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ELMOD2 Is an Arl2 GTPase-activating Protein That Also Acts on Arfs *

ELMOD2 Is an Arl2 GTPase-activating Protein That Also Acts on Arfs * THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 24, pp. 17568 –17580, June 15, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. ELMOD2 Is an Arl2 GTPase-activating Protein That Also Acts on Arfs Received for publication, February 15, 2007, and in revised form, April 13, 2007 Published, JBC Papers in Press, April 23, 2007, DOI 10.1074/jbc.M701347200 ‡ § § ‡1 J. Bradford Bowzard , Dongmei Cheng , Junmin Peng , and Richard A. Kahn ‡ § From the Department of Biochemistry and Department of Human Genetics, Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, Georgia 30322 Regulatory GTPases in the Ras superfamily employ a cycle of were initially thought to simply inactivate their cognate alternating GTP binding and hydrolysis, controlled by guanine GTPase, but more recent research suggests that GAPs can also nucleotide exchange factors and GTPase-activating proteins serve as effectors of the signaling pathways regulated by the (GAPs), as essential features of their actions in cells. Studies of GTPase (e.g. ArfGAP1 and ARAP2 (2, 3)). these GAPs and guanine nucleotide exchange factors have pro- The first GAPs were purified based on their biochemical vided important insights into our understanding of GTPase sig- activities, but sequence and mutational analyses led to the iden- naling and biology. Within the Ras superfamily, the Arf family is tification of GAP domains, which have proven highly predictive composed of 30 members in mammals, including 22 Arf-like for identifying novel GAPs within a GTPase family. More than (Arl) proteins. Much less is known about the mechanisms of cell 170 putative GAPs have been identified and are grouped regulation by Arls than by Arfs. We report the purification from according to which GTPase they act upon and which GAP bovine testis of an Arl2 GAP and its identity as ELMOD2, a domain they possess (4, 5). Typically a protein with a given GAP protein with no previously described function. ELMOD2 is one domain will be active against members of one family of of six human proteins that contain an ELMO domain, and a GTPases, or a subset within that family, but not against other second member, ELMOD1, was also found to have Arl2 GAP families. For example, a protein with an Arf GAP domain will be activity. Surprisingly, ELMOD2 also exhibited GAP activity active against one or more (but not necessarily all) Arfs but not against Arf proteins even though it does not contain the canon- against Rabs, Rhos, or Ras. Exceptions to these general rules ical Arf GAP sequence signature. The broader specificity of exist (6–8), and more complete information about the deter- ELMOD2, as well as the previously described role for ELMO1 minants of GAP specificity will be required to understand the and ELMO2 in linking Arf6 and Rac1 signaling, suggests that roles that GAPs play in the biology of the cell. Determining the ELMO family members may play a more general role in integrat- specificity of a GAP for different GTPases is further compli- ing signaling pathways controlled by Arls and other GTPases. cated by factors that can influence the results of in vitro GAP assays, including the assay used to measure GTP hydrolysis, the need for hydrophobic surfaces on which protein interactions The Ras superfamily of regulatory GTPases is composed of occur, the degree of membrane curvature (e.g. in the case of over 150 members divided into at least five families (Ras, ArfGAP1 (9–11)), or the presence of co-activators (e.g. phos- phatidylinositols (12, 13)). Rho, Rab, Ran, and Arf (1)). Although the biological function The importance of specificity is illustrated by the Arf family, of each family is distinct, all members exhibit many struc- tural and biochemical similarities. They share conserved which in mammals consists of 6 Arfs, 22 Arf-like (Arl), and 2 Sar sequences involved in guanine nucleotide binding, have low proteins (14). The Arf family is best known for the role of Arfs and Arl1 as regulators of adaptor recruitment and phospholipid intrinsic levels of GTP hydrolysis, and cycle between GTP- metabolism, predominantly at the Golgi, although members of and GDP-bound states as critical components of their bio- logical functions. It is the ability of these two states to inter- the Arf family are probably active at every membrane in act with different sets of proteins that enable these small eukaryotes (15). The remaining Arls exhibit a far greater diver- sity of functions than do the Arfs as they have been implicated GTPases to act as “molecular switches.” as regulators of microtubule-dependent processes (Arl2 and Because of the low intrinsic rates of GTP hydrolysis, the tran- sition from the GTP-bound to GDP-bound form of the GTPase Arl3 (16–20)), lysosome mobility, and microtubule binding requires an accessory GTPase-activating protein (GAP). GAPs (Arl8 (21, 22)), ciliogenesis (Arl3 and Arl6 (23–28)), and tumor- igenesis (Arl11 (29, 30)). With increasing interest in these pro- teins, it becomes more important to identify regulators and * This work was supported in part by Grants GM24680 (to R. A. K.), GM067465 effectors that can provide insights into the biological function (to J. B. B.), and AG025688 (to J. P.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the pay- of each of the Arls and to determine the extent of overlap in ment of page charges. This article must therefore be hereby marked their actions and protein partners. “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi- cate this fact. To whom correspondence should be addressed: Dept. of Biochemistry, Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322- monio]-1-propanesulfonic acid; Arl, Arf-like; MBP, maltose-binding pro- 3050. Tel.: 404-727-3561; E-mail: [email protected]. tein; LC-MS/MS, liquid chromatography-tandem mass spectrometry; The abbreviations used are: GAP, GTPase-activating protein; GEF, guanine DTT, dithiothreitol; CMC, critical micelle concentration; PH, pleckstrin nucleotide exchange factor; CHAPS, 3-[(3-cholamidopropyl)dimethylam- homology. 17568 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 24 •JUNE 15, 2007 This is an Open Access article under the CC BY license. ELMOD2 Is an Arl2 GAP The prediction of20 Arf GAPs in the human proteome (4) umn (GE Healthcare) according to the manufacturer’s instruc- is based upon the presence of the Arf GAP domain, a cysteine- tions. Purified recombinant preparations of RhoA and Rac1 (G. rich zinc finger with specific spacing (CX CX CX C, where Bokoch, Scripps Research Institute) and Ran (I. Macara, Uni- 2 16–18 2 X is any amino acid (12, 31–33)). The specificities of these pro- versity of Virginia) were generously provided by colleagues. teins have not yet been completely determined, but to date none have demonstrated activity against Arls. One of the four GAP Assays Arf GAPs in Saccharomyces cerevisiae (34) and a partially puri- Charcoal GAP Assay—Our standard GAP assay was per- fied preparation of rat spleen cytosol (35) have shown activity formed by pre-loading recombinant human Arl2 with against both Arfs and Arl1, but this is not surprising given their [- P]GTP in a reaction that contained 2 M Arl2 in 25 mM close sequence and functional relatedness. HEPES, pH 7.4, 100 mM NaCl, 2.5 mM MgCl ,1mM dithiothre- GAPs are often expressed only to low levels, and their assay in itol (DTT), and 0.3 Ci/l[- P]GTP (6000 Ci/mmol; cell or tissue lysates can be compromised by a number of fac- PerkinElmer Life Sciences) for 15 min at 30 °C. Under these tors. We took advantage of the observation that Arl2 is partially conditions, typically50% of the radionucleotide will be bound localized to mitochondria to develop an Arl2 GAP assay using to Arl2 by the end of the preincubation. The amount of bound an enriched mitochondria preparation as the source of the [- P]GTP was determined by filtration of an aliquot of the activity (36). Later modifications to the assay allowed us to pre-loaded Arl2 onto a nitrocellulose filter and scintillation purify the activity from bovine testis. The purified Arl2 GAP counting, as described below. During this incubation, 25 lof contained a domain found in other human proteins, the ELMO the GAP sample to be assayed was mixed with 20 l of reaction domain, that we propose functions as an Arl GAP domain. The buffer (62.5 mM HEPES, 6.25 mM MgCl , 2.5 mM DTT, 2.5 mM purification of this Arl2 GAP and issues surrounding the assay GTP, and 12.5 mM ATP) on ice. The reaction was started by of GAP proteins in general are discussed. The observation that adding 5 l of the pre-loaded Arl2 to the 45-l GAP/buffer this GAP also acted on Arfs provides an opportunity for cross- combination, mixing, and placing at 30 °C. The reaction was talk between Arfs and Arls. stopped 4 min later by the addition of 750 l of a suspension of ice-cold activated charcoal (5% in 50 mM NaH PO ). The char- 2 4 EXPERIMENTAL PROCEDURES coal was pelleted, and the amount of released P in 400 lof Antibodies, Cells, and Reagents the supernatant was determined by scintillation counting. A All chemicals used were purchased from commercial mock load reaction, which was identical to the pre-loading step sources. Sol-Grade CHAPS was purchased from Anatrace, Inc. but did not contain Arl2, was also performed and run in parallel (Maumee, OH). Mouse anti-Myc (9E10; Covance Research for each sample to be assayed. This control measures the Products, Berkeley, CA) was used at a 1:1000 dilution in immu- amount of GTP hydrolysis that occurs during the assay that is noblots. Bovine testicles (trimmed) were purchased from Pel- independent of Arl2-GTP. The control results were then sub- Freez Biologicals (Rogers, AR), and the tunica albuginea was tracted from those obtained with Arl2-GTP in the reaction to removed before use. obtain the Arl2-dependent GTP hydrolysis. Because Arl2 can- not hydrolyze GTP to a measurable extent by itself, this value is Expression Plasmids equal to the Arl2 GAP activity. The coding sequences of human ELMOD1 and ELMOD2 Filter Trapping GAP Assay—Each GTPase was pre-loaded were amplified from a human fetal brain cDNA library with [- P]GTP in a reaction that is 1 M GTPase in 20 mM (Clontech) using custom synthetic oligonucleotide primers HEPES, pH 7.4, 100 mM NaCl, 50 g/ml bovine serum albumin, and cloned into a pET28-based vector also containing the 1mM DTT, 0.5 mM MgCl ,1mM EDTA, 5 mM ATP, and 0.3 Escherichia coli maltose-binding protein (MBP) open read- Ci/l[- P]GTP (PerkinElmer Life Sciences, 6000 Ci/mmol) ing frame (provided by Xing Zhang and Xiaodong Cheng, for 15 min at 30 °C. The pre-load reaction was then brought to Emory University) and pcDNA3.1/myc-His (Invitrogen). a final concentration of 20 mM MgCl and placed on ice. The The coding sequence of ELMOD3 was amplified from a plas- GAP sample (15 l) to be assayed was mixed with reaction mid purchased from OriGene Technologies (Rockville, MD) buffer (25 l; 62.5 mM HEPES, 6.25 mM MgCl , 2.5 mM DTT, 2.5 and subcloned as above. mM GTP, and 12.5 mM ATP) on ice. A control reaction (GAP) was run in parallel to determine the amount of [- P]GTP or Preparation of Recombinant Proteins P released from the GTPase independent of the GAP. The reactions were started by adding 10 l of the pre-loaded Purified recombinant human Arl2, Arl3, Arf1, and Arf6 were GTPase to the 40-l GAP/buffer combination, mixing, and each prepared as described previously (36–38). pET-based placing at 30 °C. At each time point, 10 l was removed from plasmids encoding MBP-ELMOD1 or MBP-ELMOD2 were the reaction to 2 ml of ice-cold HNMD (20 mM HEPES, pH 7.4, transformed into E. coli (BL21(DE3)) and single colonies picked 100 mM NaCl, 10 mM MgCl ,1mM DTT) and immediately into LB media. Cultures were grown at 37 °C until the A filtered over a 0.45-m Protran BA85 filter (Whatman), which 0.2 at which point they were induced with isopropyl 1-thio-- D-galactopyranoside to a final concentration of 0.1 mM. After an was then washed four times with 2-ml aliquots of HNMD. The additional 15 h of growth at room temperature, the cells were amount of GTPase-[- P]GTP still bound to the filters was harvested and lysed by multiple passes through a French press. determined by scintillation counting. The amount of GAP Purification was carried out with a HiTrap chelating HP col- activity is the difference between the counts lost from the JUNE 15, 2007• VOLUME 282 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 17569 ELMOD2 Is an Arl2 GAP GTPase in the presence of the GAP sample minus the control (Beckman, Fullerton, CA) rotor for 120 min. The interface (-GAP) sample. between the 20 and 60% sucrose layers (“20/60”) was recovered by pipetting and gently vortexed to mix. Calculation of Specific Arl2 GAP Activities CHAPS (1.25 ml of 10% in 50 mM HEPES, pH 7.4) and Buffer Arl2 GAP activities were determined using the charcoal GAP A (148.75 ml; 25 mM HEPES, pH 7.4, 10 mM NaCl, 1 mM DTT, assay, performed in parallel with controls, as described above. 