact directly with and inhibit neuronal PLD2, suggesting that this inhibitory mode of regulation may play a role in neuronal pathfinding during the developmental stage. Although the functional significance of neuronal phospholipase D (PLD) is being recognized, little is known about its regulatory role in neuronal cells. To elucidate the regulatory mechanism of neuronal PLD, we investigated PLD2-binding neuronal protein from rat brain cytosol. During the fractionation of rat brain cytosol by four-column chromatography, a 62-kDa PLD2-interacting protein was detected by PLD2 overlay assay and identified as collapsin response mediator protein-2 (CRMP-2), which controls neuronal axon guidance and outgrowth. Using bacterially expressed glutathioneS-transferase fusion proteins, we found that two regions (amino acids 65–192 (the phagocytic oxidase domain) and 724–825) of PLD2 and a single region (amino acids 243–300) of CRMP-2 are required for the direct binding of both proteins. A co-immunoprecipitation study in COS-7 cells also showed anin vivo interaction between CRMP-2 and PLD2. Interestingly, CRMP-2 was found to potently inhibit PLD2activity in a concentration-dependent manner (IC50 = 30 nm). Overexpression studies also showed that CRMP-2 is an in vivo inhibitor of PLD2 in PC12 cells. Moreover, increasing the concentration of semaphorin 3A, one of the repulsive axon guidance cues, showed that PLD2 activity can be inhibited in PC12 cells. Immunocytochemistry further revealed that PLD2 is co-localized with CRMP-2 in the distal tips of neurites, its possible action site, in differentiated PC12 cells. Taken together, our results indicate that CRMP-2 may interact directly with and inhibit neuronal PLD2, suggesting that this inhibitory mode of regulation may play a role in neuronal pathfinding during the developmental stage. Phospholipase D (PLD) 1PLDphospholipase DCRMPcollapsin response mediator proteinrCRMPrat CRMPhCRMPhuman CRMPGSTglutathione S-transferasePBSphosphate-buffered saline catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid and choline in response to various signals, including hormones, neurotransmitters, and growth factors (1Exton J.H. Biochim. Biophys. Acta. 1997; 1439: 121-133Crossref Scopus (337) Google Scholar). Phosphatidic acid has been shown to be involved in multiple physiological events in various cells, including mitogenesis, vesicle trafficking, respiratory burst in immune cells, and actin cytoskeletal rearrangements (2Danniel L.W. Sciorra V.A. Ghosh S. Biochim. Biophys. Acta. 1999; 1439: 265-276Crossref PubMed Scopus (36) Google Scholar, 3Jones D. Morgan C. Cockcroft S. Biochim. Biophys. Acta. 1999; 1439: 229-244Crossref PubMed Scopus (168) 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.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar). These relationships mean that receptor-mediated PLD activity is deeply implicated in a variety of cellular responses in different cells. phospholipase D collapsin response mediator protein rat CRMP human CRMP glutathione S-transferase phosphate-buffered saline A number of reports have suggested that neuronal PLD activity may be closely associated with amyloid β-induced cytotoxicity, hormone secretion, and neuronal differentiation (6Cox D.A. Cohen M.L. Neurosci. Lett. 1997; 229: 37-40Crossref PubMed Scopus (13) Google Scholar, 7Zheng L. Krsmanovic L.Z. Vergara L.A. Catt K.J. Stojilkovic S.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1573-1578Crossref PubMed Scopus (30) Google Scholar, 8Min D.S. Ahn B. Rhie D. Yoon S. Hahn S.J. Kim M. Jo Y. Neuropharmacology. 2001; 41: 384-391Crossref PubMed Scopus (50) Google Scholar, 9Hayakawa K. Nakashima S. Ito Y. Mizuta K. Miyata H. Nozawa Y. Neurosci. Lett. 1999; 265: 127-130Crossref PubMed Scopus (19) Google Scholar). Recently, the two types of mammalian PLD isozymes, PLD1 and PLD2, were cloned. These isozymes have a sequence homology exceeding 50% and contain several domains (conserved regions I-IV) that are highly conserved in the PLD superfamily and that are dependent on phosphatidylinositol 4,5-bisphosphate for their activity. PLD1 can be activated by many cytosolic factors, including protein kinase C and small GTP-binding proteins such as Rac1, Cdc42, ADP-ribosylation factor-1, and RhoA (10Hammond S.M. Altshuller Y.M. Sung T.C. Rudge S.A. Rose K. Engebrecht J. Morris A.J. Frohman M.A. J. Biol. Chem. 1995; 270: 29640-29643Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, 11Sung T.C. Zhang Y. Morris A.J. Frohman M.A. J. Biol. Chem. 1999; 274: 3659-3666Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 12Lee T.G. Park J.B. Lee S.D. Hong S. Kim J.H. Kim Y., Yi, K.S. Bae S. Hannun Y.A. Obeid L.M. Suh P.