COMMUNICATION
Interaction of the Small G Protein RhoA with the C Terminus of Human Phospholipase D1*

Masakazu YamazakiDagger , Yue Zhang§, Hiroshi WatanabeDagger , Takeaki YokozekiDagger , Sigeo Ohnoparallel , Kozo Kaibuchi**, Hideki ShibataDagger Dagger , Hideyuki MukaiDagger Dagger , Yoshitaka OnoDagger Dagger , Michael A. Frohman§, and Yasunori KanahoDagger §§

From the Dagger  Department of Life Science, Tokyo Institute of Technology, Yokohama 226-8501, the parallel  Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, the ** Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, the Dagger Dagger  Department of Biology, Faculty of Science, Kobe University, Kobe 657-8501, Japan and the § Department of Pharmacological Sciences and the Institute for Cell and Developmental Biology and the Program for Molecular and Cellular Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-8651

    ABSTRACT
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Abstract
Introduction
References

Mammalian phosphatidylcholine-specific phospholipase D1 (PLD1) is a signal transduction-activated enzyme thought to function in multiple cell biological settings including the regulation of membrane vesicular trafficking. PLD1 is activated by the small G proteins, ADP-ribosylation factor (ARF) and RhoA, and by protein kinase C-alpha (PKC-alpha ). This stimulation has been proposed to involve direct interaction and to take place at a distinct site in PLD1 for each activator. In the present study, we employed the yeast two-hybrid system to attempt to identify these sites. Successful interaction of ARF and PKC-alpha with PLD1 was not achieved, but a C-terminal fragment of human PLD1 (denoted "D4") interacted with the active mutant of RhoA, RhoAVal-14. Deletion of the CAAX box from RhoAVal-14 decreased the strength of the interaction, suggesting that lipid modification of RhoA is important for efficient binding to PLD1. The specificity of the interaction was validated by showing that the PLD1 D4 fragment interacts with glutathione S-transferase-RhoA in vitro in a GTP-dependent manner and that it associates with RhoAVal-14 in COS-7 cells, whereas the N-terminal two-thirds of PLD1 does not. Finally, we show that recombinant D4 peptide inhibits RhoA-stimulated PLD1 activation but not ARF- or PKC-alpha -stimulated PLD1 activation. These results conclusively demonstrate that the C-terminal region of PLD1 contains the RhoA-binding site and suggest that the ARF and PKC interactions occur elsewhere in the protein.

    INTRODUCTION
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Abstract
Introduction
References

Phospholipase D (PLD)1 activity is present in many types of mammalian cells and tissues and is up-regulated in response to a wide variety of agonists (1, 2). PLD catalyzes the hydrolysis of phosphatidylcholine (PC) to yield phosphatidic acid (PA) and choline (2). PA can be further metabolized to diacylglycerol, the activator of protein kinase C (PKC), or to lyso-PA, which acts on specific cell-surface receptors (3). PA itself may play a role(s) as a second messenger; PA has been shown in vitro to stimulate phosphatidylinositol 4-phosphate 5-kinase (4), PKC-zeta (5), and protein-tyrosine phosphatase (6). It has also been reported that PA promotes coatomer binding (7) and enhances actin polymerization (8).

Studies in recent years have identified several PLD activators, including many members of the ADP-ribosylation factor (ARF) (9, 10) and RhoA (11, 12) low molecular weight GTP-binding protein (small G protein) families and PKC-alpha (13). In addition, phosphatidylinositol 4,5-bisphosphate (PIP2) has been demonstrated to be absolutely required as a cofactor for such activator-dependent PLD stimulation (9).

cDNAs encoding two mammalian PLDs, PLD1 (14-16) and PLD2 (15, 17), have recently been cloned. Two alternatively spliced forms, termed PLD1a and PLD1b, exist for PLD1 (18). PLD1a comprises 1074 amino acids, whereas PLD1b is a shorter form lacking 38 amino acids residues (585-622). PLD2 is ~50% identical to PLD1 and is constitutively active in vitro and in vivo (15). In contrast, PLD1a and PLD1b exhibit a low basal activity and are stimulated by ARF, RhoA, and PKC-alpha in the presence of PIP2. These findings were demonstrated using purified recombinant PLD1 and activators (18), which led to the conclusion that PLD1 interacts directly with the lipid cofactor PIP2 and the protein activators. Since the PKC-alpha and the small G protein activators elicit a synergistic activation when combined, it was also proposed that they would mediate PLD1 stimulation through interactions at separate sites (18). These sites of interaction, however, have not yet been defined. We address this question in the present study and show that the C-terminal region of PLD1 is the RhoA-binding domain.