2mM EDTA, 0.25% CHAPS) were added to 50 ml of 20/60 to To best quantify this assay we made an assumption, based upon bring the final protein concentration to2 mg/ml and the final prior ligand-binding data, that when Arl2 is exposed to high detergent concentration to 0.25%. After a 30-min incubation on concentrations of GTP it binds very rapidly to a stoichiometry ice, the diluted 20/60 was spun at 100,000  g for 60 min. The of 20% (determined for each preparation of Arl2 but quite supernatant was recovered by pipette and loaded onto a 140-ml consistent). Thus, during the preincubation phase the Arl2 (2.6  30 cm) Macro-Prep High Q column (Bio-Rad). The col- bound about 50% of the [- P]GTP available to it. When the umn was washed with 140 ml of Buffer A, and the bound pro- vast excess of unlabeled GTP is added at the start of the GAP teins were eluted with a gradient of 0–40% Buffer B (25 mM assay, the free GTP-binding sites on the remaining Arl2 mole- HEPES, pH 7.4, 1 M NaCl, 1 mM DTT, 2 mM EDTA, 0.25% cules quickly became occupied with cold GTP. Thus, we used in CHAPS) over 400 ml. Arl2 GAP activity was assayed using the our calculations the 20% value to estimate the concentration of charcoal assay and found to elute between 75 and 250 mM NaCl. Arl2-GTP at the start of the assay, typically 1.8 pmol per assay The peak fractions were pooled, concentrated using an Amicon point (2.5 nM). The GAP-dependent P counts, released dur- Ultra (Millipore, Billerica, MA) 15-ml 30,000-Da nominal ing the 4-min incubation, were divided by the total counts avail- molecular weight limit centrifugal concentrator, and applied to able in each assay, determined by filter trapping at the end of the a 120-ml (1.6  60 cm) HiLoad Superdex 200 prep grade (GE preincubation phase. This ratio represents the percentage of Healthcare) gel filtration column, which was developed with total Arl2-GTP present in the GAP reaction that was converted Buffer C (25 mM HEPES, pH 7.4, 50 mM NaCl, 1 mM DTT, 0.25% into Arl2-GDP during the assay. This was then multiplied by CHAPS). The peak fractions from the gel filtration column, the [Arl2-GTP] to get the amount converted in the 4-min assay. corresponding to a predicted molecular mass range of 150–300 Specific activity was reported in units/mg protein in the assay kDa, were loaded onto a 15-ml (1.6  20 cm) Macro-Prep where 1 unit is the amount of activity required to convert 1 ceramic hydroxyapatite type I column (20 m; Bio-Rad). The nmol of Arl2-GTP to Arl2-GDP/min. column was washed with 10 ml of Buffer D (25 mM HEPES, pH To calculate the specific activity of a GAP using the filter 7.4, 10 mM NaCl, 1 mM DTT, 5 mM K HPO ,5mM KH PO , 2 4 2 4 trapping assay, we again determined the GAP-dependent loss 0.25% CHAPS) and then 10 ml of 10% Buffer E (25 mM HEPES, of P trapped on the filter during the assay, as described above. pH 7.4, 10 mM NaCl, 1 mM DTT, 200 mM K HPO , 200 mM i 2 4 This number was divided by the total bound radionucleotide KH PO , 0.25% CHAPS) before being developed with a 60 ml of 2 4 available at t 0 to get the percentage hydrolyzed. Because the 10–70% gradient of Buffer E. Fractions containing activity were GTPases other than Arl2 do not bind significant amounts of pooled, concentrated, and further resolved on the 120-ml unlabeled GTP during the GAP assay, because of the presence Superdex 200 column as above. of high [Mg ], the amount of GTPase-GTP present in the Peak fractions from the gel filtration column were pooled reaction was determined directly from the specific activity of and brought to a final concentration of1% CHAPS by adding the radioligand. Note that because of the differences in the ways an amount of 10% CHAPS equal to one-ninth of the initial these two assays are performed, we estimate that the concen- volume of the pooled fractions. After 30 min on ice, this sample tration of Arl2-GTP was about 10-fold higher than the GTP- was applied to a 6-ml Resource Q column (GE Healthcare). The bound form of the other GTPases. flow-through was collected and concentrated using an Amicon Ultra 4-ml 10,000-Da nominal molecular weight limit centrif- Purification of ELMOD2 from Bovine Testis ugal concentrator to a final volume of 0.5 ml. This was then Numerous preparations of Arl2 GAP activity were per- applied to a 24-ml (1.0  30 cm) Superdex 200 gel filtration formed, with a number of variations, and a typical example (the column (GE Healthcare). Fractions (0.35 ml) from this final one that was used for mass spectroscopy analysis) is described column were collected and assayed. here. 137 g (wet weight) of bovine testis was homogenized on Mass Spectroscopy ice in 500 ml of SH Buffer (25 mM HEPES, pH 7.4, 10 mM NaCl, 1mM DTT, 320 mM sucrose) with a PT2000 Polytron with a Proteins in fraction 27 from the final gel filtration column 20-mm TSM probe (Brinkmann Instruments, Westbury, NY) (Superdex 200) were resolved on an 11% SDS-polyacrylamide three times on setting 1 for 1 min each with a 1-min pause in gel, stained with Coomassie Blue, and the band of interest was between. The homogenate was transferred to a 1-liter centri- excised and trypsinized in the gel slice. The resulting peptides fuge bottle and spun 1,000  g for 45 min. The S1 supernatant were analyzed by nanoscale reverse phase liquid chromatogra- was transferred by pipetting into 250-ml centrifuge bottles and phy coupled with tandem mass spectrometry (LC-MS/MS) as spun 10,000  g for 30 min. The S10 was then removed and described previously (39). Briefly, the digested sample was dis- layered in 26-ml aliquots onto 13 ml of sucrose (in 25 mM solved in 0.4% acetic acid, 0.005% heptafluorobutyric acid, 5% HEPES, pH 7.4, 10 mM NaCl) gradients. These gradients con- acetonitrile, 95% water and loaded onto a 75-m  12-cm sisted of 8 ml of 20% sucrose layered on top of 5 ml of 60% self-packed fused-silica C18 capillary column. The peptides sucrose. The gradients were spun 100,000  g in an SW28 were eluted during a 30-min gradient from 10 to 30% elution 17570 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 24 •JUNE 15, 2007 ELMOD2 Is an Arl2 GAP buffer (0.4% acetic acid, 0.005% heptafluorobutyric acid, 95% or in the absence of the Arl2. Because, like Arfs, Arl2 has no acetonitrile, and 5% water) at a flow rate of 0.3 l/min. intrinsic GTPase activity (43), the Arl2-dependent GTP hydrol- Eluted peptides were ionized under high voltage (1.8 kV), ysis is also a measure of the Arl2 GAP activity. It is important to detected in a survey scan from 400 to 1600 atomic mass units (2 clearly differentiate between GTP hydrolysis occurring as a microscans) followed by three data-dependent MS/MS scans (4 result of GAP activity from hydrolysis resulting from contami- microscans each, isolation width 3 atomic mass units, 35% nor- nating nucleotidases present in every protein preparation. malized collision energy, dynamic range 1 min) in a completely Thus, each assay contained two sets of parallel, control incuba- automated fashion on an LCQ-DECA XP ion trap mass spec- tions as follows: one in which no GAP was added (to determine trometer (Thermo Finnigan, San Jose, CA). Acquired MS/MS nonspecific GTPase activity of the Arl2 preparation), and one spectra were searched against combined reference data bases for each GAP sample in which the Arl2 was absent (to deter- (mouse, rat, and human) from the National Center for Biotech- mine the nonspecific GTPase activity of each GAP fraction nology Information, leading to the identification of four pep- being assayed). Although contaminating GTPase activities tides that are each present in human and bovine ELMOD2. The were low in our Arl2 preparations, this can be a substantial peptide matches were further validated by manually examining source of hydrolysis that results in low signal to noise ratios and the spectra. was why we used the filter-trapping assay for several other GTPases (see below). Transfection, SDS-PAGE, Immunoblotting, Rapid dissociation of GTP from Arl2 in the GAP assay and Northern Blotting remains a problem in that the assay underestimates the GAP HeLa cells were maintained in Dulbecco’s modified Eagle’s activity. Although there was excess unlabeled nucleotide in the medium supplemented with 10% fetal bovine serum (Invitro- assay to minimize the hydrolysis of free [- P]GTP and the gen) and transfected with Lipofectamine Plus (Invitrogen), time of the assay was kept to a minimum, substantial release or according to the manufacturer’s instructions. SDS-PAGE and exchange of GTP still occurs. In addition, we used subsaturat- immunoblotting were performed essentially as described pre- ing concentrations of nucleotide in the loading reaction. The viously (19). A Northern blot containing human poly(A) RNA maximum concentration of the labeled substrate is predicted to from brain, colon, heart, kidney, liver, lung, muscle, placenta, be2nM, well below the apparent K value of previously iden- small intestine, spleen, stomach, and testis (HB2010, OriGene tified GAPs (45–49). This further lowers the activity measured Technologies, Rockville, MD) was probed with a random hex- and compromises the quantification that can be achieved. amer primed P-labeled fragment containing the entire However, with the exception of assaying fractions during puri- ELMOD2 coding sequence. Hybridization was performed with fication, the Arl2 GAP assay is performed under conditions in ULTRAhyb (Ambion, Austin, TX) and followed standard pro- which activity decreases linearly with dilution of at least cedures, with the highest stringency washing performed at 10-fold, and the values reported are highly consistent between 50 °C in 0.1% SDS 0.1 SSC. preparations and experiments. The close similarities between specific activities of the purified bovine testis and HeLa recom- RESULTS binant Arl2 GAP preparations (see below) is another indication Development of a Specific Arl2 GAP Assay—For most that the methods adopted are valid for comparative purposes. GTPases, the dissociation of bound GTP or GDP can be slowed Two other issues that had to be overcome before purification of substantially by raising the free Mg concentration (40–42). the Arl2 GAP from tissues could be achieved were problems Thus, after preloading the GTPase with radiolabeled GTP arising because of the quaternary structure and the low stability under optimal conditions, the addition of increased free of the activity. Mg “locks in” the bound GTP to maximize stability of the Arl2 GAP Activity in Testis Extracts Is Present in a Large substrate in the GAP assay. The GAP reaction is stopped by Complex—Early attempts to measure Arl2 GAP activity in tis- dilution into cold buffer before trapping the remaining protein- sue homogenates were unsuccessful, presumably because of its bound [- P]GTP on nitrocellulose filters. Controls run in low abundance. After Arl2 and its effector, BART, were par- parallel allow discrimination between intrinsic GTPase activity, tially localized to mitochondria (19), an enriched bovine brain GAP-stimulated GTPase activity, and simple GTP dissociation mitochondria preparation was assayed and found to contain in the assay. This protocol was used to assess the specificity of Arl2 GAP activity (36). However, the levels of activity proved to our purified Arl2 GAP against other GTPases (see below) but be insufficient to allow purification to homogeneity. Further cannot be used with Arl2 because the rate of dissociation of searches indicated that bovine testis homogenates contained guanine nucleotides from Arl2 is insensitive to [Mg ] (43). 10-fold the activity of bovine brain mitochondria but that the Instead, we developed an Arl2 GAP assay that was based activity was present in a very large particle. upon those used earlier to assay GTPase activity of the  sub- During the initial stages of purification from testis, and prior units of heterotrimeric G proteins (44) and that uses the ability to the addition of any detergent, Arl2 GAP activity was only of activated charcoal to bind nucleotides but not free phos- found in a large complex of undetermined composition. This phate. Arl2 was pre-bound to [- P]GTP and exposed to the complex was large enough to be pelleted at 100,000  g, but GAP for a very limited time (typically 4 min), and the reaction activity was inefficiently recovered from the pellet. However, was stopped by the addition of a suspension of activated char- the activity was preserved and could be easily resolved from coal. GAP-dependent GTP hydrolysis is the amount of liber- smaller complexes and soluble proteins, by either gel filtration ated P over and above that released in the absence of the GAP or centrifugation through discontinuous isopycnic sucrose gra- JUNE 15, 2007• VOLUME 282 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 17571 ELMOD2 Is an Arl2 GAP activity eluted from gel filtration media with an apparent mass of 150 kDa (Fig. 1B). This decrease in apparent size was even more pronounced in 1% CHAPS, in which the activity displays an apparent mass of55 kDa (Fig. 1C). We interpret these data as evidence that the Arl2 GAP can assemble into larger com- plexes via interactions that are likely hydrophobic in nature. We also noted that the Arl2 GAP activity was relatively unstable in cholate above its CMC (Fig. 2A), and so we used CHAPS for the remainder of the purification. CHAPS was found to inhibit the Arl2 GAP assay at concentrations above 0.1% (Fig. 2B), so during purification in CHAPS each fraction had to be diluted to a point where this inhibitory effect did not interfere with the assay. During tests of the effects of CHAPS on the Arl2 GAP assay (Fig. 2B), we noticed that the specific activity of the crude GAP preparation (20/60) was actually increased in the presence of low amounts of detergent. To separate the effects of the deter- gent on our assay from effects on the large GAP complex, we treated the 20/60 with increasing concentrations of CHAPS on ice and then diluted the samples so that the detergent was less than 0.03% in the assay (Fig. 2C). The effects were relatively small, only 2-fold, and could be explained by either an effect of the detergent to increase the stability of a labile Arl2 GAP activ- ity or to dissociate it from the effects of an inhibitory compo- nent in the complex. Tests of these two possibilities indicated that perhaps both are true. The stability of Arl2 GAP activity in 20/60 was assayed at different times at 37 °C in the presence of 0, 0.25, or 1% CHAPS (Fig. 2D). The activity became more sta- ble with increasing concentrations of the detergent. Also, treat- ment of 20/60 with 0.25% CHAPS followed by centrifugation at FIGURE 1. Arl2 GAP activity behaves as a species of decreasing size with 100,000  g resulted in release of 85% of the activity from the increasing concentrations of CHAPS. The elution profiles of Arl2 GAP activ- ity (squares) and total protein (A , diamonds) are shown after resolution by complex. Only 70% of the total protein was released, yielding Superdex 200 gel filtration chromatography. Relative migration values (R )of a 1.2-fold increase in specific activity. More importantly, the molecular mass markers were calculated by dividing the volume at which the exposure to sub-CMC levels of detergent produced activity that marker eluted by the total volume of the column and are indicated by arrows. A, activity in 20/60, in the absence of detergent, migrates in the void volume was smaller, as judged by gel filtration (see Fig. 1B), and could be with a predicted molecular mass of 600 kDa. B, peak fractions from the further resolved by additional steps of chromatography (see Macro-Prep High Q column from an Arl2 GAP purification were concentrated by ultrafiltration prior to resolution in 0.25% CHAPS. The peak of activity Table 1). migrated with an apparent molecular mass of150 kDa. C, concentration of Purification of Arl2 GAP Activity—The Arl2 GAP was ulti- CHAPS was increased to 1%, and the sample was then resolved by gel filtra- mately purified 1600-fold from bovine testis homogenates to tion chromatography. This is the final step in the purification outlined in Table 1. The apparent molecular mass of the peak fraction is55 kDa. an estimated 20% purity with an overall yield of 0.08% (Table 1), as described under “Experimental Procedures.” After produc- dients. These two steps yielded very similar results so prepara- tion of 20/60 and treatment with 0.25% CHAPS to give a 10-fold tive discontinuous sucrose gradients were used as the first step increase in specific activity over the starting material, clarifica- of the purification for ease of scale up. tion by centrifugation at 100,000  g for 1 h yielded a prepara- The Arl2 GAP activity collected at the interface of the tion that was 12-fold enriched over homogenates with close to 20–60% sucrose layers (referred to as 20/60) eluted in the void 100% recovery. Sequential chromatography using ion exchange volume of both a Sephacryl S-300 column and a Superdex 200 (Macro-Prep High Q column), hydroxyapatite, and gel filtra- column (Fig. 1A), indicating an apparent molecular mass of tion media (Superdex 200) was performed in 0.25% CHAPS and 1.3 MDa. The activity present in this large complex proved resulted in a net 500-fold purification with 4% yield. The yield refractory to further purification using a number of conven- was low as a result of both taking only peak fractions forward at tional chromatography resins, so a means of dissociating the each step and denaturation of the protein. Elution of Arl2 GAP complex with retention