-G. Ryu S.H. Biochim. Biophys. Acta. 1997; 1347: 199-204Crossref PubMed Scopus (65) Google Scholar, 13Min D.S. Park S.K. Exton J.H. J. Biol. Chem. 1998; 273: 7044-7051Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 14Frohman M.A. Sung T.C. Morris A.J. Biochim. Biophys. Acta. 1999; 1439: 175-186Crossref PubMed Scopus (277) Google Scholar, 15Kim J.H. 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). On the other hand, PLD2 activity in the same setting is relatively insensitive to the PLD1-activating factors (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, 17Colley 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 (639) Google Scholar). However, the manner in which agonist-induced PLD isozymes are regulated in neuronal cells remains substantially unknown, although it has been reported that PLD mRNA and protein are more highly expressed in brain than other tissues (18Colley W.C. Altshuller Y.M. Sue-Ling C.K. Copeland N.G. Gilbert D.J. Jenkins N.A. Branch K.D. Tsirka S.E. Bollag R.J. Bollag W.B. Frohman M.A. Biochem. J. 1997; 326: 745-753Crossref PubMed Scopus (115) Google Scholar, 19Park S.K. Provost J.J. Bae C.D., Ho, W.-T. Exton J.H. J. Biol. Chem. 1997; 272: 29263-29271Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 20Brown H.A. Gutowski S. Kahn R.A. Sternweis P.C. J. Biol. Chem. 1995; 270: 14935-14943Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). In this regard, we believed an investigation of neuronal PLD regulation was warranted to further determine its function. Collapsin response mediator protein-2 (CRMP-2) is a cytosolic phosphoprotein that is exclusively expressed in the nervous system (21Kamata T. Subleski M. Hara Y. Yuhki N. Kung H. Copeland N.G. Jenkins N.A. Yoshimura T. Modi W. Copeland T.D. Brain Res. Mol. Brain Res. 1998; 54: 219-236Crossref PubMed Scopus (49) Google Scholar). It has been suggested to be a key mediator of semaphorin 3A action, one of the repulsive guidance cues that leads to growth cone collapse during neuronal axon guidance (22Goshima Y. Nakamura F. Strittmatter P. Strittmatter S.M. Nature. 1995; 376: 509-514Crossref PubMed Scopus (641) Google Scholar). In contrast to the rapid progress made in identifying and characterizing axon guidance molecules and their receptors (34He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (973) Google Scholar, 35Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 36Takahashi T. Fournier A. Nakamura F. Wang L.H. Murakami Y. Kalb R.G. Fujisawa H. Strittmatter S.M. Cell. 1999; 99: 59-69Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar, 55Nakamura F. Kalb R.G. Strittmatter S.M. J. Neurobiol. 2000; 44: 219-229Crossref PubMed Scopus (248) Google Scholar, 56Reza J.N. Gavazzi I.I. Cohen J. Mol. Cell. Neurosci. 1999; 14: 317-326Crossref PubMed Scopus (58) Google Scholar, 57Raper J.A. Curr. Opin. Neurobiol. 2000; 10: 88-94Crossref PubMed Scopus (405) Google Scholar), much remains to be discovered about the intracellular mechanism by which signals are transduced into the eventual response of the growth cone. In this study, we found for the first time that CRMP-2 specifically inhibits PLD2 activity by direct interaction. In addition, we tried to elucidate the role of CRMP-2 in the regulation of PLD2 in neuronal cells using PLD2-overexpressing PC12 cells as a model system. Thus, we hope that this study will provide several clues concerning the involvement of PLD2 in growth cone collapse. V8 protease, dipalmitoylphosphatidyl[methyl-3H]choline, chelating Sepharose, Q-Sepharose, phenyl-Sepharose, the HiTrap heparin column, and the enhanced chemiluminescence kit (ECL system) were purchased from Amersham Biosciences, Inc. Horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgA, IgM, and IgG were from Kirkegaard & Perry Laboratories (Gaithersburg, MD). The Bio-Gel HTP column and the polyvinylidene difluoride membrane were from Bio-Rad. Anti-actin antibody was from ICN Pharmaceuticals (Costa Mesa, CA). β-Octyl glucopyranoside was obtained from Calbiochem. Dipalmitoylphosphatidylcholine, phosphatidylinositol 4,5-bisphosphate, dioleoylphosphatidylethanolamine, paraformaldehyde, poly-l-lysine, anti-FLAG