    EXPERIMENTAL PROCEDURES

Reagents-- Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma, and phosphatidylethanolamine (PE), 1,2-dipalmitoyl-PC (DPPC), PIP2, [choline-methyl-3H]DPPC, and GTPgamma S were from sources previously reported (12).

Yeast Two-hybrid Analysis-- A template for PCR to generate cDNA fragments for truncation mutants of human (h) PLD1a was prepared from total RNA of HL60 cells by reverse transcription with random hexamer mixed primers, followed by PCR with a set of primers designed to amplify the entire open reading frame of hPLD1a. The PCR product was cloned into pT7Blue(R) vector (Novagen). cDNA fragments encoding amino acid residues 1-415 (D1), 365-727 (D2), 663-1074 (D3), 712-1074 (D4), and 763-1074 (D5) of hPLD1a were generated by PCR with primers containing 5'-BclI and 3'-NotI sites, digested with BclI and NotI, and purified. A cDNA fragment encoding amino acid residues 712-1015 (D6) of hPLD1a was obtained from the D4 cDNA by digestion with PvuII. The cDNA fragments for D1-D6 were ligated into the yeast expression vector pVP16 containing the VP16 transcription activation domain (a generous gift of Dr. Stanley Fields).

The cDNAs encoding wild type and mutants of RhoA were ligated into the vector pBTM116 containing LexA-binding domain as described previously (19). The expression plasmids were co-transfected into Saccharomyces cerevisiae strain L40 cells and plated on synthetic complete media lacking tryptophan and leucine. Interaction was basically assessed by a filter assay for beta -galactosidase activity (20).

Preparation of GST-RhoA and FLAG-tagged D4 Peptide-- Wild type RhoA was expressed in Escherichia coli as a glutathione S-transferase fusion protein (GST-RhoA) and purified by glutathione-Sepharose 4B column chromatography. After removal of free glutathione by Q-Sepharose column chromatography, the purified GST-RhoA was stored at -80 °C until use.

The D4 peptide was expressed in E. coli as a FLAG epitope-tagged protein (FLAG-D4). cDNA of the D4 peptide was amplified by PCR with a sense primer containing 5'-XhoI/EcoRI sites and an antisense primer containing a 3'-BglII site using pT7Blue(R)-hPLD1a as a template, digested with XhoI and BglII, and inserted into the pFLAG MAC vector (Eastman Kodak Co.). FLAG-D4 was inducibly expressed in E. coli strain BL21 cells at 30 °C for 3 h with 0.3 mM isopropyl-beta -D-thiogalactopyranoside. After disruption of cells by sonication and centrifugation (100,000 × g, 30 min), solubilized FLAG-D4 was purified with an anti-FLAG M2 antibody affinity resin (Kodak) and stored at -80 °C until use.

In Vitro Association Assay-- Five µg of GST-RhoA was incubated at 30 °C for 10 min with 5 µg of FLAG-D4 in the presence of 40 µM GDP or GTPgamma S in a total volume of 50 µl. GST-RhoA was then precipitated with a glutathione-Sepharose 4B resin in the presence of 0.3% n-octyl-beta -D-glucopyranoside. FLAG-D4 co-precipitated with GST-RhoA was detected by immunoblotting with an anti-FLAG M2 antibody (Kodak).