of activity was sought. The complex activity from gel filtration chromatography in the presence of was not disrupted by treatment with DNase, RNase, or phos- 0.25% CHAPS was consistent with an apparent molecular mass pholipases A or C (data not shown) but was dissociated by the of 150 kDa (Fig. 1B). At this point in the purification the addition of CHAPS or sodium cholate. After more detailed concentration of the CHAPS was increased to 1%, and the activ- studies of the effects of CHAPS on the apparent size, we discov- ity no longer bound to anion exchange resins, e.g. it eluted in the ered that with addition of the detergent to levels below the flow-through of the Resource Q column. Substantial loss of critical micelle concentration (CMC; 0.6%) the Arl2 GAP activity resulted from the increase in [CHAPS] but allowed for 17572 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 24 •JUNE 15, 2007 ELMOD2 Is an Arl2 GAP FIGURE 2. Effects of detergents on Arl2 GAP activity. Each data point in the figure is the average of triplicate measurements, and each panel is a single experiment that is representative of at least two repeats. A, 20/60 preparation was exposed to detergents at different concentrations and for the indicated lengths of time, all at 4 °C. The samples were diluted 20-fold at the indicated times and assayed as described under “Experimental Procedures.” Activities shown are relative to the 20/60 starting material that had not been exposed to detergent. Note the loss of activity in 1% cholate (triangles). B, effect of CHAPS on the Arl2 GAP assay was determined by inclusion of detergent at the indicated concentrations in the assay. Activities of 20/60 shown are relative to that found in the absence of detergent. C, exposure to high concentrations of CHAPS increases Arl2 GAP activity. To determine the effect of CHAPS on the specific activity of the large GAP complex, 20/60 was treated with the indicated concentration of CHAPS for 30 min on ice. The detergent/GAP mixture was then diluted 20-fold before assaying to reduce the level of CHAPS to a concentration that does not interfere with the assay. D, Arl2 GAP activity in 20/60 becomes more thermostable with increasing concentration of CHAPS. Samples of 20/60 were brought to a final CHAPS concentration of either 0 (triangles), 0.25% (squares), or 1.0% (diamonds) and immediately moved to 37 °C for the length of time indicated. The samples were then cooled briefly on ice, diluted 20-fold, and assayed. Results are reported relative to identical samples that had not been placed at 37 °C. TABLE 1 Summary of the steps used to purify Arl2 GAP activity from bovine testis As detailed under “Experimental Procedures,” 137 g of bovine testis tissue was homogenized and clarified by centrifugation at 1,000  g for 45 min. The supernatant was recovered and spun at 10,000  g for 30 min to yield the material in the 1st line of the table. All table entries represent pools of peak fractions except for the final one that is a single fraction. 1 unit is the amount of Arl2 GAP activity required to convert 1 nmol of Arl2-GTP into 1 nmol of Arl2-GDP in 1 min. Purification step Total activity Total protein Specific activity Yield Purification units mg units/mg % -fold 10,000  g supernatant 9.74 3340 0.00292 100 1 Isopycnic sucrose gradient (20/60 interface) 4.16 401 0.0104 42.7 3.56 20/60 in 0.25% CHAPS 100,000  g supernatant 9.47 274 0.0345 97.2 11.8 Macro-Prep High Q peak 4.22 53.9 0.0782 43.3 26.8 Hydroxyapatite peak 1.38 2.98 0.464 14.2 159 Superdex 200 peak 0.405 0.264 1.54 4.16 527 Resource Q flow-through 0.0852 0.0589 1.45 0.87 496 Superdex 200 fraction 27 0.00819 0.00175 4.68 0.08 1600 Protein concentration was estimated by Coomassie and silver staining. resolution of the GAP from many proteins that remained able that peak in fractions 28 and 29, respectively. This band was to bind to the ion exchange column. In addition, the increase in estimated to be one-fifth of the total protein present in the peak CHAPS concentration also resulted in an activity that eluted fraction indicating that the specific activity of fully purified from the gel filtration column with an apparent molecular mass bovine Arl2 GAP is 23 units/mg (Table 1) and would require of 55 kDa (Fig. 1C). Fractions from this final column were 8,000-fold enrichment to achieve purity. analyzed by SDS-PAGE (Fig. 3A). Four major bands (one a dou- The region surrounding the 32-kDa band of the gel shown in blet) were seen by silver staining, but only one, migrating to a Fig. 3A was excised and subjected to tryptic digestion and tan- predicted mass of32 kDa by comparison to standards, peaked dem MS/MS analysis for identification, as described under in fraction 27 and showed staining intensity that correlated “Experimental Procedures.” Seventeen peptides were identified completely with GAP activity across the fractions (Fig. 3A). in this sample, which mapped to four different proteins. Four of This is in contrast, for example, to the bands at 50 and 25 kDa the 17 peptides were 100% identical to regions of bovine JUNE 15, 2007• VOLUME 282 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 17573 ELMOD2 Is an Arl2 GAP FIGURE 3. ELMOD2 is the Arl2 GAP purified from bovine testis. A, fractions from the final gel filtration column were resolved by SDS-PAGE and stained with silver, as described under “Experimental Procedures.” Migration of molecular mass markers (size in kDa) is shown at left. The relative amount of Arl2-GAP activity is indicated below each lane. The indicated band was excised and analyzed by mass spectroscopy to identify the protein. B, amino acid sequences of human and bovine ELMOD2 are shown. Human and bovine ELMOD2 sequences are 94% identical (asterisks mark the nonidentical residues), and the four peptides identified by tandem MS analysis (bars) are completely conserved in the human sequence. ELMOD2 (Fig. 3B). The bovine and human ELMOD2 proteins tion and misfolding. Thus, we conclude that ELMOD2 pos- are each 293 residues in length with only 17 amino acid differ- sesses Arl2 GAP activity but cannot exclude (and indeed think ences (94% identity), none of which were within the identified very likely) that other proteins will be found that bind and sta- peptides. The open reading frame of the human ortholog was bilize the protein and activity in cells. amplified by PCR from a cDNA library and subcloned into a The open reading frames of the human orthologs of the three pET28 vector for expression in bacterial (BL21(DE3)) cells. A other proteins identified by MS analyses were also expressed in large amount of recombinant protein was expressed upon bacteria and HeLa cells, but none of the expressed proteins induction, but it was all insoluble and pelleted during centrifu- either had any detectable Arl2 GAP activity or increased the gation at 100,000 g after lysis of bacteria in a French press. No half-life or GAP activity of purified recombinant human Arl2 GAP activity was detected in the S100, and a variety of ELMOD2. attempts to solubilize the protein from the pellet were unsuc- Because another possibility for the low specific activity of the cessful. However, expression of the E. coli MBP as an N-termi- bacterially expressed ELMOD2 was the absence of post-trans- nal fusion with human ELMOD2 resulted in small amounts of lational modifications, we also expressed C-terminal epitope- soluble protein that could be purified by affinity chromatogra- tagged (c-Myc/His) human ELMOD2 in cultured human cells phy. Both the S100 and the purified preparation of MBP- and estimated its specific activity. HeLa cells were transiently ELMOD2 were active in the Arl2 GAP assay, thus confirming transfected with pCDNA3.1-ELMOD2, and cell lysates were ELMOD2 as an Arl2 GAP. The protein was found to precipitate collected 24 h post-transfection. Lysates were cleared by cen- progressively over a few days when stored at 4 °C or completely trifugation at 2,000 g for 3 min, and tagged ELMOD2 expres- upon either one cycle of freeze/thaw or removal of the MBP tag sion levels were determined by immunoblotting, using a puri- by thrombin cleavage. Furthermore, the specific activity of the fied Myc-tagged protein (Arf1) of known concentration as a bacterially expressed fusion protein was1000-fold lower than standard. Control HeLa lysates prepared in this way yield Arl2 that of the purified bovine protein, likely the result of aggrega- GAP activities that are close to the lower limit of detection of 17574 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 24 •JUNE 15, 2007 ELMOD2 Is an Arl2 GAP our assay. When ELMOD2-Myc/His is expressed, we found a MOtility or phagocytosis of apoptotic cells. The mammalian 10–20-fold increase in activity in the lysates. Combination of ortholog was confirmed to play a similar role in higher results from the two assays yielded an estimate that the recom- eukaryotes (53). Although ELMO1 and ELMO2 are thought to binant human ELMOD2 in HeLa lysates has a specific activity be functionally interchangeable (53–55), they do exhibit differ- of24 nmol of Arl2-GTP hydrolyzed per min/mg protein, very ential expression during development (56). ELMO1–3 share close to that determined for the purified bovine testis Arl2 GAP high sequence identities (50%) overall (Fig. 4B)andaPH (23 units/mg, see above), further confirming the identity of the domain in their C termini (Fig. 4A) that is absent in Arl2 GAP as ELMOD2. For comparison, the specific activity of ELMOD1–3. The three ELMO proteins are much less closely ArfGAP1 is 6.7 (50) or 10–50 nmol (51) of Arf1-GTP hydro- related to the three ELMO domain proteins, sharing 20% lyzed per min/mg protein. These results prove that ELMOD2 identity that is limited to the ELMO domain. ELMOD1 and encodes an Arl2 GAP but leave open the question of whether ELMOD2 share 53% identity, whereas ELMOD3 (also called any post-translational modifications or protein interactions are RBED1) shares no more than 20% identity with any of the other important modulators of activity. family members and again only in the ELMO domain (Fig. 4B). ELMOD2 Protein and Message Are Low in Abundance—Our Although the peptides identified by MS analysis were exclu- purification from bovine testis indicated that ELMOD2 makes sive to ELMOD2, we determined whether any other ELMO up0.02% of total testis protein. This is similar to our estimate family members exhibited Arl2 GAP activity by expressing of endogenous levels of the Arl2 GAP in HeLa cells (0.04%), ELMO1–3 in both E. coli and HeLa cells and assaying cell although each of these numbers is close to the lower limit of lysates. The plasmids encoding ELMO1–3 were provided by detection of the assay and each is compromised by difficulties in Kodi Ravichandran (University of Virginia) and directed assaying Arl2 GAP activity in lysates. The low level of expres- expression of glutathione S-transferase-tagged bacterial pro- sion, apparent presence in larger complexes, and lability during teins and FLAG-tagged proteins in HeLa cells. In contrast to purification combine to account for most of the difficulties in ELMOD2, ELMO1–3 all expressed well and remained soluble its isolation. in bacterial and HeLa lysates, but Arl2 GAP activity was not We also found extremely low levels of ELMOD2 mRNA mes- detected in any of these lysates. sage in our probe of a multitissue mRNA Northern blot (Ori- The ELMOD1 that we amplified and subcloned from a Gene Technologies). This filter contained mRNA from human TM cDNA library was based upon the sequence in GenBank brain, colon, heart, kidney, liver, lung, muscle, placenta, small (entry GI 34192051 dated 17 July 2006) and encoded a protein intestine, spleen, stomach, and testis and was probed with a of 283 amino acids. During the course of our studies this entry hexamer primed probe generated using the entire open reading was updated to one predicting a protein with an additional 51 frame. ELMOD2 was barely detected, and only after 72hof amino acids at the N terminus. In the intervening time, we exposure to a PhosphorImager screen, as a faint band of 1.3 expressed the shorter open reading frame in bacteria and found kb in the lane containing mRNA from stomach. The only other that truncated human MBP-ELMOD1 possesses Arl2 GAP tissue yielding detectable signal was testis, in which a faint, activity with a specific activity 23% that of MBP-ELMOD2. broad smear was noted (data not shown). In contrast, strong This fusion protein was also unstable, like MBP-ELMOD2, but signals were detected in all lanes with the control -actin it has been less thoroughly studied at this point. Thus, the pro- probe after only a brief (0.5 h) exposure to film. Previously tein with the highest sequence identity to ELMOD2 also pos- published work showed that human ELMOD2 RNA was in sesses Arl2 GAP activity. all tissues examined by reverse transcription-PCR, although A plasmid purported to encode full-length human ELMOD3 no details about levels were reported (52). Thus, the was purchased from OriGene but was later found to encode a ELMOD2 message is likely expressed to only low levels but is TM variant (GenBank entry GI 85662659). This variant (referred widespread, perhaps ubiquitous, in its expression in differ- TM to here as ELMOD3_C) is identical to the current GenBank ent human tissues. Arl2 is both abundantly and ubiquitously entry for ELMOD3 (GI 34147693) through amino acid 314 expressed (19). The low expression of ELMOD2 likely but differs at the C terminus and presumably is an alterna- explains the limited number of ESTs in public data bases and tively spliced version. The C terminus of this variant exhibits limits its likelihood of discovery in functional screening of similarity to the ELMOD3 proteins from chimpanzee, cDNA libraries. macaque, cow, dog, and mouse, whereas the C terminus of the ELMOD1 Also Has Arl2 GAP Activity—Bovine and human TM official ELMOD3 GenBank entry only exhibits sequence ELMOD2 are each 293 residues in length and are 94% identical similarity to a chimpanzee ortholog. Therefore, we expressed and 98% homologous. Orthologs of human ELMOD2 were TM ELMOD3_C in HeLa cells as a Myc/His-tagged protein and also found in primates (GenBank accession number TM failed to detect any Arl2 GAP activity in cell lysates. These tests XP_001090344, 98% identity), rodents (GenBank acces- TM for Arl2 GAP activity among the human ELMO domain-con- sion number NP_848851, 87% identity), chicken (GenBank taining proteins reveal that only ELMOD1 and ELMOD2, accession number XP_420415, 70% identity), and Zebrafish TM which share 53% identity (Fig. 4C), are active. It is possible that (GenBank accession number XP_691971, 61% identity). ELMOD2 is so named because it is one of six human proteins other family members have activity that was not detected as a that contain the ELMO domain, a “domain of unknown func- result of the need for orienting the protein on a membrane tion” (DUF609). ELMO1 was first identified as CED-12 in Cae- surface, because of the presence of the PH domain (57), or pos- norhabditis elegans, a protein required for EnguLfment and cell sibly interference of the tags used. It is also possible that the JUNE 15, 2007• VOLUME 282 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 17575 ELMOD2 Is an Arl2 GAP FIGURE 4. ELMOD2 is one of six human family members