M2 antibody-agarose, anti-FLAG antibody, tetramethylrhodamine B isothiocyanate-conjugated goat anti-mouse IgG, and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG were from Sigma. Dulbecco's modified Eagle's medium was from Invitrogen. Anti-neuropilin-1 polyclonal antibody was from Oncogene Research Products (Cambridge, MA). Immobilized protein A was from Pierce. A polyclonal antibody that recognizes both PLD1 and PLD2 was generated as described previously (23Kim 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 preparations were performed at 4 °C in a refrigerated room or on ice. Adult rat brains (total of 30 g) were homogenized using a Polytron homogenizer in homogenization buffer containing 20 mm Tris (pH 7.6), 1 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, and 0.1 mm dithiothreitol. The homogenate was centrifuged at 100,000 × g for 1 h, and the resulting supernatant (cytosolic fraction) was collected. The cytosolic fraction (900 mg) was applied to a Q-Sepharose anion-exchange column (13 × 3 cm) equilibrated with buffer A (20 mm Tris (pH 7.6), 1 mm EDTA, 1 mm EGTA, and 0.1 mm dithiothreitol). Unbound proteins (flow-through fractions) were collected, and NaCl was added as salt to 2 m. After centrifugation at 50,000 ×g for 20 min, the resulting supernatant was loaded onto a phenyl-Sepharose column (70 × 2 cm). Proteins were eluted at a flow rate of 2 ml/min by applying a decreasing gradient of NaCl (2 to 0m) over 60 min. Fractions were collected and tested by PLD2 overlay assay. Peak fractions were pooled and diluted with buffer A to adjust the salt concentration to 50 mmNaCl and then loaded onto a HiTrap heparin column (1 ml) equilibrated with buffer A containing 50 mm NaCl. Bound proteins were eluted at a flow rate of 0.5 ml/min using a linear gradient of 0.05–1m NaCl over 30 min. Fractions were collected and also tested by PLD2 overlay assay. Fractions containing PLD2-interacting proteins were pooled and continuously loaded onto a Bio-Gel HTP column (1 ml) equilibrated with buffer containing 20 mm Tris (pH 7.6), 50 mm NaCl, and 0.1 mm dithiothreitol. Bound proteins were then eluted at a flow rate of 0.3 ml/min using an increasing gradient of KH2PO4 (0–0.25 m). Fractions were collected and tested by PLD2 overlay assay. The fractions containing p62 were collected and stored at −70 °C for further study. Hexahistidine-tagged human PLD2 was purified from detergent extracts of baculovirus-infected Sf9 cells by chelating Sepharose affinity column chromatography as described previously (15Kim J.H. 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). The assay method used was a modification of one described previously (24Park 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, rat brain cytosolic proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The blots were preincubated overnight with PLD assay buffer (50 mm HEPES (pH 7.3), 3 mmEGTA, 3 mm CaCl2, 3 mmMgCl2, and 80 mm KCl) containing 0.1 mm dithiothreitol and 5% (w/v) skimmed milk at room temperature and reincubated with the same buffer containing 1 μg/ml purified PLD2 for 3 h at room temperature. The membranes were then washed several times with Tris-buffered saline (20 mm Tris-HCl (pH 7.5) and 150 mm NaCl) containing 0.05% Tween 20 and reacted with polyclonal antibodies directed against PLD for 3 h. After washing with TTBS (10 mm Tris-HCl (pH 7.5), 150 mm NaCl, and 0.05% Tween 20), membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies for 1 h and developed using the enhanced chemiluminescence kit as described by the manufacturer. Purified PLD2-interacting protein from the hydroxylapatite column was digested for 2 h at 37 °C with V8 protease obtained from Staphylococcus aureus and then subjected to 15% SDS-PAGE to separate the cleaved peptides. After transferring the peptides to a polyvinylidene difluoride membrane, they were stained with Coomassie Brilliant Blue, rinsed several times with 30% methanol, excised, and subjected to Edman degradation. The candidate protein was identified by sequencing (ABI473 Sequencer) at the Institute of Basic Science (Busan, Korea) and by comparing the results obtained from the Swiss Protein Database using the BlastP algorithm. Affinity-purified anti-PLD2 antibodies immobilized with protein A beads were first incubated with purified PLD2 for 2 h at 4 °C. After a brief centrifugation, the