In Vivo Association Assay-- For the expression of FLAG-D4 in COS-7 cells, the D4 cDNA amplified by PCR as described above was digested with EcoRI and BglII and cloned into pTB701-FLAG vector (a generous gift of Drs. S. Kuroda and U. Kikkawa) (21). To express a FLAG-tagged peptide (FLAG-D(1+2)) which complemented the FLAG-D3/D4 peptides, a cDNA encoding amino acid residues 1-670 was generated. In brief, an allele of hPLD1b altered through the insertion of a stop codon at amino acid 671 was used. The allele was generated using a pentapeptide mutagenesis protocol (22) that will be described in detail elsewhere. pTB701-FLAG-D(1+2) was constructed by insertion of the 1.8-kilobase pair BglII-BamHI fragment of pCGN-D(1+2) into the BglII-BglII gap of pTB701-FLAG-hPLD1a (see below). pEF-BOS-HA-RhoA and pEF-BOS-HA-RhoAVal-14 were prepared as described previously (23). COS-7 cells were co-transfected with pTB701-FLAG-D4 or pTB701-FLAG-D(1+2) and either pEF-BOS-HA-RhoA or pEF-BOS-HA-RhoAVal-14 by electroporation. After 48 h, the cells were lysed in a buffer containing 0.5% Nonidet P-40 and centrifuged at 10,000 × g for 20 min. FLAG-D4 or FLAG-D(1+2) was immunoprecipitated from the supernatant with the anti-FLAG M2 antibody affinity resin, and co-immunoprecipitated HA-tagged RhoA or RhoAVal-14 was detected by immunoblotting with an anti-HA monoclonal antibody (16B12, BAbCO).

Preparation of FLAG-hPLD1a, RhoA, ARF, and PKC-alpha -- To prepare FLAG-hPLD1a, the cDNA encoding the full-length of hPLD1a was amplified by PCR with primers containing an MunI site using pT7Blue(R)-hPLD1a as a template, digested with MunI, and inserted into the pTB701-FLAG vector. The plasmid was transfected into COS-7 cells by electroporation. After being cultured for 48 h, cells were lysed in a buffer containing 1% Nonidet P-40 and centrifuged (50,000 × g, 30 min), and solubilized FLAG-hPLD1a was purified with the anti-FLAG M2 antibody affinity resin.

For RhoA purification, bovine brain cytosol was subjected to the DEAE-Toyopearl 650S and the phenyl-Toyopearl 650M column chromatography (12). The major peak of RhoA eluted from the latter column (12) was re-chromatographed on a phenyl-Toyopearl 650M column. To highly purify ARF, the partially purified ARF from bovine brain cytosol by DEAE-Sepharose and Sephacryl S-200 column chromatography (12) was then sequentially subjected to chromatography on a heparin-Sepharose CL-6B and a phenyl-Toyopearl 650M column. Both the purified small G proteins were estimated to be greater than 90% pure by SDS-PAGE (data not shown). Recombinant PKC-alpha was expressed in and purified from Sf21 cells as previously reported (24).

PLD Assay-- FLAG-hPLD1a was reconstituted with the purified RhoA or ARF or recombinant PKC-alpha in the presence of PE/PIP2/[choline-methyl-3H]DPPC vesicles (158.64 µM) in a molar ratio of 16:1.4:1 (9). The reaction also contained the indicated concentrations of the FLAG-D4 peptide. For the activation of FLAG-hPLD1a by PKC-alpha , 100 nM PMA was included in the reaction. The mixture was incubated at 37 °C for 30 min in 45 mM Na-Hepes, pH 7.4, 3 mM EGTA, 150 mM KCl, 1 mM dithiothreitol, 3 mM MgCl2, 2 mM CaCl2, 40 µM GTPgamma S, and 1 mg/ml bovine serum albumin, and then the production of [3H]choline was determined (12).

Immunoblotting-- Proteins in samples were separated by SDS-PAGE on 12% gel and transferred to polyvinylidene difluoride membranes. The membrane was blocked and incubated with the first antibodies and then with peroxidase-conjugated rabbit anti-mouse IgG (12). Immunoreactive proteins were detected with an ECL immunoblotting detection reagent (Amersham Pharmacia Biotech).

Protein Assay-- Protein concentration was determined by the method of Bradford (25) using bovine serum albumin as a standard.