containing the ELMO domain. A, all six human ELMO domain-containing proteins are depicted with ELMO domains (black box) aligned. ELMO1–3 also contain a PH domain (hatched box) located C-terminal to the ELMO domain. B, amino acid sequence identities between human ELMO domain family members are shown. The percent identity of pairwise combinations is shown above the diagonal line. The number of identical residues and the total number of residues compared between each pair is indicated below the diagonal line. ELMOD2 has 53% identity to ELMOD1 over 294 amino acids but 20% or lower identity with the remaining family members. C, amino acid sequences of the Arl2 GAP activity containing ELMOD1 and ELMOD2 proteins are shown with conserved residues identified. The ELMO domain region is boxed. other family members are active as GAPs for GTPases other otidase activity, we used the filter-binding assay described than Arl2. above and under “Experimental Procedures.” Because each ELMOD2 Also Possesses GAP Activity Against Arfs but Not GTPase has a different rate of release of bound nucleotide, in Ran, Rac1, or RhoA—To determine the specificity of the each case the relevant parameter is not the absolute rate of GAP activity of ELMOD2 toward GTPases other than Arl2, loss of radioactivity but the difference in the rate of loss we examined the activity of recombinant human MBP- between the sample with and without (buffer alone) the ELMOD2 against Arl3, Arf1, Arf6, RhoA, Rac1, and Ran. GAP. ArfGAP1 and 20/60 were included as positive controls, Because of the nucleotide handling properties of these as the latter is a crude sample predicted to contain a number GTPases and the varying presence of contaminating nucle- of GAP activities. Note that the rate of dissociation of GTP 17576 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 24 •JUNE 15, 2007 ELMOD2 Is an Arl2 GAP active against Arl3 (Fig. 5A). As expected, both MBP-ELMOD2 and 20/60 were active against Arl3, but ArfGAP1 was not. None of the GTPase activities of proteins out- side the Arf family (RhoA (Fig. 5D), Rac1 (Fig. 5E), and Ran (Fig. 5F)) were increased upon exposure to MBP-ELMOD2 or ArfGAP1, although each was increased by 20/60. Thus, MBP-ELMOD2 is active as a GAP for Arl2 and Arl3 but not for these GTPases outside the Arf family. Surprisingly, MBP-ELMOD2 ex- hibited substantial GAP activity for Arf1 (Fig. 5B) and Arf6 (Fig. 5C), although less for the latter. This is despite the fact that ELMOD2 lacks the consensus Arf GAP domain (32, 59) and clearly provides opportuni- ties for cross-signaling between Arl and Arf proteins, which are thought to have quite distinct functions. To further test the ability of MBP- ELMOD2 to act as a GAP for Arfs, we assayed Arf1 and Arf6 in the charcoal assay. Despite the varying FIGURE 5. ELMOD2 has GAP activity against Arl3, Arf1, and Arf6. The filter-trapping GAP assay was per- signal to noise ratios in these formed using different GTPases, as described under “Experimental Procedures.” Each data point in A–F is the assays, the measured specific average of triplicate measurements, and each panel is a single experiment that is representative of at least two ELMOD2-dependent GAP activ- repeats. Note that the GAP activity is the difference between the loss of bound radionucleotide in the presence of the GAP sample from that found in its absence (diamonds). ArfGAP1 (triangles) is included as a positive ity for the Arfs was very similar in control for the Arfs, and 20/60 (circles) is a crude preparation predicted to contain several GAPs and is also each assay (Fig. 6). included as a positive control. Relative GAP activities of ELMOD2 against all GTPases tested are shown in Fig. 6. These relative activities were obtained using the same concentration of each GTPase and [- P]GTP, the same time points, and the same buffer conditions and were repeated at least three times with similar results. However, because the conditions of the assay were developed and therefore optimized for Arl2, differ- ences in the binding stoichiometries of each GTPase yield dif- ferent substrate (GTPase-GTP) concentrations at t 0. Specif- ically, because of differences in the nucleotide binding properties of the different GTPases and in the charcoal and filter trapping assays, the concentrations of substrate (GTPase- GTP) are predicted to be about 10-fold higher for Arl2 than for FIGURE 6. Relative activities of ELMOD2 toward Ras family GTPases. Spe- cific activities of purified recombinant MBP-ELMOD2 against seven different the other GTPases, making it likely that we are underestimating GTPases were determined, as described under “Experimental Procedures,” the activity of ELMOD2 toward the other GTPases. More rig- and normalized to that against Arl2 (set to 100%). The light bars are numbers generated from the experiments in A–F of Fig. 5. and the dark bars are gener- orous kinetics, measured under Michaelis-Menten conditions ated in the charcoal assay as described under “Experimental Procedures.” and with more stable protein preparations, will be required Note the similarities in the results of the different assays for Arf1 and Arf6. before detailed comparisons of rate constants and relative affin- Because of differences in the nucleotide binding properties of Arl2, it is pre- dicted that the concentration of GTP-bound Arl2 in these assays was 10- ities can be determined. fold higher than the GTP-bound form of the other GTPases, making it likely that activity toward these other GTPases is underestimated. DISCUSSION from Arl2 under these conditions is too rapid to allow it to be We report the purification, identification, expression, and assayed in this way. initial characterization of ELMOD2 as the first mammalian Arl Arl2 is more closely related to Arl3 than to any other GTPase GAP. ELMOD2 was found to exist in cells as part of a large (20, 58), so it was not surprising to see that ELMOD2 was also protein complex, the components of which are likely to con- JUNE 15, 2007• VOLUME 282 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 17577 ELMOD2 Is an Arl2 GAP tribute to the stability, activities, and biological function of reactants, particularly for the ELMO proteins that contain PH ELMOD2. Surprisingly, ELMOD2 was also found to be active as domains (57). The more challenging, but also more important, an Arf GAP. The presence of an ELMO domain in this and five question is the extent to which ELMO domain proteins serve other human proteins, one of which we showed also possesses as regulators or effectors of Arl2 or other Arf family GTPases Arl2 GAP activity, leads us to speculate that the ELMO domain in vivo. is a GAP domain for Arl2 and other members of the Arf family, An intriguing hint that ELMOs may act to link Arf and other with specificities yet to be determined. The biological functions GTPase signaling pathways is found in the role of Arf6 to stim- of the ELMO proteins, their locations, and their ability to act as ulate actin reorganization in lamellipodia formation. The Arf terminators or effectors of Arf family GTPases are currently guanine nucleotide exchange factor ARNO activates cell migra- unknown but will likely provide new insights into signaling by tion through activated Arf6 in a Rac1-dependent manner (61). these GTPases and the potential for cross-talk between Arfs The Rac1 dependence was recently shown to require the co- and Arls. localization of DOCK180/ELMO1 Rac GEF activity (62). It is Human ELMOD2 was shown to be an Arl2 GAP by purifica- not yet known how Arf6 is inactivated along the sides and trail- tion of the activity from bovine testis and the demonstration ing edge of the forming protrusions, although recruitment of that the purified recombinant protein is active. However, Arl2 Arf GAPs has been proposed as a possibility (62). But another GAP activity is most stable when part of a large cellular complex intriguing possibility is that the Arf GAP activity is already there and exhibits a shortened half-life when monomeric. The find- in the form of an ELMO domain protein. Although ELMO1 did ings that sub-CMC concentrations of CHAPS allowed partial not have Arf GAP activity in our in vitro assays, it is certainly dissociation of the large complex and increased the thermal possible that the activity may be found under different condi- stability of Arl2 GAP activity suggest that hydrophobic residues tions. Thus, the position of ELMO1 between Arf6 and Rac1 are involved in ELMOD2 interactions. Because lability of the signaling may be a prototype for other ELMO domain proteins. recombinant and purified testis proteins contributed to diffi- Because ELMOD2 was found to be active against Arl3, it is culties in working with each preparation and limited the accu- also tempting to speculate that it functions in the regulation racy of quantification that can be achieved, development of a of cytokinesis. We recently showed (20) that knockdown of more stable preparation is a high priority. This will likely Arl3 by short interfering RNA leads to inhibition of cytoki- require the identification of ELMOD2-binding partners. In nesis in HeLa cells and ELMOD2 was one of only a few genes addition to providing access to a more stable preparation of discovered in an RNA interference screen in flies for genes Arl2 GAP activity, interaction of ELMOD2 with these binding required for cytokinesis (63). partners in vitro may result in changes in catalytic rates or spec- Although opportunities for cross-talk between GTPase sig- ificity among GTPases. Knowledge of the composition of naling pathways are evident, it is still most likely that the bio- ELMOD2 complexes should also provide additional insights logical role of ELMOD2 will result from its actions as GAP and into its cellular locations and biological functions. potential effector of Arl2 signaling. Arl2 was first found to be The finding that ELMOD2 had GAP activity toward Arf1 was involved in tubulin and microtubule dynamics in a yeast genetic surprising because it lacks the canonical, cysteine-rich, zinc fin- screen (17, 64) and later biochemically through its binding to ger Arf GAP signature, CX CX CX C (where X is any tubulin folding chaperone cofactor D (16). Although mono- 2 16–18 2 amino acid) (32), that is present in every previously reported meric Arl2 binds GTP readily in the absence of a GEF and protein with Arf GAP activity (e.g. see Refs. 12, 31, and 33) and would therefore be expected to exist in the activated form in found in 20 proteins in the human proteome. ELMOD2 has cells, the bulk of cellular Arl2 is bound to cofactor D and in this five cysteines, but their arrangement does not resemble the Arf form cannot bind GTP (65). We recently showed that an excess GAP signature and only one of them is completely conserved of activated Arl2 causes the loss of microtubules and cell cycle among metazoan orthologs. Thus, we predict a novel structure arrest (20), and so cofactor D may act as a sink for Arl2, keeping and mechanism of promoting GTP hydrolysis by Arf family it locked in the inactive form. It is likely that ELMOD2, as an members will be found for ELMOD2. Arl2 GAP, provides an additional level of control in the critical We tested all six human ELMO domain proteins and function of regulating the levels of activated Arl2. Although found that ELMOD1 also had Arl2 GAP activity, whereas the most cellular Arl2 is in the cytosol, a smaller pool is found in other proteins did not. We cannot exclude the possibility mitochondria where it can bind BART and the adenine nucle- that other ELMO domain proteins possess GAP activity otide transporter 1 (19). The function of Arl2 in mitochondria is against other members of the Arf family. Indeed, we speculate not clear, but it may be involved in regulating energy metabo- that the ELMO domain is an Arf family GAP domain that may lism (19). Thus Arl2 and ELMOD2 may act together to sense provide cross-talk between Arf and Arl proteins in cells. A more and transmit information about the general health of the cell systematic screening of GAP activities of ELMO domain pro- between mitochondria and the cell division apparatus. Further teins for Arf family members is planned but must await more studies of the localization of ELMOD2 and the identification of stable preparations of ELMOD1 and ELMOD2, as well as iden- its binding partners and regulators will contribute significantly tification of the complexes that exist in cells. It will also be to our understanding of these pathways. important to evaluate potential roles of other molecules Finally, the fact that Arl2 is an ancient protein, with orthologs (including lipids and phosphoinositides) to serve as cofactors in in the earliest eukaryotes, whereas orthologs of ELMOD2 and the GAP assay, as has been shown for some Arf GAPs (60), and ELMOD1 are not evident in early eukaryotes, suggests that the need for a membrane surface to assist in the orientation of other Arl2 GAPs may exist in early eukaryotes that perhaps are 17578 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 24 •JUNE 15, 2007 ELMOD2 Is an Arl2 GAP J. Cell Sci. 117, 4705–4715 also expressed in mammals. Detailed analyses of the key func- 23. Avidor-Reiss, T., Maer, A. M., Koundakjian, E., Polyanovsky, A., Keil, T., tional residues and structures of the ELMO domain proteins Subramaniam, S., and Zuker, C. S. (2004) Cell 117, 527–539 may allow the detection of more divergent proteins, from 24. Chiang, A. P., Nishimura, D., Searby, C., Elbedour, K., Carmi, R., Ferguson, potentially any eukaryote, that share Arl GAP activities. A. L., Secrist, J., Braun, T., Casavant, T., Stone, E. M., and Sheffield, V. C. (2004) Am. J. Hum. Genet. 75, 475–484 Acknowledgments—We gratefully acknowledge the assistance of cur- 25. Fan, Y., Esmail, M. A., Ansley, S. J., Blacque, O. E., Boroevich, K., Ross, A. J., Moore, S. J., Badano, J. L., May-Simera, H., Compton, D. S., Green, J. S., rent and former members of the laboratory in discussions of the work Lewis, R. A., van Haelst, M. M., Parfrey, P. S., Baillie, D. L., Beales, P. L., presented. We thank John Hepler for critical reading of the manu- Katsanis, N., Davidson, W. S., and Leroux, M. R. (2004) Nat. Genet. 36, script and members of the Daniel Reines laboratory for providing 989–993 expert technical assistance in performing Northern blots. The gener- 26. Li, J. B., Gerdes, J. M., Haycraft, C. J., Fan, Y., Teslovich, T. M., May- ous gifts of purified recombinant GTPases from Gary Bokoch (The Simera, H., Li, H., Blacque, O. E., Li, L., Leitch, C. C., Lewis, R. A., Green, Scripps Research Institute), Ian Macara (University of Virginia), and J. S., Parfrey, P. S., Leroux, M. R., Davidson, W. S., Beales, P. L., Guay- Sharon Campbell (University of North Carolina, Chapel Hill) greatly Woodford, L. M., Yoder, B. K., Stormo, G. D., Katsanis, N., and Dutcher, facilitated our studies of specificity. Kodi Ravichandran and James S. K. (2004) Cell 117, 541–552 Casanova (University of Virginia) provided helpful and interesting 27. Pazour, G. J., Agrin, N., Leszyk, J., and Witman, G. B. (2005) J. Cell Biol. discussions of ELMO proteins as well as constructs for their expression 170, 103–113 28. Schrick, J. J., Vogel, P., Abuin, A., Hampton, B., and Rice, D. S. (2006) Am. J. in bacterial and mammalian cells. 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ELMOD2 Is an Arl2 GTPase-activating Protein That Also Acts on Arfs *