precipitates were reincubated with the indicated amounts of purified CRMP-2 for 15 min at 37 °C in PLD assay buffer containing 1% Triton X-100. Binding site mapping between PLD2 and CRMP-2 was performed by incubating the indicated amounts of glutathione S-transferase (GST) fusion proteins with purified PLD2 or rat CRMP-2, respectively, under the same buffer conditions for 15 min at 37 °C. After a brief centrifugation, the pellets were washed three times with the same buffer before being loaded onto a polyacrylamide gel. The full-length coding region of human CRMP-2 was ligated into the EcoRI site of the pGEX-4T1 vector to make a GST fusion protein (Amersham Biosciences, Inc.). To construct the deletion mutants of CRMP-2, the full-length coding region of human CRMP-2 was also digested into N- and C-terminal serial deletion mutants by random cleavage. The deletion mutants were then ligated into the 5′-EcoRI and 3′-SalI sites of the same vector. The phagocytic oxidase and pleckstrin homology domains were constructed by digesting the PCR products using the restriction enzymes 5′-EcoRI and 3′-SalI and inserted into the pGEX-4T1 vector. The pCMV vector was used as the mammalian expression vector of CRMP-2. The full-length coding region of human CRMP-2 was ligated into the 5′-EcoRI and 3′-XbaI sites of the pCMV vector (CLONTECH). Standard methods were used for subcloning and PCR to produce expression vectors encoding the respective GST fusion proteins (25Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Escherichia coli BL21 cells were transfected with individual expression vectors encoding the GST fusion proteins and grown. The GST fusion proteins thus obtained were purified by standard methods (26Lee 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) using glutathione-Sepharose 4B (Amersham Biosciences, Inc.). The PLD activity assay was performed by measuring choline release from phosphatidylcholine as described previously (27Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (823) Google Scholar), but with minor modifications. In brief, the reaction was carried out at 37 °C for 15 min in a 125-μl assay mixture containing PLD assay buffer, the PLD preparation, and 25 μl of phospholipid vesicles composed of dioleoylphosphatidylethanolamine, phosphatidylinositol 4,5-bisphosphate, and dipalmitoylphosphatidylcholine at a molar ratio of 16:1.4:1 and dipalmitoylphosphatidyl[methyl-3H]choline (total of 150,000 cpm/assay). The reactions were terminated by adding 0.3 ml of a solution containing 1 n HCl and 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. PLD2-overexpressing PC12 cells were prepared using the tetracycline-regulated expression system (TET-OFF, Invitrogen) as described previously (28Lee 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 usually maintained in Dulbecco's modified Eagle's medium supplemented with 0.5 μg/ml tetracycline, 10% (v/v) equine serum, and 5% fetal bovine serum. The cells were differentiated by incubation in Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum and nerve growth factor (100 ng/ml) for 4 days. The COS-7 cells were maintained in growth medium composed of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a humidified CO2 (5%)-controlled incubator. To induce the transient expression of human PLD2 or human CRMP-2, COS-7 or PC12 cells were plated at densities of 1 × 106 cells/well in 100-mm dishes and 1 × 105 cells/well in six-well plates, respectively, and transfected using LipofectAMINE (Invitrogen) as described previously (29Kim J.H. Lee B.D. Kim Y. Lee S.D. Suh P.-G. Ryu S.H. J. Immunol. 1999; 163: 5462-5470PubMed Google Scholar). After transfecting COS-7 cells with human PLD2 or human CRMP-2 cDNA, the cells were harvested and lysed by brief sonication in PLD assay buffer containing 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 5 μg/ml aprotinin. The cells were then centrifuged at 100, 000 × g for 1 h, and the resulting supernatant was incubated on anti-FLAG M2 antibody-agarose for 4 h at 4 °C. After three washings with the same incubation buffer, the final pellet was loaded onto a polyacrylamide gel for immunoblot analysis. Proteins were denatured by boiling for 5 min at 95 °C in Laemmli sample buffer (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), separated by SDS-PAGE, and transferred to nitrocellulose membranes by electroblotting using the Bio-Rad wet transfer