    RESULTS AND DISCUSSION

RhoA Interacts with the C Terminus of PLD1-- To identify the PLD1 domains mediating interaction with each activator, we employed the LexA version of the yeast two-hybrid system (26). Since reporter gene activation in this system requires import of the fusion proteins into the nucleus and this nuclear import is often unsuccessful for large proteins (26), fragments of hPLD1 and the activators (RhoA, ARF, and PKC-alpha ) were fused to the binding domain of LexA and the herpes VP16/GAL4 activation domain, respectively. Consistent with our previous report (27), RhoAVal-14, the active form of RhoA, successfully interacted with the C terminus of PLD1. However, this was the only combination that succeeded (data not shown). The RhoAVal-14/PLD1 C terminus interaction appears likely to be specific, since the corresponding C-terminal regions of mammalian PLD2 and yeast PLD, which are related in sequence to mammalian PLD1 but do not respond to RhoA (15), did not activate the yeast reporter genes when co-expressed with RhoAVal-14 (data not shown).

To begin to validate the RhoA-PLD1 interaction, we switched the PLD fragments to the VP16 activation vector and RhoA to the LexA DNA binding vector and attempted to subdivide the interacting PLD1 region (Fig. 1A). Confirming the results described above, the N-terminal (D1) and central (D2) fragments did not interact with RhoAVal-14, but the C terminus (D3, amino acid residues 663-1074, and D4, amino acid residues 712-1074) did. Further truncation of the D4 fragment from either end (D5, amino acid residues 763-1074, and D6, amino acid residues 712-1015) eliminated the interaction. These results demonstrate that a 362-amino acid C-terminal fragment of hPLD1 constitutes essentially the minimal fragment capable of interacting with RhoAVal-14 in the yeast two-hybrid system.


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Fig. 1.   Dissection of the PLD1 interaction with RhoAVal-14. Yeast L40 cells were co-transfected with expression vectors encoding fragments of hPLD1a fused to the VP16 activation domain and wild type or mutants of RhoA fused to the LexA DNA-binding domain. Interaction was assessed using a filter assay for beta -galactosidase activity. A, confirmation and refinement of the RhoA-binding region in hPLD1 using RhoAVal-14 and a series of hPLD1a peptides. Hatched boxes (I-IV) in PLD1a (top line) indicate the regions highly conserved in the PLD superfamily (43). B, interaction of the hPLD1a D4 fragment with wild type and mutant RhoA. The results shown are from a single experiment representative of seven.

The C-terminal motif CAAX (C, cysteine; A, aliphatic amino acid; X, any amino acid) found in RhoA family members is post-translationally modified by geranylgeranylation on the cysteine residue, which leads to subsequent proteolysis and carboxylmethylation (28, 29). These post-translational modifications of the Rho GTPases are important for promoting interaction with their GDP/GTP exchange proteins and effectors (30-32). To examine the role of C-terminal modification of RhoA in the interaction with PLD1 in the yeast two-hybrid system, four types of RhoA (wild type, the CAAX deletion mutant RhoACLVL-, the active mutant RhoAVal-14, and the CAAX-deleted active mutant RhoAVal-14CLVL-) were tested (Fig. 1B). Wild type RhoA and RhoACLVL- both failed to interact, confirming that PLD1 specifically interacts with the active form of RhoA. RhoAVal-14CLVL- interacted with PLD1, but the interaction was clearly weaker than that observed with RhoAVal-14, suggesting that geranylgeranylation of RhoA strengthens the interaction with PLD1.

RhoA Interacts with the D4 Fragment in Vitro-- An important control for two-hybrid interactions is to demonstrate successful association in vitro. This was addressed by mixing bacterially expressed GST-RhoA with FLAG-D4 in the presence of GDP or GTPgamma S and precipitating the GST-RhoA using glutathione-Sepharose (Fig. 2). In the presence of GTPgamma S, FLAG-D4 was readily co-precipitated (lane 4), whereas very little FLAG-D4 was co-precipitated in the presence of GDP (lane 3). GST itself did not interact with FLAG-D4 at all (lanes 1 and 2). Thus, the D4 fragment specifically interacts only with the active form of RhoA in vitro as well.