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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 24, pp. 17568 –17580, June 15, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. ELMOD2 Is an Arl2 GTPase-activating Protein That Also Acts on Arfs Received for publication, February 15, 2007, and in revised form, April 13, 2007 Published, JBC Papers in Press, April 23, 2007, DOI 10.1074/jbc.M701347200 ‡ § § ‡1 J. Bradford Bowzard , Dongmei Cheng , Junmin Peng , and Richard A. Kahn ‡ § From the Department of Biochemistry and Department of Human Genetics, Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, Georgia 30322 Regulatory GTPases in the Ras superfamily employ a cycle of were initially thought to simply inactivate their cognate alternating GTP binding and hydrolysis, controlled by guanine GTPase, but more recent research suggests that GAPs can also nucleotide exchange factors and GTPase-activating proteins serve as effectors of the signaling pathways regulated by the (GAPs), as essential features of their actions in cells. Studies of GTPase (e.g. ArfGAP1 and ARAP2 (2, 3)). these GAPs and guanine nucleotide exchange factors have pro- The first GAPs were purified based on their biochemical vided important insights into our understanding of GTPase sig- activities, but sequence and mutational analyses led to the iden- naling and biology. Within the Ras superfamily, the Arf family is tification of GAP domains, which have proven highly predictive composed of 30 members in mammals, including 22 Arf-like for identifying novel GAPs within a GTPase family. More than (Arl) proteins. Much less is known about the mechanisms of cell 170 putative GAPs have been identified and are grouped regulation by Arls than by Arfs. We report the purification from according to which GTPase they act upon and which GAP bovine testis of an Arl2 GAP and its identity as ELMOD2, a domain they possess (4, 5). Typically a protein with a given GAP protein with no previously described function. ELMOD2 is one domain will be active against members of one family of of six human proteins that contain an ELMO domain, and a GTPases, or a subset within that family, but not against other second member, ELMOD1, was also found to have Arl2 GAP families. For example, a protein with an Arf GAP domain will be activity. Surprisingly, ELMOD2 also exhibited GAP activity active against one or more (but not necessarily all) Arfs but not against Arf proteins even though it does not contain the canon- against Rabs, Rhos, or Ras. Exceptions to these general rules ical Arf GAP sequence signature. The broader specificity of exist (6–8), and more complete information about the deter- ELMOD2, as well as the previously described role for ELMO1 minants of GAP specificity will be required to understand the and ELMO2 in linking Arf6 and Rac1 signaling, suggests that roles that GAPs play in the biology of the cell. Determining the ELMO family members may play a more general role in integrat- specificity of a GAP for different GTPases is further compli- ing signaling pathways controlled by Arls and other GTPases. cated by factors that can influence the results of in vitro GAP assays, including the assay used to measure GTP hydrolysis, the need for hydrophobic surfaces on which protein interactions The Ras superfamily of regulatory GTPases is composed of occur, the degree of membrane curvature (e.g. in the case of over 150 members divided into at least five families (Ras, ArfGAP1 (9–11)), or the presence of co-activators (e.g. phos- phatidylinositols (12, 13)). Rho, Rab, Ran, and Arf (1)). Although the biological function The importance of specificity is illustrated by the Arf family, of each family is distinct, all members exhibit many struc- tural and biochemical similarities. They share conserved which in mammals consists of 6 Arfs, 22 Arf-like (Arl), and 2 Sar sequences involved in guanine nucleotide binding, have low proteins (14). The Arf family is best known for the role of Arfs and Arl1 as regulators of adaptor recruitment and phospholipid intrinsic levels of GTP hydrolysis, and cycle between GTP- metabolism, predominantly at the Golgi, although members of and GDP-bound states as critical components of their bio- logical functions. It is the ability of these two states to inter- the Arf family are probably active at every membrane in act with different sets of proteins that enable these small eukaryotes (15). The remaining Arls exhibit a far greater diver- sity of functions than do the Arfs as they have been implicated GTPases to act as “molecular switches.” as regulators of microtubule-dependent processes (Arl2 and Because of the low intrinsic rates of GTP hydrolysis, the tran- sition from the GTP-bound to GDP-bound form of the GTPase Arl3 (16–20)), lysosome mobility, and microtubule binding requires an accessory GTPase-activating protein (GAP). GAPs (Arl8 (21, 22)), ciliogenesis (Arl3 and Arl6 (23–28)), and tumor- igenesis (Arl11 (29, 30)). With increasing interest in these pro- teins, it becomes more important to identify regulators and * This work was supported in part by Grants GM24680 (to R. A. K.), GM067465 effectors that can provide insights into the biological function (to J. B. B.), and AG025688 (to J. P.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the pay- of each of the Arls and to determine the extent of overlap in ment of page charges. This article must therefore be hereby marked their actions and protein partners. “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi- cate this fact. To whom correspondence should be addressed: Dept. of Biochemistry, Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322- monio]-1-propanesulfonic acid; Arl, Arf-like; MBP, maltose-binding pro- 3050. Tel.: 404-727-3561; E-mail: [email protected]. tein; LC-MS/MS, liquid chromatography-tandem mass spectrometry; The abbreviations used are: GAP, GTPase-activating protein; GEF, guanine DTT, dithiothreitol; CMC, critical micelle concentration; PH, pleckstrin nucleotide exchange factor; CHAPS, 3-[(3-cholamidopropyl)dimethylam- homology. 17568 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 24 •JUNE 15, 2007 This is an Open Access article under the CC BY license. ELMOD2 Is an Arl2 GAP The prediction of20 Arf GAPs in the human proteome (4) umn (GE Healthcare) according to the manufacturer’s instruc- is based upon the presence of the Arf GAP domain, a cysteine- tions. Purified recombinant preparations of RhoA and Rac1 (G. rich zinc finger with specific spacing (CX CX CX C, where Bokoch, Scripps Research Institute) and Ran (I. Macara, Uni- 2 16–18 2 X is any amino acid (12, 31–33)). The specificities of these pro- versity of Virginia) were generously provided by colleagues. teins have not yet been completely determined, but to date none have demonstrated activity against Arls. One of the four GAP Assays Arf GAPs in Saccharomyces cerevisiae (34) and a partially puri- Charcoal GAP Assay—Our standard GAP assay was per- fied preparation of rat spleen cytosol (35) have shown activity formed by pre-loading recombinant human Arl2 with against both Arfs and Arl1, but this is not surprising given their [- P]GTP in a reaction that contained 2 M Arl2 in 25 mM close sequence and functional relatedness. HEPES, pH 7.4, 100 mM NaCl, 2.5 mM MgCl ,1mM dithiothre- GAPs are often expressed only to low levels, and their assay in itol (DTT), and 0.3 Ci/l[- P]GTP (6000 Ci/mmol; cell or tissue lysates can be compromised by a number of fac- PerkinElmer Life Sciences) for 15 min at 30 °C. Under these tors. We took advantage of the observation that Arl2 is partially conditions, typically50% of the radionucleotide will be bound localized to mitochondria to develop an Arl2 GAP assay using to Arl2 by the end of the preincubation. The amount of bound an enriched mitochondria preparation as the source of the [- P]GTP was determined by filtration of an aliquot of the activity (36). Later modifications to the assay allowed us to pre-loaded Arl2 onto a nitrocellulose filter and scintillation purify the activity from bovine testis. The purified Arl2 GAP counting, as described below. During this incubation, 25 lof contained a domain found in other human proteins, the ELMO the GAP sample to be assayed was mixed with 20 l of reaction domain, that we propose functions as an Arl GAP domain. The buffer (62.5 mM HEPES, 6.25 mM MgCl , 2.5 mM DTT, 2.5 mM purification of this Arl2 GAP and issues surrounding the assay GTP, and 12.5 mM ATP) on ice. The reaction was started by of GAP proteins in general are discussed. The observation that adding 5 l of the pre-loaded Arl2 to the 45-l GAP/buffer this GAP also acted on Arfs provides an opportunity for cross- combination, mixing, and placing at 30 °C. The reaction was talk between Arfs and Arls. stopped 4 min later by the addition of 750 l of a suspension of ice-cold activated charcoal (5% in 50 mM NaH PO ). The char- 2 4 EXPERIMENTAL PROCEDURES coal was pelleted, and the amount of released P in 400 lof Antibodies, Cells, and Reagents the supernatant was determined by scintillation counting. A All chemicals used were purchased from commercial mock load reaction, which was identical to the pre-loading step sources. Sol-Grade CHAPS was purchased from Anatrace, Inc. but did not contain Arl2, was also performed and run in parallel (Maumee, OH). Mouse anti-Myc (9E10; Covance Research for each sample to be assayed. This control measures the Products, Berkeley, CA) was used at a 1:1000 dilution in immu- amount of GTP hydrolysis that occurs during the assay that is noblots. Bovine testicles (trimmed) were purchased from Pel- independent of Arl2-GTP. The control results were then sub- Freez Biologicals (Rogers, AR), and the tunica albuginea was tracted from those obtained with Arl2-GTP in the reaction to removed before use. obtain the Arl2-dependent GTP hydrolysis. Because Arl2 can- not hydrolyze GTP to a measurable extent by itself, this value is Expression Plasmids equal to the Arl2 GAP activity. The coding sequences of human ELMOD1 and ELMOD2 Filter Trapping GAP Assay—Each GTPase was pre-loaded were amplified from a human fetal brain cDNA library with [- P]GTP in a reaction that is 1 M GTPase in 20 mM (Clontech) using custom synthetic oligonucleotide primers HEPES, pH 7.4, 100 mM NaCl, 50 g/ml bovine serum albumin, and cloned into a pET28-based vector also containing the 1mM DTT, 0.5 mM MgCl ,1mM EDTA, 5 mM ATP, and 0.3 Escherichia coli maltose-binding protein (MBP) open read- Ci/l[- P]GTP (PerkinElmer Life Sciences, 6000 Ci/mmol) ing frame (provided by Xing Zhang and Xiaodong Cheng, for 15 min at 30 °C. The pre-load reaction was then brought to Emory University) and pcDNA3.1/myc-His (Invitrogen). a final concentration of 20 mM MgCl and placed on ice. The The coding sequence of ELMOD3 was amplified from a plas- GAP sample (15 l) to be assayed was mixed with reaction mid purchased from OriGene Technologies (Rockville, MD) buffer (25 l; 62.5 mM HEPES, 6.25 mM MgCl , 2.5 mM DTT, 2.5 and subcloned as above. mM GTP, and 12.5 mM ATP) on ice. A control reaction (GAP) was run in parallel to determine the amount of [- P]GTP or Preparation of Recombinant Proteins P released from the GTPase independent of the GAP. The reactions were started by adding 10 l of the pre-loaded Purified recombinant human Arl2, Arl3, Arf1, and Arf6 were GTPase to the 40-l GAP/buffer combination, mixing, and each prepared as described previously (36–38). pET-based placing at 30 °C. At each time point, 10 l was removed from plasmids encoding MBP-ELMOD1 or MBP-ELMOD2 were the reaction to 2 ml of ice-cold HNMD (20 mM HEPES, pH 7.4, transformed into E. coli (BL21(DE3)) and single colonies picked 100 mM NaCl, 10 mM MgCl ,1mM DTT) and immediately into LB media. Cultures were grown at 37 °C until the A filtered over a 0.45-m Protran BA85 filter (Whatman), which 0.2 at which point they were induced with isopropyl 1-thio-- D-galactopyranoside to a final concentration of 0.1 mM. After an was then washed four times with 2-ml aliquots of HNMD. The additional 15 h of growth at room temperature, the cells were amount of GTPase-[- P]GTP still bound to the filters was harvested and lysed by multiple passes through a French press. determined by scintillation counting. The amount of GAP Purification was carried out with a HiTrap chelating HP col- activity is the difference between the counts lost from the JUNE 15, 2007• VOLUME 282 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 17569 ELMOD2 Is an Arl2 GAP GTPase in the presence of the GAP sample minus the control (Beckman, Fullerton, CA) rotor for 120 min. The interface (-GAP) sample. between the 20 and 60% sucrose layers (“20/60”) was recovered by pipetting and gently vortexed to mix. Calculation of Specific Arl2 GAP Activities CHAPS (1.25 ml of 10% in 50 mM HEPES, pH 7.4) and Buffer Arl2 GAP activities were determined using the charcoal GAP A (148.75 ml; 25 mM HEPES, pH 7.4, 10 mM NaCl, 1 mM DTT, assay, performed in parallel with controls, as described above. 