system. After blocking in TTBS containing 5% skim milk powder, the membranes were incubated with individual monoclonal or polyclonal antibodies and then further incubated with anti-mouse or anti-rabbit IgG coupled to horseradish peroxidase. Blots were detected using the enhanced chemiluminescence kit according to the manufacturer's instructions. Immunocytochemistry was performed based on a procedure reported previously (31Kim 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, FLAG-CRMP-2-transfected PLD2-overexpressing PC12 cells in the presence of nerve growth factor were grown on coverslips for 4 days, rinsed four times with phosphate-buffered saline (PBS), and fixed with 4% (w/v) paraformaldehyde overnight at 4 °C. After rinsing with PBS and blocking with PBS containing 1% goat serum and 0.1% Triton X-100 for 30 min at room temperature, the cells were incubated with 2 μg/ml primary polyclonal and monoclonal antibodies specific to PLD and FLAG for 2 h at room temperature. After washing six times with PBS, fluorescein isothiocyanate-labeled goat anti-rabbit antibody and tetramethylrhodamine B isothiocyanate-conjugated goat anti-mouse antibody were incubated with the cells for 1 h to allow the visualization of PLD2 and CRMP-2. After six washings with PBS, the slides were examined under a Zeiss confocal microscope. In vivo PLD activity was determined as described previously (32Lee S.D. Lee B.D. Kim Y. Suh P.-G. Ryu S.H. Neurosci. Lett. 2000; 294: 130-132Crossref PubMed Scopus (19) Google Scholar). In brief, PLD2-overexpressing PC12 cells were cultured in the presence or absence of tetracycline for 48 h. The cells were loaded with [3H]myristic acid (2 μCi/ml) for 4 h and then washed twice with Dulbecco's modified Eagle's medium. The loaded cells were incubated with 0.4% butanol for 5 min in the presence of the indicated concentrations of semaphorin 3A, scraped into 0.8 ml of methanol and 1 m NaCl (1:1), and mixed with 0.4 ml of chloroform. The organic phases were dried, and the lipids were separated by thin-layer chromatography on silica gel plates. The PLD activity of the vector or human CRMP-2-transfected PLD2-overexpressing PC12 cells was determined using the same procedure. The amount of [3H]phosphatidylbutanol formed was expressed as a percentage of the total 3H-lipid to account for cell labeling efficiency differences. The cytosolic fraction (900 mg of proteins) of rat brain was fractionated by sequential column chromatography, and the fractions thus obtained were subjected to a blot overlay assay using PLD2 to explore the major PLD2-binding proteins. Initially, the flow-through fraction from the Q-Sepharose column was subjected to phenyl-Sepharose column chromatography. The blot overlay assay showed that the proteins with relative molecular masses of 62 kDa (p62) and 40 kDa (p40), which coeluted in a linear gradient of 1.8 to 1.6 m NaCl from the phenyl-Sepharose column, were major PLD2-binding proteins (data not shown). A further purification step was performed to purify the PLD2-binding proteins on a HiTrap heparin column. p62 and p40 were also coeluted in 0.2 m NaCl as determined by the overlay assay, but other minor bands were removed by column chromatography (data not shown). Using a hydroxylapatite column, p62 and p40 were separately eluted by 0.15–0.2 m and 0.2–0.25m KH2PO4, respectively (Fig.1 A). SDS-PAGE showed that the p62 yield was 0.7 mg, assuming proportionality between the band densities and the protein quantities after Coomassie Brilliant Blue staining (Fig. 1 B). The p62 protein was also detected on the overlay blot (Fig. 1 C). These results suggest that the 62-kDa protein purified from rat brain cytosol binds directly with PLD2. The identity of p62 in fractions containing nearly homogeneous 62-kDa protein (>90%) was determined in two ways. First, the p62 peptide produced by V8 protease was sequenced, and its N-terminal sequence was identified as KKNIPRITSDDLLIK. This sequence was then searched for in the Swiss Protein Database using the BlastP peptide program. The search showed that this sequence is almost identical to an internal sequence (amino acids 6–20) of rat CRMP (rCRMP), especially of CRMP-2 and CRMP-4 (Fig.2 A). However, CRMP-4 has an apparent molecular mass of 39 kDa, which does not match that of purified p62 and which suggests that p62 might be a CRMP-2. p62 was then identified as CRMP-2 by immunoblot