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Identification of a Novel GTPase, the Inducibly Expressed GTPase, That Accumulates in Response to Interferon γ

Identification of a Novel GTPase, the Inducibly Expressed GTPase, That Accumulates in Response to... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 34, Issue of August 23, pp. 20399–20405, 1996 Printed in U.S.A. Identification of a Novel GTPase, the Inducibly Expressed GTPase, That Accumulates in Response to Interferon g* (Received for publication, April 16, 1996, and in revised form, May 31, 1996) Gregory A. Taylor, Michael Jeffers, David A. Largaespada‡, Nancy A. Jenkins, Neal G. Copeland, and George F. Vande Woude§ From the ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 factor a, markedly increases expression of major histocompat- Interferon g is a pleiotropic cytokine that regulates many immune functions. We have identified a novel pro- ibility class I molecules and the cell adhesion molecules, tein, inducibly expressed GTPase (IGTP), whose expres- ICAM-1 and ELAM-1, thereby promoting recruitment of im- sion was regulated by interferon g in macrophages. In mune cells to areas of inflammation (10). These pleiotropic mouse RAW 264.7 macrophages, IGTP mRNA levels were responses are initiated by binding of IFNg to its receptor, almost undetectable but increased within1hof expo- followed by receptor dimerization and a cascade of primary and sure to interferon g, peaked at very high levels within secondary events (1). However, these molecular events have 3 h, and remained at high levels to at least 48 h; pretreat- not been completely defined, and identification of proteins ment of the cells with cycloheximide blocked the major- whose expression is regulated by IFNg is the subject of active ity of mRNA accumulation. In the mouse, the mRNA was investigation. highly expressed in thymus, spleen, lung, and small in- We report here the identification of a 48-kDa protein, desig- testine. Using interspecific backcross analysis, the Igtp nated IGTP, whose expression was rapidly and dramatically gene was mapped to mouse chromosome 11. The IGTP increased by IFNg in macrophages and fibroblasts. The protein cDNA encoded a putative polypeptide of M 48,507 and was first recognized as the product of a differentially displayed pI 7.79 that contained three consensus GTP binding mo- cDNA in hepatocyte growth factor (HGF)-treated C127 mouse tifs, GXXXXGK(S/T), DXXG, and NTKXD. Both IGTP that fibroblasts. In these cells, the IGTP mRNA levels showed a had been immunoprecipitated from RAW cells and a slight transient increase following HGF exposure; in addition, glutathione S-transferase IGTP fusion protein were able mRNA levels were constitutively elevated in C127 and NIH/ to convert GTP to GDP in vitro. Subcellular protein 3T3 cells that were transformed by coexpression of HGF and its fractionation and Western blotting localized IGTP to the receptor Met. These observations and their significance will be cytosol of RAW cells. In addition, the protein was homol- ogous to proteins encoded by three previously cloned discussed elsewhere. In the present work, we characterize the cDNAs, IRG-47, TGTP/Mg21, and LRG-47, and thus may IFNg-induced IGTP gene expression, and we demonstrate that be representative of a new family of interferon g-regu- the protein product is a GTPase. Because of its expression lated GTPases. pattern and its biochemical activity, the protein has been des- ignated inducibly-expressed GTPase (IGTP). The protein had high sequence homology with the proteins encoded by three Interferon g (IFNg) is a 20-kDa protein that regulates a other cDNAs, IRG-47 (11), TGTP(12)/Mg21(13), and LRG-47(14), wide variety of immunological and inflammatory processes (for for which no biochemical or physiological function had been de- review see Ref. 1). While production of IFNg is limited to CD8 scribed previously, but which also contained putative GTP binding T cells, some CD4 T cell subsets (2), and natural killer cells sequences. IGTP, therefore, may be representative of a new family (3), its receptor is found on almost all cell types where it elicits of GTPases that could potentially mediate the effects of IFNg in many diverse physiological responses. In macrophages for ex- macrophages and other cells. ample, IFNg induces major histocompatibility class II expres- EXPERIMENTAL PROCEDURES sion (4), increases F receptor-mediated phagocytosis (5), and Cells and Culture—C127 cells (ATCC CRL-1616, American Type mediates removal of neoplastic cells and virally and parasiti- Culture Collection), Met-transfected C127 cells (C127/Met), and RAW cally infected cells by initiating secretion of cytosidal com- 264.7 cells (ATCC TIB-71) were maintained in Dulbecco’s modified pounds and tumor necrosis factor a (6–9). In endothelial cells Eagle’s medium (Life Technologies, Inc.) supplemented with 10% (v/v) on the other hand, IFNg, in combination with tumor necrosis fetal bovine serum (Life Technologies, Inc.), 0.292 mg/ml L-glutamine (Life Technologies, Inc.), 100 units/ml penicillin (Life Technologies, Inc.), and 100 units/ml streptomycin (Life Technologies, Inc.). Hepato- * This research is sponsored by the National Cancer Institute, cyte growth factor was purified from the supernatant of transformed DHHS, under contract with ABL. The costs of publication of this article NIH/3T3 cells that overproduce the factor (15); it was added directly to were defrayed in part by the payment of page charges. This article must the growth medium of C127 cells at 200 units/ml. Recombinant mouse therefore be hereby marked “advertisement” in accordance with 18 interferon g (Boehringer Mannheim) and lipopolysaccharide (serotype U.S.C. Section 1734 solely to indicate this fact. 055:B5, Sigma) were added directly to the culture media of C127 and ‡ Leukemia Society of America Fellow. RAW 264.7 cells at the concentrations indicated in the text. § To whom correspondence should be addressed: ABL-Basic Research Northern Blotting—Total RNA was prepared from cultured cells (16) Program, NCI-Frederick Cancer Research & Development Center, P. O. and from mouse tissues (17) using standard acidic phenol/chloroform Box B, Frederick, MD 21702. Tel.: 301-846-1584; Fax: 301-846-5038. extraction protocols. 15 mg of RNA samples were separated on 1.2% The abbreviations used are: IFNg, interferon g; HGF, hepatocyte agarose/formaldehyde gels and used for Northern blot analysis as de- growth factor; GST, glutathione S-transferase; IGTP, inducibly ex- pressed GTPase; PBS, phosphate-buffered saline; PEI, polyethylenei- mine; kb, kilobase pair(s); SRP, signal recognition particle; RFLPs, restriction fragment length polymorphisms; CHAPSO, 3-[(3-cholamido- M. Jeffers, S. Rong, M. Oskarsson, M. Anver, and G. F. Vande propyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid. Woude, submitted for publication. This is an open access article under the CC BY license. 20399 20400 IGTP, an IFNg-induced GTPase scribed previously (18). The blots were probed with a P-labeled human rotator at 4 °C for 1 h, and with 6 mg of protein A-Sepharose (Pharma- glyceraldehyde-phosphate dehydrogenase probe isolated as a 1.2-kb cia, Uppsala, Sweden) for an additional hour at 4 °C. Following a 2-min PstI fragment of pHcGAP (19), a mouse IP10 probe isolated as a 0.5-kb centrifugation at 500 3 g to pellet the protein A-Sepharose, the super- EcoRI fragment of the mouse C7–1 (ATCC 63135), a human b-actin natant was transfered to a new microcentrifuge tube and incubated for cDNA probe (Clontech; supplied with product 7760–1), or an IGTP 3–4 h with anti-IGTP polyclonal rabbit serum that had been generated probe isolated as a 0.28-kb EcoRI fragment of the IGTP cDNA (bases against the peptide CSLRKALKDSVLPPEIH-OH corresponding to the 1645–1927, GenBank accession U53219). The IGTP probe contained 16 carboxyl-terminal amino acids of IGTP. Next, 6 mg of protein A- entirely 39-untranslated sequences that were not conserved with the Sepharose were added, and the slurry was incubated for an additional related cDNAs referred to in the text; thus the probe was specific for 1 h at 4 °C. Finally, the protein A-Sepharose was washed four times IGTP. with ice-cold lysis buffer and 0.1 ml of SDS buffer (24% (w/v) sucrose, Differential Display Screening and Library Screening—C127/Met 0.36 M dithiothreitol, 0.048 M EDTA, 4.8% (w/v) sodium dodecyl sulfate, cells were exposed to 200 units/ml hepatocyte growth factor for various 0.05% (w/v) bromphenol blue) that had been diluted 1:3 with PBS was times, and total RNA was isolated and used for differential display added; the suspension was mixed vigorously and boiled for 5 min, and screening with an RNAimage kit 1 (GenHunter Corp., Brookline, MA) the resin was pelleted by centrifugation for 5 min at 15,000 3 g. The according to the protocols supplied by the manufacturer. One 