2mM EDTA, 0.25% CHAPS) were added to 50 ml of 20/60 to To best quantify this assay we made an assumption, based upon bring the final protein concentration to2 mg/ml and the final prior ligand-binding data, that when Arl2 is exposed to high detergent concentration to 0.25%. After a 30-min incubation on concentrations of GTP it binds very rapidly to a stoichiometry ice, the diluted 20/60 was spun at 100,000  g for 60 min. The of 20% (determined for each preparation of Arl2 but quite supernatant was recovered by pipette and loaded onto a 140-ml consistent). Thus, during the preincubation phase the Arl2 (2.6  30 cm) Macro-Prep High Q column (Bio-Rad). The col- bound about 50% of the [- P]GTP available to it. When the umn was washed with 140 ml of Buffer A, and the bound pro- vast excess of unlabeled GTP is added at the start of the GAP teins were eluted with a gradient of 0–40% Buffer B (25 mM assay, the free GTP-binding sites on the remaining Arl2 mole- HEPES, pH 7.4, 1 M NaCl, 1 mM DTT, 2 mM EDTA, 0.25% cules quickly became occupied with cold GTP. Thus, we used in CHAPS) over 400 ml. Arl2 GAP activity was assayed using the our calculations the 20% value to estimate the concentration of charcoal assay and found to elute between 75 and 250 mM NaCl. Arl2-GTP at the start of the assay, typically 1.8 pmol per assay The peak fractions were pooled, concentrated using an Amicon point (2.5 nM). The GAP-dependent P counts, released dur- Ultra (Millipore, Billerica, MA) 15-ml 30,000-Da nominal ing the 4-min incubation, were divided by the total counts avail- molecular weight limit centrifugal concentrator, and applied to able in each assay, determined by filter trapping at the end of the a 120-ml (1.6  60 cm) HiLoad Superdex 200 prep grade (GE preincubation phase. This ratio represents the percentage of Healthcare) gel filtration column, which was developed with total Arl2-GTP present in the GAP reaction that was converted Buffer C (25 mM HEPES, pH 7.4, 50 mM NaCl, 1 mM DTT, 0.25% into Arl2-GDP during the assay. This was then multiplied by CHAPS). The peak fractions from the gel filtration column, the [Arl2-GTP] to get the amount converted in the 4-min assay. corresponding to a predicted molecular mass range of 150–300 Specific activity was reported in units/mg protein in the assay kDa, were loaded onto a 15-ml (1.6  20 cm) Macro-Prep where 1 unit is the amount of activity required to convert 1 ceramic hydroxyapatite type I column (20 m; Bio-Rad). The nmol of Arl2-GTP to Arl2-GDP/min. column was washed with 10 ml of Buffer D (25 mM HEPES, pH To calculate the specific activity of a GAP using the filter 7.4, 10 mM NaCl, 1 mM DTT, 5 mM K HPO ,5mM KH PO , 2 4 2 4 trapping assay, we again determined the GAP-dependent loss 0.25% CHAPS) and then 10 ml of 10% Buffer E (25 mM HEPES, of P trapped on the filter during the assay, as described above. pH 7.4, 10 mM NaCl, 1 mM DTT, 200 mM K HPO , 200 mM i 2 4 This number was divided by the total bound radionucleotide KH PO , 0.25% CHAPS) before being developed with a 60 ml of 2 4 available at t 0 to get the percentage hydrolyzed. Because the 10–70% gradient of Buffer E. Fractions containing activity were GTPases other than Arl2 do not bind significant amounts of pooled, concentrated, and further resolved on the 120-ml unlabeled GTP during the GAP assay, because of the presence Superdex 200 column as above. of high [Mg ], the amount of GTPase-GTP present in the Peak fractions from the gel filtration column were pooled reaction was determined directly from the specific activity of and brought to a final concentration of1% CHAPS by adding the radioligand. Note that because of the differences in the ways an amount of 10% CHAPS equal to one-ninth of the initial these two assays are performed, we estimate that the concen- volume of the pooled fractions. After 30 min on ice, this sample tration of Arl2-GTP was about 10-fold higher than the GTP- was applied to a 6-ml Resource Q column (GE Healthcare). The bound form of the other GTPases. flow-through was collected and concentrated using an Amicon Ultra 4-ml 10,000-Da nominal molecular weight limit centrif- Purification of ELMOD2 from Bovine Testis ugal concentrator to a final volume of 0.5 ml. This was then Numerous preparations of Arl2 GAP activity were per- applied to a 24-ml (1.0  30 cm) Superdex 200 gel filtration formed, with a number of variations, and a typical example (the column (GE Healthcare). Fractions (0.35 ml) from this final one that was used for mass spectroscopy analysis) is described column were collected and assayed. here. 137 g (wet weight) of bovine testis was homogenized on Mass Spectroscopy ice in 500 ml of SH Buffer (25 mM HEPES, pH 7.4, 10 mM NaCl, 1mM DTT, 320 mM sucrose) with a PT2000 Polytron with a Proteins in fraction 27 from the final gel filtration column 20-mm TSM probe (Brinkmann Instruments, Westbury, NY) (Superdex 200) were resolved on an 11% SDS-polyacrylamide three times on setting 1 for 1 min each with a 1-min pause in gel, stained with Coomassie Blue, and the band of interest was between. The homogenate was transferred to a 1-liter centri- excised and trypsinized in the gel slice. The resulting peptides fuge bottle and spun 1,000  g for 45 min. The S1 supernatant were analyzed by nanoscale reverse phase liquid chromatogra- was transferred by pipetting into 250-ml centrifuge bottles and phy coupled with tandem mass spectrometry (LC-MS/MS) as spun 10,000  g for 30 min. The S10 was then removed and described previously (39). Briefly, the digested sample was dis- layered in 26-ml aliquots onto 13 ml of sucrose (in 25 mM solved in 0.4% acetic acid, 0.005% heptafluorobutyric acid, 5% HEPES, pH 7.4, 10 mM NaCl) gradients. These gradients con- acetonitrile, 95% water and loaded onto a 75-m  12-cm sisted of 8 ml of 20% sucrose layered on top of 5 ml of 60% self-packed fused-silica C18 capillary column. The peptides sucrose. The gradients were spun 100,000  g in an SW28 were eluted during a 30-min gradient from 10 to 30% elution 17570 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 24 •JUNE 15, 2007 ELMOD2 Is an Arl2 GAP buffer (0.4% acetic acid, 0.005% heptafluorobutyric acid, 95% or in the absence of the Arl2. Because, like Arfs, Arl2 has no acetonitrile, and 5% water) at a flow rate of 0.3 l/min. intrinsic GTPase activity (43), the Arl2-dependent GTP hydrol- Eluted peptides were ionized under high voltage (1.8 kV), ysis is also a measure of the Arl2 GAP activity. It is important to detected in a survey scan from 400 to 1600 atomic mass units (2 clearly differentiate between GTP hydrolysis occurring as a microscans) followed by three data-dependent MS/MS scans (4 result of GAP activity from hydrolysis resulting from contami- microscans each, isolation width 3 atomic mass units, 35% nor- nating nucleotidases present in every protein preparation. malized collision energy, dynamic range 1 min) in a completely Thus, each assay contained two sets of parallel, control incuba- automated fashion on an LCQ-DECA XP ion trap mass spec- tions as follows: one in which no GAP was added (to determine trometer (Thermo Finnigan, San Jose, CA). Acquired MS/MS nonspecific GTPase activity of the Arl2 preparation), and one spectra were searched against combined reference data bases for each GAP sample in which the Arl2 was absent (to deter- (mouse, rat, and human) from the National Center for Biotech- mine the nonspecific GTPase activity of each GAP fraction nology Information, leading to the identification of four pep- being assayed). Although contaminating GTPase activities tides that are each present in human and bovine ELMOD2. The were low in our Arl2 preparations, this can be a substantial peptide matches were further validated by manually examining source of hydrolysis that results in low signal to noise ratios and the spectra. was why we used the filter-trapping assay for several other GTPases (see below). Transfection, SDS-PAGE, Immunoblotting, Rapid dissociation of GTP from Arl2 in the GAP assay and Northern Blotting remains a problem in that the assay underestimates the GAP HeLa cells were maintained in Dulbecco’s modified Eagle’s activity. Although there was excess unlabeled nucleotide in the medium supplemented with 10% fetal bovine serum (Invitro- assay to minimize the hydrolysis of free [- P]GTP and the gen) and transfected with Lipofectamine Plus (Invitrogen), time of the assay was kept to a minimum, substantial release or according to the manufacturer’s instructions. SDS-PAGE and exchange of GTP still occurs. In addition, we used subsaturat- immunoblotting were performed essentially as described pre- ing concentrations of nucleotide in the loading reaction. The viously (19). A Northern blot containing human poly(A) RNA maximum concentration of the labeled substrate is predicted to from brain, colon, heart, kidney, liver, lung, muscle, placenta, be2nM, well below the apparent K value of previously iden- small intestine, spleen, stomach, and testis (HB2010, OriGene tified GAPs (45–49). This further lowers the activity measured Technologies, Rockville, MD) was probed with a random hex- and compromises the quantification that can be achieved. amer primed P-labeled fragment containing the entire However, with the exception of assaying fractions during puri- ELMOD2 coding sequence. Hybridization was performed with fication, the Arl2 GAP assay is performed under conditions in ULTRAhyb (Ambion, Austin, TX) and followed standard pro- which activity decreases linearly with dilution of at least cedures, with the highest stringency washing performed at 10-fold, and the values reported are highly consistent between 50 °C in 0.1% SDS 0.1 SSC. preparations and experiments. The close similarities between specific activities of the purified bovine testis and HeLa recom- RESULTS binant Arl2 GAP preparations (see below) is another indication Development of a Specific Arl2 GAP Assay—For most that the methods adopted are valid for comparative purposes. GTPases, the dissociation of bound GTP or GDP can be slowed Two other issues that had to be overcome before purification of substantially by raising the free Mg concentration (40–42). the Arl2 GAP from tissues could be achieved were problems Thus, after preloading the GTPase with radiolabeled GTP arising because of the quaternary structure and the low stability under optimal conditions, the addition of increased free of the activity. Mg “locks in” the bound GTP to maximize stability of the Arl2 GAP Activity in Testis Extracts Is Present in a Large substrate in the GAP assay. The GAP reaction is stopped by Complex—Early attempts to measure Arl2 GAP activity in tis- dilution into cold buffer before trapping the remaining protein- sue homogenates were unsuccessful, presumably because of its bound [- P]GTP on nitrocellulose filters. Controls run in low abundance. After Arl2 and its effector, BART, were par- parallel allow discrimination between intrinsic GTPase activity, tially localized to mitochondria (19), an enriched bovine brain GAP-stimulated GTPase activity, and simple GTP dissociation mitochondria preparation was assayed and found to contain in the assay. This protocol was used to assess the specificity of Arl2 GAP activity (36). However, the levels of activity proved to our purified Arl2 GAP against other GTPases (see below) but be insufficient to allow purification to homogeneity. Further cannot be used with Arl2 because the rate of dissociation of searches indicated that bovine testis homogenates contained guanine nucleotides from Arl2 is insensitive to [Mg ] (43). 10-fold the activity of bovine brain mitochondria but that the Instead, we developed an Arl2 GAP assay that was based activity was present in a very large particle. upon those used earlier to assay GTPase activity of the  sub- During the initial stages of purification from testis, and prior units of heterotrimeric G proteins (44) and that uses the ability to the addition of any detergent, Arl2 GAP activity was only of activated charcoal to bind nucleotides but not free phos- found in a large complex of undetermined composition. This phate. Arl2 was pre-bound to [- P]GTP and exposed to the complex was large enough to be pelleted at 100,000  g, but GAP for a very limited time (typically 4 min), and the reaction activity was inefficiently recovered from the pellet. However, was stopped by the addition of a suspension of activated char- the activity was preserved and could be easily resolved from coal. GAP-dependent GTP hydrolysis is the amount of liber- smaller complexes and soluble proteins, by either gel filtration ated P over and above that released in the absence of the GAP or centrifugation through discontinuous isopycnic sucrose gra- JUNE 15, 2007• VOLUME 282 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 17571 ELMOD2 Is an Arl2 GAP activity eluted from gel filtration media with an apparent mass of 150 kDa (Fig. 1B). This decrease in apparent size was even more pronounced in 1% CHAPS, in which the activity displays an apparent mass of55 kDa (Fig. 1C). We interpret these data as evidence that the Arl2 GAP can assemble into larger com- plexes via interactions that are likely hydrophobic in nature. We also noted that the Arl2 GAP activity was relatively unstable in cholate above its CMC (Fig. 2A), and so we used CHAPS for the remainder of the purification. CHAPS was found to inhibit the Arl2 GAP assay at concentrations above 0.1% (Fig. 2B), so during purification in CHAPS each fraction had to be diluted to a point where this inhibitory effect did not interfere with the assay. During tests of the effects of CHAPS on the Arl2 GAP assay (Fig. 2B), we noticed that the specific activity of the crude GAP preparation (20/60) was actually increased in the presence of low amounts of detergent. To separate the effects of the deter- gent on our assay from effects on the large GAP complex, we treated the 20/60 with increasing concentrations of CHAPS on ice and then diluted the samples so that the detergent was less than 0.03% in the assay (Fig. 2C). The effects were relatively small, only 2-fold, and could be explained by either an effect of the detergent to increase the stability of a labile Arl2 GAP activ- ity or to dissociate it from the effects of an inhibitory compo- nent in the complex. Tests of these two possibilities indicated that perhaps both are true. The stability of Arl2 GAP activity in 20/60 was assayed at different times at 37 °C in the presence of 0, 0.25, or 1% CHAPS (Fig. 2D). The activity became more sta- ble with increasing concentrations of the detergent. Also, treat- ment of 20/60 with 0.25% CHAPS followed by centrifugation at FIGURE 1. Arl2 GAP activity behaves as a species of decreasing size with 100,000  g resulted in release of 85% of the activity from the increasing concentrations of CHAPS. The elution profiles of Arl2 GAP activ- ity (squares) and total protein (A , diamonds) are shown after resolution by complex. Only 70% of the total protein was released, yielding Superdex 200 gel filtration chromatography. Relative migration values (R )of a 1.2-fold increase in specific activity. More importantly, the molecular mass markers were calculated by dividing the volume at which the exposure to sub-CMC levels of detergent produced activity that marker eluted by the total volume of the column and are indicated by arrows. A, activity in 20/60, in the absence of detergent, migrates in the void volume was smaller, as judged by gel filtration (see Fig. 1B), and could be with a predicted molecular mass of 600 kDa. B, peak fractions from the further resolved by additional steps of chromatography (see Macro-Prep High Q column from an Arl2 GAP purification were concentrated by ultrafiltration prior to resolution in 0.25% CHAPS. The peak of activity Table 1). migrated with an apparent molecular mass of150 kDa. C, concentration of Purification of Arl2 GAP Activity—The Arl2 GAP was ulti- CHAPS was increased to 1%, and the sample was then resolved by gel filtra- mately purified 1600-fold from bovine testis homogenates to tion chromatography. This is the final step in the purification outlined in Table 1. The apparent molecular mass of the peak fraction is55 kDa. an estimated 20% purity with an overall yield of 0.08% (Table 1), as described under “Experimental Procedures.” After produc- dients. These two steps yielded very similar results so prepara- tion of 20/60 and treatment with 0.25% CHAPS to give a 10-fold tive discontinuous sucrose gradients were used as the first step increase in specific activity over the starting material, clarifica- of the purification for ease of scale up. tion by centrifugation at 100,000  g for 1 h yielded a prepara- The Arl2 GAP activity collected at the interface of the tion that was 12-fold enriched over homogenates with close to 20–60% sucrose layers (referred to as 20/60) eluted in the void 100% recovery. Sequential chromatography using ion exchange volume of both a Sephacryl S-300 column and a Superdex 200 (Macro-Prep High Q column), hydroxyapatite, and gel filtra- column (Fig. 1A), indicating an apparent molecular mass of tion media (Superdex 200) was performed in 0.25% CHAPS and 1.3 MDa. The activity present in this large complex proved resulted in a net 500-fold purification with 4% yield. The yield refractory to further purification using a number of conven- was low as a result of both taking only peak fractions forward at tional chromatography resins, so a means of dissociating the each step and denaturation of the protein. Elution of Arl2 GAP complex with retention of activity was sought. The complex activity from gel filtration