analysis using monoclonal antibody specific to CRMP-2 (Fig. 2 B). Therefore, we suggest that the 62-kDa protein identified as a PLD2 direct binder is a rCRMP-2. In vitro binding and immunoblot analyses were used to determine whether CRMP-2 associates directly with PLD2. As shown in Fig.3 A, purified rCRMP-2 interacted specifically with PLD2, but not with the anti-PLD antibody-protein A-Sepharose complexes alone, confirming a direct interaction between CRMP-2 and PLD2. To identify the region in PLD2 responsible for this CRMP-2 binding, we constructed and prepared GST fusion proteins of PLD2fragments as indicated in Fig. 3 B. The GST fusion proteins were then tested for their ability to bind to rCRMP-2. As shown in Fig.3 C, rCRMP-2 interacted with both the N-terminal (amino acids 1–314) and C-terminal (amino acids 724–825) regions of PLD2, whereas no interaction was observed with the other regions. To identify the PLD2 N-terminal site responsible for CRMP-2 binding, we constructed GST-tagged phagocytic oxidase- and pleckstrin homology-like domains for in vitro binding analysis. The results obtained showed that CRMP-2 bound to the phagocytic oxidase domain (amino acids 65–192) in the N-terminal region of PLD2 (Fig. 3 D). These results suggest that two regions (amino acids 65–192 and 724–825) of PLD2are responsible for its binding to CRMP-2. To identify the region of CRMP-2 responsible for binding to PLD2, we generated N- and C-terminal deletion mutants of human CRMP-2 (hCRMP-2). It should also be noted that hCRMP-2 is almost identical to rCRMP-2 (99% sequence identity) (33Wang L.H. Strittmatter S.M. J. Neurosci. 1996; 16: 6197-6207Crossref PubMed Google Scholar). As shown in Fig.4 A, N- and C-terminal deletion mutants were constructed by the stepwise removal of hCRMP-2. Wild-type hCRMP-2 and all of its deletion mutants were tagged with GST, affinity-purified, and examined in terms of their ability to bind to PLD2. The results obtained using C-terminal deletion mutants showed that PLD2 interacted directly with C2 (amino acids 1–300) of hCRMP-2, but not with C3 (amino acids 1–143) of hCRMP-2. This suggests that amino acids 144–300 of hCRMP-2 may be important for binding to PLD2 (Fig. 4 B). The purity and quantity of the C-terminal deletion mutants of hCRMP-2 were verified by Ponceau staining (Fig. 4 B, lower panel). To further identify the PLD2-binding site in CRMP-2, GST-tagged N-terminal serial deletion mutants of hCRMP-2 were subjected to a pull-down assay and immunoblot analysis. Fig.4 C shows that amino acids 243–300 of hCRMP-2 were involved in the binding of PLD2. Our results suggest that a region (amino acids 243–300) of CRMP-2 may be important for PLD2binding. To verify that PLD2 and CRMP-2 interactin vivo, we cotransfected hCRMP-2 and human PLD2cDNAs into COS-7 cells and performed immunoblot analysis after immunoprecipitating FLAG-tagged hCRMP-2 using anti-FLAG antibodies. As shown in Fig. 5, PLD2 was found to interact specifically with CRMP-2 in these cells, suggesting that PLD2 forms a complex with CRMP-2 in COS-7 cells. We next examined whether CRMP-2 plays a role in the regulation of PLD2 by measuring the effect of CRMP-2 on PLD2 activity in vitro. As shown in Fig. 6, purified rCRMP-2 was found to specifically inhibit PLD2 activity in a concentration-dependent manner. Under these conditions, the IC50 of this CRMP-2-mediated inhibition was ∼30 nm, and inhibition was complete at 150 nm. To further evaluate the effect of CRMP-2 on PLD2 activity, GST-tagged wild-type hCRMP-2 and its deletion mutants N3 (amino acids 243–573) and N4 (amino acids 301–573) were reconstituted in a PLD activity assay. As expected, N3 (amino acids 243–573) showed an inhibitory potency comparable to that of wild-type hCRMP-2. However, GST alone or N4 (amino acids 301–573) did not inhibit PLD2activity, although they were added to the assay up to 150 nm, at which level PLD2 activity was completely inhibited by wild-type hCRMP-2 (Fig. 6). This result is consistent with the in vitro binding analysis (Fig. 4, B andC). In summary, we suggest that amino acids 243–300 of CRMP-2 may be essential for binding to and inhibiting PLD2. To determine the effect of CRMP-2 in neuronal cells, we transfected hCRMP-2 into PLD2-overexpressing PC12 cells, which are able to induce PLD2 expression upon tetracycline withdrawal (28Lee S.D. Lee B.D. Han J.M. Kim J.H. Kim Y.
Referência(s)