150-base proteins in the SDS buffer were separated by 9% SDS-polyacrylamide cDNA fragment, designated A2c, was isolated using primers H-T A gel electrophoresis. and H-AP2; levels of A2c were slightly increased in cDNA generated GTP Hydrolysis Assays—Protein was immunoprecipitated from cell from C127/Met cells exposed to HGF for 3–8 h. The cDNA fragment was lysates as described above, except that the cells were lysed in a lysis used to screen a mouse spleen cDNA library (Stratagene, La Jolla, CA), buffer of 10 mM CHAPSO, 50 mM Tris, pH 8.0, and 0.15 M NaCl; from which a 1927-base cDNA clone was isolated and later designated immunoprecipitation using this buffer results in slightly higher as the IGTP cDNA. The clone was sequenced on both strands using the GTPase activities for some GTP binding proteins (26). a-[ P]GTP hy- Applied Biosystems (Foster City, CA) Prism DyeDeoxy Sequencing drolysis and separation of the nucleotide products by polyethyleneimine System. One end of this cDNA corresponded to the 150-base A2c clone. (PEI) chromatography were carried out as described previously (26). Based on a partial poly(A) tail present in A2c, the IGTP cDNA was Subcellular Fractionation and Western Blotting—Cells on 60-mm oriented, and a 1419-base open reading frame was identified. The DNA plates were washed three times with ice-cold PBS, gently scraped into sequence and translated protein were analyzed using Genetics Com- 0.5 ml of PBS, and pelleted by centrifugation at 500 3 g for 5 min at puter Group (Madison, WI) software. 4 °C. Next, cellular protein was fractionated by either of two methods. Plasmid Subcloning and Preparation of GST Fusion Proteins—The In one method, cells were gently resuspended in a hypotonic buffer (10 plasmid encoding GST-IGTP, pGEX/IGTP, was created by ligating a mM Tris, pH 7.5, 10 mM NaCl, 1.5 mM MgCl , 0.06 mg/ml aprotinin, 0.5 1.6-kb NcoI-XhoI fragment of the IGTP CDNA into the NcoI-XhoI site of mg/ml leupeptin, and 0.4 mM Pefabloc (Boehringer Mannheim)) and pGEX-KG (20); this produced an in-frame fusion of the GST gene and recentrifuged at 500 3 g for 5 min at 4 °C. Then the cells were resus- the IGTP cDNA. pGEX-KG and pGEX/IGTP were transformed into the pended in 0.5 ml of hypotonic buffer and incubated on ice for 5 min. The Escherichia coli strain BL21/DE3, and the GST fusion proteins were cells were lysed by passing through a 25-gauge needle attached to a purified using glutathione-Sepharose (Pharmacia) as described previ- syringe 15–20 times. The broken cell suspension was centrifuged at ously (21). 3000 3 g for 5 min at 4 °C, producing a nuclear/particulate pellet and a Interspecific Backcross Mapping—Interspecific backcross progeny cytosolic supernatant. The pellet was resuspended in 1 ml of hypotonic were generated by mating (C57BL/6J 3 Mus spretus)F females and buffer, recentrifuged at 500 3 g for 5 min, and finally resuspended in C57BL/6J males as described previously (22). A total of 205 N mice 0.5 ml of hypotonic buffer; 0.25 volume of an SDS buffer (24% (w/v) were used to map the Igtp locus (see the text for details). DNA isolation, sucrose, 0.36 M dithiothreitol, 4.8% (w/v) sodium dodecyl sulfate, 0.048 restriction enzyme digestion, agarose gel electrophoresis, and Southern M EDTA, and 0.05% (w/v) bromphenol blue) was added to the suspen- blotting were performed as described previously (23). All blots were sion. The cytosolic fraction was centrifuged at 15,000 3 g to remove prepared with Hybond-N nylon membranes (Amersham Corp.). The residual nuclei and particulates; an aliquot was removed for protein probe, a 0.28-kb EcoRI fragment of the 39-untranslated region of the quantification using the Bio-Rad assay (Hercules, CA), and 0.25 volume IGTP cDNA, was labeled with an a-[ P]dCTP random primer labeling of SDS buffer was added to the remainder. system (number 300385, Stratagene, La Jolla, CA); washing was done Alternatively, cellular protein was fractionated by gently resuspend- to a final stringency of 0.25 3 SSCP, 0.1% (w/v) SDS, 65 °C. A fragment ing the pelleted cells in a Nonidet P-40 buffer (0.5% (v/v) Nonidet P-40, of 2.5-kb was detected in SacI-digested C57BL/6J DNA, and a 3.0- or 0.06 mg/ml aprotinin, 0.5 mg/ml leupeptin, and 0.4 mM Pefabloc in PBS) 4.5-kb fragment was detected in SacI-digested M. spretus DNA. The and incubating them on ice for 5 min. The nuclear/particulate and presence or absence of the 3.0- or 4.5-kb SacI M. spretus-specific