chromatography in the presence of was not disrupted by treatment with DNase, RNase, or phos- 0.25% CHAPS was consistent with an apparent molecular mass pholipases A or C (data not shown) but was dissociated by the of 150 kDa (Fig. 1B). At this point in the purification the addition of CHAPS or sodium cholate. After more detailed concentration of the CHAPS was increased to 1%, and the activ- studies of the effects of CHAPS on the apparent size, we discov- ity no longer bound to anion exchange resins, e.g. it eluted in the ered that with addition of the detergent to levels below the flow-through of the Resource Q column. Substantial loss of critical micelle concentration (CMC; 0.6%) the Arl2 GAP activity resulted from the increase in [CHAPS] but allowed for 17572 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 24 •JUNE 15, 2007 ELMOD2 Is an Arl2 GAP FIGURE 2. Effects of detergents on Arl2 GAP activity. Each data point in the figure is the average of triplicate measurements, and each panel is a single experiment that is representative of at least two repeats. A, 20/60 preparation was exposed to detergents at different concentrations and for the indicated lengths of time, all at 4 °C. The samples were diluted 20-fold at the indicated times and assayed as described under “Experimental Procedures.” Activities shown are relative to the 20/60 starting material that had not been exposed to detergent. Note the loss of activity in 1% cholate (triangles). B, effect of CHAPS on the Arl2 GAP assay was determined by inclusion of detergent at the indicated concentrations in the assay. Activities of 20/60 shown are relative to that found in the absence of detergent. C, exposure to high concentrations of CHAPS increases Arl2 GAP activity. To determine the effect of CHAPS on the specific activity of the large GAP complex, 20/60 was treated with the indicated concentration of CHAPS for 30 min on ice. The detergent/GAP mixture was then diluted 20-fold before assaying to reduce the level of CHAPS to a concentration that does not interfere with the assay. D, Arl2 GAP activity in 20/60 becomes more thermostable with increasing concentration of CHAPS. Samples of 20/60 were brought to a final CHAPS concentration of either 0 (triangles), 0.25% (squares), or 1.0% (diamonds) and immediately moved to 37 °C for the length of time indicated. The samples were then cooled briefly on ice, diluted 20-fold, and assayed. Results are reported relative to identical samples that had not been placed at 37 °C. TABLE 1 Summary of the steps used to purify Arl2 GAP activity from bovine testis As detailed under “Experimental Procedures,” 137 g of bovine testis tissue was homogenized and clarified by centrifugation at 1,000  g for 45 min. The supernatant was recovered and spun at 10,000  g for 30 min to yield the material in the 1st line of the table. All table entries represent pools of peak fractions except for the final one that is a single fraction. 1 unit is the amount of Arl2 GAP activity required to convert 1 nmol of Arl2-GTP into 1 nmol of Arl2-GDP in 1 min. Purification step Total activity Total protein Specific activity Yield Purification units mg units/mg % -fold 10,000  g supernatant 9.74 3340 0.00292 100 1 Isopycnic sucrose gradient (20/60 interface) 4.16 401 0.0104 42.7 3.56 20/60 in 0.25% CHAPS 100,000  g supernatant 9.47 274 0.0345 97.2 11.8 Macro-Prep High Q peak 4.22 53.9 0.0782 43.3 26.8 Hydroxyapatite peak 1.38 2.98 0.464 14.2 159 Superdex 200 peak 0.405 0.264 1.54 4.16 527 Resource Q flow-through 0.0852 0.0589 1.45 0.87 496 Superdex 200 fraction 27 0.00819 0.00175 4.68 0.08 1600 Protein concentration was estimated by Coomassie and silver staining. resolution of the GAP from many proteins that remained able that peak in fractions 28 and 29, respectively. This band was to bind to the ion exchange column. In addition, the increase in estimated to be one-fifth of the total protein present in the peak CHAPS concentration also resulted in an activity that eluted fraction indicating that the specific activity of fully purified from the gel filtration column with an apparent molecular mass bovine Arl2 GAP is 23 units/mg (Table 1) and would require of 55 kDa (Fig. 1C). Fractions from this final column were 8,000-fold enrichment to achieve purity. analyzed by SDS-PAGE (Fig. 3A). Four major bands (one a dou- The region surrounding the 32-kDa band of the gel shown in blet) were seen by silver staining, but only one, migrating to a Fig. 3A was excised and subjected to tryptic digestion and tan- predicted mass of32 kDa by comparison to standards, peaked dem MS/MS analysis for identification, as described under in fraction 27 and showed staining intensity that correlated “Experimental Procedures.” Seventeen peptides were identified completely with GAP activity across the fractions (Fig. 3A). in this sample, which mapped to four different proteins. Four of This is in contrast, for example, to the bands at 50 and 25 kDa the 17 peptides were 100% identical to regions of bovine JUNE 15, 2007• VOLUME 282 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 17573 ELMOD2 Is an Arl2 GAP FIGURE 3. ELMOD2 is the Arl2 GAP purified from bovine testis. A, fractions from the final gel filtration column were resolved by SDS-PAGE and stained with silver, as described under “Experimental Procedures.” Migration of molecular mass markers (size in kDa) is shown at left. The relative amount of Arl2-GAP activity is indicated below each lane. The indicated band was excised and analyzed by mass spectroscopy to identify the protein. B, amino acid sequences of human and bovine ELMOD2 are shown. Human and bovine ELMOD2 sequences are 94% identical (asterisks mark the nonidentical residues), and the four peptides identified by tandem MS analysis (bars) are completely conserved in the human sequence. ELMOD2 (Fig. 3B). The bovine and human ELMOD2 proteins tion and misfolding. Thus, we conclude that ELMOD2 pos- are each 293 residues in length with only 17 amino acid differ- sesses Arl2 GAP activity but cannot exclude (and indeed think ences (94% identity), none of which were within the identified very likely) that other proteins will be found that bind and sta- peptides. The open reading frame of the human ortholog was bilize the protein and activity in cells. amplified by PCR from a cDNA library and subcloned into a The open reading frames of the human orthologs of the three pET28 vector for expression in bacterial (BL21(DE3)) cells. A other proteins identified by MS analyses were also expressed in large amount of recombinant protein was expressed upon bacteria and HeLa cells, but none of the expressed proteins induction, but it was all insoluble and pelleted during centrifu- either had any detectable Arl2 GAP activity or increased the gation at 100,000 g after lysis of bacteria in a French press. No half-life or GAP activity of purified recombinant human Arl2 GAP activity was detected in the S100, and a variety of ELMOD2. attempts to solubilize the protein from the pellet were unsuc- Because another possibility for the low specific activity of the cessful. However, expression of the E. coli MBP as an N-termi- bacterially expressed ELMOD2 was the absence of post-trans- nal fusion with human ELMOD2 resulted in small amounts of lational modifications, we also expressed C-terminal epitope- soluble protein that could be purified by affinity chromatogra- tagged (c-Myc/His) human ELMOD2 in cultured human cells phy. Both the S100 and the purified preparation of MBP- and estimated its specific activity. HeLa cells were transiently ELMOD2 were active in the Arl2 GAP assay, thus confirming transfected with pCDNA3.1-ELMOD2, and cell lysates were ELMOD2 as an Arl2 GAP. The protein was found to precipitate collected 24 h post-transfection. Lysates were cleared by cen- progressively over a few days when stored at 4 °C or completely trifugation at 2,000 g for 3 min, and tagged ELMOD2 expres- upon either one cycle of freeze/thaw or removal of the MBP tag sion levels were determined by immunoblotting, using a puri- by thrombin cleavage. Furthermore, the specific activity of the fied Myc-tagged protein (Arf1) of known concentration as a bacterially expressed fusion protein was1000-fold lower than standard. Control HeLa lysates prepared in this way yield Arl2 that of the purified bovine protein, likely the result of aggrega- GAP activities that are close to the lower limit of detection of 17574 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 24 •JUNE 15, 2007 ELMOD2 Is an Arl2 GAP our assay. When ELMOD2-Myc/His is expressed, we found a MOtility or phagocytosis of apoptotic cells. The mammalian 10–20-fold increase in activity in the lysates. Combination of ortholog was confirmed to play a similar role in higher results from the two assays yielded an estimate that the recom- eukaryotes (53). Although ELMO1 and ELMO2 are thought to binant human ELMOD2 in HeLa lysates has a specific activity be functionally interchangeable (53–55), they do exhibit differ- of24 nmol of Arl2-GTP hydrolyzed per min/mg protein, very ential expression during development (56). ELMO1–3 share close to that determined for the purified bovine testis Arl2 GAP high sequence identities (50%) overall (Fig. 4B)andaPH (23 units/mg, see above), further confirming the identity of the domain in their C termini (Fig. 4A) that is absent in Arl2 GAP as ELMOD2. For comparison, the specific activity of ELMOD1–3. The three ELMO proteins are much less closely ArfGAP1 is 6.7 (50) or 10–50 nmol (51) of Arf1-GTP hydro- related to the three ELMO domain proteins, sharing 20% lyzed per min/mg protein. These results prove that ELMOD2 identity that is limited to the ELMO domain. ELMOD1 and encodes an Arl2 GAP but leave open the question of whether ELMOD2 share 53% identity, whereas ELMOD3 (also called any post-translational modifications or protein interactions are RBED1) shares no more than 20% identity with any of the other important modulators of activity. family members and again only in the ELMO domain (Fig. 4B). ELMOD2 Protein and Message Are Low in Abundance—Our Although the peptides identified by MS analysis were exclu- purification from bovine testis indicated that ELMOD2 makes sive to ELMOD2, we determined whether any other ELMO up0.02% of total testis protein. This is similar to our estimate family members exhibited Arl2 GAP activity by expressing of endogenous levels of the Arl2 GAP in HeLa cells (0.04%), ELMO1–3 in both E. coli and HeLa cells and assaying cell although each of these numbers is close to the lower limit of lysates. The plasmids encoding ELMO1–3 were provided by detection of the assay and each is compromised by difficulties in Kodi Ravichandran (University of Virginia) and directed assaying Arl2 GAP activity in lysates. The low level of expres- expression of glutathione S-transferase-tagged bacterial pro- sion, apparent presence in larger complexes, and lability during teins and FLAG-tagged proteins in HeLa cells. In contrast to purification combine to account for most of the difficulties in ELMOD2, ELMO1–3 all expressed well and remained soluble its isolation. in bacterial and HeLa lysates, but Arl2 GAP activity was not We also found extremely low levels of ELMOD2 mRNA mes- detected in any of these lysates. sage in our probe of a multitissue mRNA Northern blot (Ori- The ELMOD1 that we amplified and subcloned from a Gene Technologies). This filter contained mRNA from human TM cDNA library was based upon the sequence in GenBank brain, colon, heart, kidney, liver, lung, muscle, placenta, small (entry GI 34192051 dated 17 July 2006) and encoded a protein intestine, spleen, stomach, and testis and was probed with a of 283 amino acids. During the course of our studies this entry hexamer primed probe generated using the entire open reading was updated to one predicting a protein with an additional 51 frame. ELMOD2 was barely detected, and only after 72hof amino acids at the N terminus. In the intervening time, we exposure to a PhosphorImager screen, as a faint band of 1.3 expressed the shorter open reading frame in bacteria and found kb in the lane containing mRNA from stomach. The only other that truncated human MBP-ELMOD1 possesses Arl2 GAP tissue yielding detectable signal was testis, in which a faint, activity with a specific activity 23% that of MBP-ELMOD2. broad smear was noted (data not shown). In contrast, strong This fusion protein was also unstable, like MBP-ELMOD2, but signals were detected in all lanes with the control -actin it has been less thoroughly studied at this point. Thus, the pro- probe after only a brief (0.5 h) exposure to film. Previously tein with the highest sequence identity to ELMOD2 also pos- published work showed that human ELMOD2 RNA was in sesses Arl2 GAP activity. all tissues examined by reverse transcription-PCR, although A plasmid purported to encode full-length human ELMOD3 no details about levels were reported (52). Thus, the was purchased from OriGene but was later found to encode a ELMOD2 message is likely expressed to only low levels but is TM variant (GenBank entry GI 85662659). This variant (referred widespread, perhaps ubiquitous, in its expression in differ- TM to here as ELMOD3_C) is identical to the current GenBank ent human tissues. Arl2 is both abundantly and ubiquitously entry for ELMOD3 (GI 34147693) through amino acid 314 expressed (19). The low expression of ELMOD2 likely but differs at the C terminus and presumably is an alterna- explains the limited number of ESTs in public data bases and tively spliced version. The C terminus of this variant exhibits limits its likelihood of discovery in functional screening of similarity to the ELMOD3 proteins from chimpanzee, cDNA libraries. macaque, cow, dog, and mouse, whereas the C terminus of the ELMOD1 Also Has Arl2 GAP Activity—Bovine and human TM official ELMOD3 GenBank entry only exhibits sequence ELMOD2 are each 293 residues in length and are 94% identical similarity to a chimpanzee ortholog. Therefore, we expressed and 98% homologous. Orthologs of human ELMOD2 were TM ELMOD3_C in HeLa cells as a Myc/His-tagged protein and also found in primates (GenBank accession number TM failed to detect any Arl2 GAP activity in cell lysates. These tests XP_001090344, 98% identity), rodents (GenBank acces- TM for Arl2 GAP activity among the human ELMO domain-con- sion number NP_848851, 87% identity), chicken (GenBank taining proteins reveal that only ELMOD1 and ELMOD2, accession number XP_420415, 70% identity), and Zebrafish TM which share 53% identity (Fig. 4C), are active. It is possible that (GenBank accession number XP_691971, 61% identity). ELMOD2 is so named because it is one of six human proteins other family members have activity that was not detected as a that contain the ELMO domain, a “domain of unknown func- result of the need for orienting the protein on a membrane tion” (DUF609). ELMO1 was first identified as CED-12 in Cae- surface, because of the presence of the PH domain (57), or pos- norhabditis elegans, a protein required for EnguLfment and cell sibly interference of the tags used. It is also possible that the JUNE 15, 2007• VOLUME 282 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 17575 ELMOD2 Is an Arl2 GAP FIGURE 4. ELMOD2 is one of six human family members containing the ELMO domain. A, all six human ELMO domain-containing proteins