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Fig. 2.   In vitro interaction of the PLD1 C terminus with the active form of RhoA. FLAG-D4 expressed in and purified from E. coli was incubated with GST or GST-RhoA in the presence of 40 µM GDP or GTPgamma S, following which the GST-RhoA was precipitated as described under "Experimental Procedures." The FLAG-D1 and -D2 peptides were expressed poorly in E. coli, which prevented us from confirming that these peptides failed to interact with RhoA in vitro. The amount of FLAG-D4 co-precipitated with GST or the GST-RhoA was detected by immunoblotting with an anti-FLAG M2 antibody. The results shown are from a single experiment representative of six performed with independent preparations of FLAG-D4 and GST-RhoA.

We previously reported that non-geranylgeranylated RhoA does not activate rat brain PLD, whereas geranylgeranylated RhoA does (12), which was recently confirmed by Bae et al. (33). The results shown in Figs. 1B and 2 nonetheless provide evidence that non-geranylgeranylated RhoA interacts with hPLD1a, although seemingly not as well as geranylgeranylated RhoA (Fig. 1B). These observations, taken together, suggest that the post-translational modification of RhoA plays a role not only in the interaction with PLD1 but also, as a separate event, in the subsequent activation of PLD1.

RhoA Interacts with the D4 Fragment in COS-7 Cells-- Another important control for two-hybrid interactions is to demonstrate association in a physiological setting. We addressed this by examining interaction of the D4 peptide with RhoA in COS-7 cells. When FLAG-D4 was co-expressed with HA-RhoA or -RhoAVal-14 in COS-7 cells and immunoprecipitated, HA-RhoAVal-14 readily co-immunoprecipitated, whereas there was very little evidence for interaction of FLAG-D4 with wild type RhoA (Fig. 3A, lanes 5 and 6 in the upper panel). Equivalent amounts of FLAG-D4 and of RhoA were expressed in each sample, ruling out differences in protein stability or expression as an explanation for the finding (Fig. 3A, middle and lower panels, respectively). The expression levels of FLAG-D1 and -D2 in COS-7 cells were extremely low as compared with FLAG-D4, which prevented subsequent analysis of their interaction or lack of interaction with RhoA. However, FLAG-D(1+2), which encodes amino acids 1-670 of hPLD1b and is thus complementary to FLAG-D3/D4, was expressed in COS-7 cells to a level comparable with that of FLAG-D4 (Fig. 3B, middle panel). As anticipated, FLAG-D(1+2) failed to interact with both wild RhoA and RhoAVal-14 (Fig. 3B, upper panel). These results in a physiological setting strengthen the premise that the PLD1 D4 fragment specifically interacts with the active form of RhoA.


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Fig. 3.   In vivo association of the PLD1 C terminus with the active form of RhoA. FLAG-D4 (A) and FLAG-D(1+2) (B) were co-expressed with HA-RhoA or -RhoAVal-14 in COS-7 cells. After immunoprecipitation of FLAG-D4 and FLAG-D(1+2) using an anti-FLAG M2 affinity resin, the amount of RhoA co-immunoprecipitated was detected by immunoblotting with an anti-HA antibody (upper panel). As a control, the amounts of FLAG-D4 and FLAG-D(1+2) immunoprecipitated were visualized in a parallel blot using an anti-FLAG M2 antibody (middle panel), and the amounts of wild type RhoA and RhoAVal-14 expressed in the cell lysates were detected by immunoblotting using an anti-HA antibody (lower panel). The results shown are from a single experiment representative of three.

The D4 Peptide Blocks RhoA Stimulation of PLD1 in Vitro-- Although the results presented thus far conclusively demonstrated an interaction between the D4 peptide and the active form of RhoA, they did not indicate whether ARF or PKC-alpha might also activate PLD1 through interaction with the C terminus. We addressed this by examining the effects of the D4 peptide on PLD1 stimulation by ARF, RhoA, and PKC-alpha . The D4 peptide inhibited RhoA-stimulated PLD1 activation in a dose-dependent manner, but not ARF- and PKC-alpha -stimulated activation (Fig. 4). These results suggest that ARF and PKC-alpha interact with other regions of PLD1 and also rule out possible sources of nonspecific inhibition that might otherwise represent alternative mechanisms through which the D4 peptide could be causing inhibition of RhoA-mediated activation of PLD1.