frag- cytosolic protein fractions were then separated by centrifugation as in ment was followed in backcross mice. the first fractionation method. With both methods, lysis of the cells and A description of the probes and restriction fragment length polymor- integrity of the nuclei were monitored by light microscopy. phisms (RFLPs) for Il3 and Trp53, two of the loci linked to Igtp, has 25 mg of cytosolic protein and the equivalent volume of the nuclear/ been published (24). Recombination distances were calculated using the particulate protein fraction were separated on a 9% SDS- polyacryl- computer program SPRETUS MADNESS (25). Gene order was deter- amide gel; the gel was transfered to an Immobilon-P membrane (Mil- mined by minimizing the number of recombination events required to lipore, Bedford, MA) with a TEX50 wet transfer apparatus (Hoefer, San explain the allele distribution patterns. Francisco, CA) using 25 mM Tris, 480 mM glycine, 0.1%(w/v) SDS, and Immunoprecipitation—Cells were plated on 60-mm plates and grown 50%(v/v) methanol buffer. Western blotting and detection were carried to confluence. In some experiments, the cells were labeled by washing out using the ECL Detection System (Amersham Int., Buckingham- them three times with Dulbecco’s modified Eagle’s medium without shire, UK) according to the manufacturer’s protocols. The rabbit poly- methionine (Life Technologies, Inc.) and then incubating them in the clonal anti-IGTP antiserum and the mouse monoclonal anti-b-tubulin same medium supplemented with 10% fetal calf serum (v/v) and 0.5 antibody (Sigma) were used at a 1:1000 dilutions; the peroxidase- mCi of [ S]methionine (DuPont NEN) for 4 h. Cells were exposed to 100 conjugated goat anti-mouse IgG and anti-rabbit IgG secondary antibod- units/ml mouse interferon g (Boehringer Mannheim) for various times ies (Boehringer Mannheim) were used at 1:10,000 dilutions. during the labeling period. RESULTS Total cellular protein lysates were prepared by washing the cells three times with ice-cold phosphate-buffered saline and then scraping The IGTP cDNA was first identified as a 150-base cDNA them into 0.5-ml ice-cold lysis buffer (1%(v/v) Triton X-100, 0.1%(w/v) fragment that was differentially displayed in hepatocyte sodium dodecyl sulfate, 50 mM Tris, pH 7.5, 0.15 M NaCl, 5 mM EDTA) growth factor-treated C127 mouse fibroblasts (data not shown). with plastic cell scrapers. Following sonication for 15 s in a cup-horn However, it was observed during these experiments that IGTP sonicator (Sonifier 450, Branson, Danbury, CT), the lysates were cleared by centrifugation in microcentrifuge tubes in a refrigerated mRNA levels were dramatically increased by 100 units/ml microcentrifuge (MTX-150, Tomy Seiko, Tokyo) for 10 min at 15,000 3 IFNg, in both this cell line (data not shown) and in RAW 264.7 g. The samples were matched for trichloroacetic acid-precipitated ra- mouse macrophages (Fig. 1). While basal IGTP mRNA levels dioactive counts and then incubated with normal rabbit serum on a were almost undetectable, they were easily detectable within 1hofIFNg exposure, peaking at very high levels at 3 h, and remaining at high levels to at least 48 h (Fig. 1). Longer Program manual for the Wisconsin Package, Version 8, September, 1994, Genetics Computer Group, 575 Science Dr., Madison, WI 53711. exposures of the blot showed that trace amounts of IGTP IGTP, an IFNg-induced GTPase 20401 FIG.1. Effects of interferon g and lipopolysaccharide on IGTP mRNA accumulation in RAW 264.7 cells. Cells were exposed to 100 units/ml interferon g or 1 mg/ml lipopolysaccharide for the indicated times. The cells were then used for preparation of total cellular RNA and Northern blotting with an IGTP probe or a glyceraldehyde-3-phos- phate dehydrogenase probe. The positions of the major ribosomal RNA species, as determined by the stained gel, are indicated. Other details are described in the text. FIG.2. Effect of cycloheximide on interferon g-induced accu- mulation of IGTP mRNA in RAW 264.7 cells. Cells were exposed to mRNA were present before exposure to IFNg (data not shown). 