are depicted with ELMO domains (black box) aligned. ELMO1–3 also contain a PH domain (hatched box) located C-terminal to the ELMO domain. B, amino acid sequence identities between human ELMO domain family members are shown. The percent identity of pairwise combinations is shown above the diagonal line. The number of identical residues and the total number of residues compared between each pair is indicated below the diagonal line. ELMOD2 has 53% identity to ELMOD1 over 294 amino acids but 20% or lower identity with the remaining family members. C, amino acid sequences of the Arl2 GAP activity containing ELMOD1 and ELMOD2 proteins are shown with conserved residues identified. The ELMO domain region is boxed. other family members are active as GAPs for GTPases other otidase activity, we used the filter-binding assay described than Arl2. above and under “Experimental Procedures.” Because each ELMOD2 Also Possesses GAP Activity Against Arfs but Not GTPase has a different rate of release of bound nucleotide, in Ran, Rac1, or RhoA—To determine the specificity of the each case the relevant parameter is not the absolute rate of GAP activity of ELMOD2 toward GTPases other than Arl2, loss of radioactivity but the difference in the rate of loss we examined the activity of recombinant human MBP- between the sample with and without (buffer alone) the ELMOD2 against Arl3, Arf1, Arf6, RhoA, Rac1, and Ran. GAP. ArfGAP1 and 20/60 were included as positive controls, Because of the nucleotide handling properties of these as the latter is a crude sample predicted to contain a number GTPases and the varying presence of contaminating nucle- of GAP activities. Note that the rate of dissociation of GTP 17576 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 24 •JUNE 15, 2007 ELMOD2 Is an Arl2 GAP active against Arl3 (Fig. 5A). As expected, both MBP-ELMOD2 and 20/60 were active against Arl3, but ArfGAP1 was not. None of the GTPase activities of proteins out- side the Arf family (RhoA (Fig. 5D), Rac1 (Fig. 5E), and Ran (Fig. 5F)) were increased upon exposure to MBP-ELMOD2 or ArfGAP1, although each was increased by 20/60. Thus, MBP-ELMOD2 is active as a GAP for Arl2 and Arl3 but not for these GTPases outside the Arf family. Surprisingly, MBP-ELMOD2 ex- hibited substantial GAP activity for Arf1 (Fig. 5B) and Arf6 (Fig. 5C), although less for the latter. This is despite the fact that ELMOD2 lacks the consensus Arf GAP domain (32, 59) and clearly provides opportuni- ties for cross-signaling between Arl and Arf proteins, which are thought to have quite distinct functions. To further test the ability of MBP- ELMOD2 to act as a GAP for Arfs, we assayed Arf1 and Arf6 in the charcoal assay. Despite the varying FIGURE 5. ELMOD2 has GAP activity against Arl3, Arf1, and Arf6. The filter-trapping GAP assay was per- signal to noise ratios in these formed using different GTPases, as described under “Experimental Procedures.” Each data point in A–F is the assays, the measured specific average of triplicate measurements, and each panel is a single experiment that is representative of at least two ELMOD2-dependent GAP activ- repeats. Note that the GAP activity is the difference between the loss of bound radionucleotide in the presence of the GAP sample from that found in its absence (diamonds). ArfGAP1 (triangles) is included as a positive ity for the Arfs was very similar in control for the Arfs, and 20/60 (circles) is a crude preparation predicted to contain several GAPs and is also each assay (Fig. 6). included as a positive control. Relative GAP activities of ELMOD2 against all GTPases tested are shown in Fig. 6. These relative activities were obtained using the same concentration of each GTPase and [- P]GTP, the same time points, and the same buffer conditions and were repeated at least three times with similar results. However, because the conditions of the assay were developed and therefore optimized for Arl2, differ- ences in the binding stoichiometries of each GTPase yield dif- ferent substrate (GTPase-GTP) concentrations at t 0. Specif- ically, because of differences in the nucleotide binding properties of the different GTPases and in the charcoal and filter trapping assays, the concentrations of substrate (GTPase- GTP) are predicted to be about 10-fold higher for Arl2 than for FIGURE 6. Relative activities of ELMOD2 toward Ras family GTPases. Spe- cific activities of purified recombinant MBP-ELMOD2 against seven different the other GTPases, making it likely that we are underestimating GTPases were determined, as described under “Experimental Procedures,” the activity of ELMOD2 toward the other GTPases. More rig- and normalized to that against Arl2 (set to 100%). The light bars are numbers generated from the experiments in A–F of Fig. 5. and the dark bars are gener- orous kinetics, measured under Michaelis-Menten conditions ated in the charcoal assay as described under “Experimental Procedures.” and with more stable protein preparations, will be required Note the similarities in the results of the different assays for Arf1 and Arf6. before detailed comparisons of rate constants and relative affin- Because of differences in the nucleotide binding properties of Arl2, it is pre- dicted that the concentration of GTP-bound Arl2 in these assays was 10- ities can be determined. fold higher than the GTP-bound form of the other GTPases, making it likely that activity toward these other GTPases is underestimated. DISCUSSION from Arl2 under these conditions is too rapid to allow it to be We report the purification, identification, expression, and assayed in this way. initial characterization of ELMOD2 as the first mammalian Arl Arl2 is more closely related to Arl3 than to any other GTPase GAP. ELMOD2 was found to exist in cells as part of a large (20, 58), so it was not surprising to see that ELMOD2 was also protein complex, the components of which are likely to con- JUNE 15, 2007• VOLUME 282 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 17577 ELMOD2 Is an Arl2 GAP tribute to the stability, activities, and biological function of reactants, particularly for the ELMO proteins that contain PH ELMOD2. Surprisingly, ELMOD2 was also found to be active as domains (57). The more challenging, but also more important, an Arf GAP. The presence of an ELMO domain in this and five question is the extent to which ELMO domain proteins serve other human proteins, one of which we showed also possesses as regulators or effectors of Arl2 or other Arf family GTPases Arl2 GAP activity, leads us to speculate that the ELMO domain in vivo. is a GAP domain for Arl2 and other members of the Arf family, An intriguing hint that ELMOs may act to link Arf and other with specificities yet to be determined. The biological functions GTPase signaling pathways is found in the role of Arf6 to stim- of the ELMO proteins, their locations, and their ability to act as ulate actin reorganization in lamellipodia formation. The Arf terminators or effectors of Arf family GTPases are currently guanine nucleotide exchange factor ARNO activates cell migra- unknown but will likely provide new insights into signaling by tion through activated Arf6 in a Rac1-dependent manner (61). these GTPases and the potential for cross-talk between Arfs The Rac1 dependence was recently shown to require the co- and Arls. localization of DOCK180/ELMO1 Rac GEF activity (62). It is Human ELMOD2 was shown to be an Arl2 GAP by purifica- not yet known how Arf6 is inactivated along the sides and trail- tion of the activity from bovine testis and the demonstration ing edge of the forming protrusions, although recruitment of that the purified recombinant protein is active. However, Arl2 Arf GAPs has been proposed as a possibility (62). But another GAP activity is most stable when part of a large cellular complex intriguing possibility is that the Arf GAP activity is already there and exhibits a shortened half-life when monomeric. The find- in the form of an ELMO domain protein. Although ELMO1 did ings that sub-CMC concentrations of CHAPS allowed partial not have Arf GAP activity in our in vitro assays, it is certainly dissociation of the large complex and increased the thermal possible that the activity may be found under different condi- stability of Arl2 GAP activity suggest that hydrophobic residues tions. Thus, the position of ELMO1 between Arf6 and Rac1 are involved in ELMOD2 interactions. Because lability of the signaling may be a prototype for other ELMO domain proteins. recombinant and purified testis proteins contributed to diffi- Because ELMOD2 was found to be active against Arl3, it is culties in working with each preparation and limited the accu- also tempting to speculate that it functions in the regulation racy of quantification that can be achieved, development of a of cytokinesis. We recently showed (20) that knockdown of more stable preparation is a high priority. This will likely Arl3 by short interfering RNA leads to inhibition of cytoki- require the identification of ELMOD2-binding partners. In nesis in HeLa cells and ELMOD2 was one of only a few genes addition to providing access to a more stable preparation of discovered in an RNA interference screen in flies for genes Arl2 GAP activity, interaction of ELMOD2 with these binding required for cytokinesis (63). partners in vitro may result in changes in catalytic rates or spec- Although opportunities for cross-talk between GTPase sig- ificity among GTPases. Knowledge of the composition of naling pathways are evident, it is still most likely that the bio- ELMOD2 complexes should also provide additional insights logical role of ELMOD2 will result from its actions as GAP and into its cellular locations and biological functions. potential effector of Arl2 signaling. Arl2 was first found to be The finding that ELMOD2 had GAP activity toward Arf1 was involved in tubulin and microtubule dynamics in a yeast genetic surprising because it lacks the canonical, cysteine-rich, zinc fin- screen (17, 64) and later biochemically through its binding to ger Arf GAP signature, CX CX CX C (where X is any tubulin folding chaperone cofactor D (16). Although mono- 2 16–18 2 amino acid) (32), that is present in every previously reported meric Arl2 binds GTP readily in the absence of a GEF and protein with Arf GAP activity (e.g. see Refs. 12, 31, and 33) and would therefore be expected to exist in the activated form in found in 20 proteins in the human proteome. ELMOD2 has cells, the bulk of cellular Arl2 is bound to cofactor D and in this five cysteines, but their arrangement does not resemble the Arf form cannot bind GTP (65). We recently showed that an excess GAP signature and only one of them is completely conserved of activated Arl2 causes the loss of microtubules and cell cycle among metazoan orthologs. Thus, we predict a novel structure arrest (20), and so cofactor D may act as a sink for Arl2, keeping and mechanism of promoting GTP hydrolysis by Arf family it locked in the inactive form. It is likely that ELMOD2, as an members will be found for ELMOD2. Arl2 GAP, provides an additional level of control in the critical We tested all six human ELMO domain proteins and function of regulating the levels of activated Arl2. Although found that ELMOD1 also had Arl2 GAP activity, whereas the most cellular Arl2 is in the cytosol, a smaller pool is found in other proteins did not. We cannot exclude the possibility mitochondria where it can bind BART and the adenine nucle- that other ELMO domain proteins possess GAP activity otide transporter 1 (19). The function of Arl2 in mitochondria is against other members of the Arf family. Indeed, we speculate not clear, but it may be involved in regulating energy metabo- that the ELMO domain is an Arf family GAP domain that may lism (19). Thus Arl2 and ELMOD2 may act together to sense provide cross-talk between Arf and Arl proteins in cells. A more and transmit information about the general health of the cell systematic screening of GAP activities of ELMO domain pro- between mitochondria and the cell division apparatus. Further teins for Arf family members is planned but must await more studies of the localization of ELMOD2 and the identification of stable preparations of ELMOD1 and ELMOD2, as well as iden- its binding partners and regulators will contribute significantly tification of the complexes that exist in cells. It will also be to our understanding of these pathways. important to evaluate potential roles of other molecules Finally, the fact that Arl2 is an ancient protein, with orthologs (including lipids and phosphoinositides) to serve as cofactors in in the earliest eukaryotes, whereas orthologs of ELMOD2 and the GAP assay, as has been shown for some Arf GAPs (60), and ELMOD1 are not evident in early eukaryotes, suggests that the need for a membrane surface to assist in the orientation of other Arl2 GAPs may exist in early eukaryotes that perhaps are 17578 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 24 •JUNE 15, 2007 ELMOD2 Is an Arl2 GAP J. Cell Sci. 117, 4705–4715 also expressed in mammals. Detailed analyses of the key func- 23. Avidor-Reiss, T., Maer, A. M., Koundakjian, E., Polyanovsky, A., Keil, T., tional residues and structures of the ELMO domain proteins Subramaniam, S., and Zuker, C. S. (2004) Cell 117, 527–539 may allow the detection of more divergent proteins, from 24. Chiang, A. P., Nishimura, D., Searby, C., Elbedour, K., Carmi, R., Ferguson, potentially any eukaryote, that share Arl GAP activities. A. L., Secrist, J., Braun, T., Casavant, T., Stone, E. M., and Sheffield, V. C. (2004) Am. J. Hum. Genet. 75, 475–484 Acknowledgments—We gratefully acknowledge the assistance of cur- 25. Fan, Y., Esmail, M. A., Ansley, S. J., Blacque, O. E., Boroevich, K., Ross, A. J., Moore, S. J., Badano, J. L., May-Simera, H., Compton, D. S., Green, J. S., rent and former members of the laboratory in discussions of the work Lewis, R. A., van Haelst, M. M., Parfrey, P. S., Baillie, D. L., Beales, P. L., presented. We thank John Hepler for critical reading of the manu- Katsanis, N., Davidson, W. S., and Leroux, M. R. (2004) Nat. Genet. 36, script and members of the Daniel Reines laboratory for providing 989–993 expert technical assistance in performing Northern blots. The gener- 26. Li, J. B., Gerdes, J. M., Haycraft, C. J., Fan, Y., Teslovich, T. M., May- ous gifts of purified recombinant GTPases from Gary Bokoch (The Simera, H., Li, H., Blacque, O. E., Li, L., Leitch, C. C., Lewis, R. A., Green, Scripps Research Institute), Ian Macara (University of Virginia), and J. S., Parfrey, P. S., Leroux, M. R., Davidson, W. S., Beales, P. L., Guay- Sharon Campbell (University of North Carolina, Chapel Hill) greatly Woodford, L. M., Yoder, B. K., Stormo, G. D., Katsanis, N., and Dutcher, facilitated our studies of specificity. Kodi Ravichandran and James S. K. (2004) Cell 117, 541–552 Casanova (University of Virginia) provided helpful and interesting 27. Pazour, G. J., Agrin, N., Leszyk, J., and Witman, G. B. (2005) J. Cell Biol. discussions of ELMO proteins as well as constructs for their expression 170, 103–113 28. Schrick, J. J., Vogel, P., Abuin, A., Hampton, B., and Rice, D. S. (2006) Am. J. in bacterial and mammalian cells. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Jun 15, 2007

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