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Fig. 4.   Effects of the D4 peptide on activation of hPLD1a by RhoA, ARF, and PKC-alpha . Purified FLAG-hPLD1a (8 nM) was incubated at 37 °C for 30 min with 0.14 µM RhoA, 0.3 µM ARF, or 5.3 nM PKC-alpha in the presence of the indicated concentrations of the FLAG-D4 peptide, and PLD activity was determined as described under "Experimental Procedures." The PLD activities stimulated by RhoA, ARF, and PKC-alpha were 84.12, 38.31, and 12.12 pmol/30 min/assay, respectively. The PLD activities are presented as a percentage of the activity stimulated by each activator in the absence of the FLAG-D4 peptide. The error bars represent the differences of duplicate determinations. The results shown are from a single experiment representative of three.

Various proteins, including Rhofillin, PKN, Rhotekin, PRK2, citron, Rho kinase/p160ROCK/ROK, p140mDia, and myosin-binding subunit, have been identified as downstream effectors for RhoA (34). The amino acid sequences of the RhoA-binding domains of Rhofillin, PKN, Rhotekin, and PRK2 share about 50% homology and have therefore been denoted as the RhoA effector motif class 1 (REM-1) (35). The RhoA-binding motif of the other effectors, termed REM-2, does not share homology with REM-1 (36). Thus, two RhoA-binding motifs have thus far been identified. The nature of the interaction of PLD1 with the Rho family GTPases seems to be different from those of the already reported Rho-binding effectors. PLD1 appears to be able to interact with many subsets of Rho family members at the same site, as is inferred from the observation that Rac1 and Cdc42 also activate hPLD1a, and their effects are not synergistic with RhoA or with each other (18). On the other hand, citron and PRK2 interact with RhoA and Rac1, but not with Cdc42 (34, 37, 38), and other RhoA target proteins, PKN, Rhotekin, p160ROCK, and p140Dia, interact only with RhoA (35, 39-41). These observations suggest that the mechanism employed by the PLD1 RhoA-binding motif is different from that used by REM-1 and REM-2. Not surprisingly, computer analysis using the CLUSTAL W program (42) reveals that the amino acid sequence of the RhoA-binding region in PLD1 defined in this study is not significantly similar to either REM-1 or REM-2 (data not shown), confirming that the PLD1 interacting site is unique.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Shun'ichi Kuroda and Ushio Kikkawa for providing the pTB701-FLAG vector and to Dr. Stanley Fields for providing the pVP16 vector. We also thank Dr. Akira Matsuura for helpful discussions.

    FOOTNOTES

* This work was supported in part by research grants from the Ministry of Education, Science, Sports and Culture, Japan, the ONO Medical Research Foundation, and the Sankyo Foundation of Life Science (to Y. K.) and by National Institutes of Health Grant GM54813 (to M. A. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Research Fellow of the Japan Society for the Promotion of Science.

§§ To whom correspondence should be addressed. Tel.: 81-45-924-5717; Fax: 81-45-924-5774; E-mail: ykanaho{at}bio.titech.ac.jp.

    ABBREVIATIONS

The abbreviations used are: PLD, phospholipase D; PC, phosphatidylcholine; DPPC, 1,2-dipalmitoyl-PC; PE, phosphatidylethanolamine; PIP2, phosphatidylinositol 4,5-bisphosphate; PA, phosphatidic acid; PMA, phorbol 12-myristate 13-acetate; small G proteins, low molecular weight GTP-binding proteins; ARF, ADP-ribosylation factor; PKC, protein kinase C; PCR, polymerase chain reaction; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); PAGE, polyacrylamide gel electrophoresis; REM-1 and REM-2, Rho effector motif class 1 and 2; GST, glutathione S-transferase; HA, hemagglutinin.

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