0.1 mM cycloheximide for the indicated times, to 100 units/ml interferon In RAW cells, IGTP mRNA levels were also induced by 1 mg/ml g for the indicated times, or to 0.1 mM cycloheximide for 30 min and lipopolysaccharide with similar kinetics but to a lesser extent then to 100 units/ml interferon g for the indicated times. The cells were (Fig. 1). With increased lipopolysaccharide exposure times, the then used for preparation of total cellular RNA and Northern blotting with an IGTP probe, an IP-10 probe, or a glyceraldehyde-3-phosphate mRNA showed a slightly increased mobility which could have dehydrogenase probe. The positions of the major ribosomal mRNA resulted from shortening of the poly(A) tail; however, this pos- species, as determined by the stained gel, are indicated. sibility was not addressed experimentally. The half-life of the IGTP mRNA was determined by exposing RAW cells to 100 units/ml IFNg for 3 h and then to the tran- scriptional inhibitor actinomycin D (4 mM) for various times; the decay of the accumulated mRNA was then followed by North- ern blotting. Under these conditions, the mRNA decayed with a half-life of about 4.5 h (data not shown). Inducibly expressed genes are often classified as primary response genes if induction requires only previously translated transcription factors or as secondary response genes if newly translated factors are required (for review see Ref. 27). To determine if IGTP accumulation required protein synthesis, RAW cells were exposed to 0.1 mM cycloheximide for 30 min and then to 100 units/ml IFNg; mRNA accumulation was fol- lowed by Northern blotting (Fig. 2). Inhibition of protein syn- thesis in this manner blocked the majority of the IGTP mRNA FIG.3. Expression of IGTP mRNA in various mouse tissues. accumulation, although a small amount of mRNA did accumu- Total cellular RNA was prepared from a 3-month-old male C57BL/6 late (Fig. 2). Conversely, under the same conditions, cyclohex- mouse. 15-mg samples were subjected to Northern blotting with an IGTP cDNA probe or a b-actin cDNA probe. The positions of the major imide did not affect the accumulation of IP10 mRNA, which is ribosomal mRNA species, as determined by the stained gel, are a primary IFNg response gene in macrophages (28) (Fig. 2). indicated. Therefore, because IGTP mRNA levels did not show detectable increases until 1 h following IFNg exposure, and because full induction required protein synthesis, IGTP accumulation may This interspecific backcross mapping panel has been typed for be classified as a secondary IFNg response. over 2100 loci that are well distributed among the autosomes, Hybridization of the IGTP cDNA to a total RNA blot contain- as well as the X chromosome (22). C57BL/6J and M. spretus ing RNA from several mouse tissues revealed very high expres- DNAs were digested with several enzymes and analyzed by sion in the thymus and slightly lower expression in the spleen, Southern blot hybridization for informative restriction map lung, and small intestine; expression in brain, heart, kidney, polymorphisms (RFLPs) using a fragment from the 39-untrans- liver, skeletal muscle, and testes was very low or undetectable lated region of the IGTP cDNA as probe. The 3.0- or 4.5-kb SacI (Fig. 3). The tissue expression pattern suggested that IGTP M. spretus RFLP (see “Experimental Procedures”) was used to may be highly expressed in immune cell populations. follow the segregation of the Igtp locus in backcross mice. The The mouse chromosomal location of Igtp was determined by mapping results indicated that Igtp was located in the central interspecific backcross analysis using progeny derived from region of mouse chromosome 11 linked to Il3 and Trp53. One matings of ((C57BL/6J 3 Mus spretus)F and C57BL/6J) mice. hundred and twenty-two mice were analyzed for every marker 1 20402 IGTP, an IFNg-induced GTPase (14), respectively. In IFNg-treated RAW cells, levels of the three mRNAs increased markedly with kinetics similar to the increases in IGTP mRNA levels (data not shown). In addition, each mRNA was highly expressed in mouse thymus, spleen, lung, and small intestine (data not shown). Thus, expression of IGTP and these three related cDNAs was nearly identical in several cell types and tissues. Comparison of the amino acid sequences showed that in at least three of the four proteins 36% of the amino acids were absolutely conserved or were replaced with conservative substitutions (Fig. 5). Sequence homology was found throughout the proteins and was particularly high in the regions of three conserved GTP binding motifs, GXXXXGKS/T, DXXG, and N/TKXD (29); these motifs are found in many functionally diverse GTP-binding proteins in- cluding Ras (29) and dynamin (30). In the case of the proteins encoded by IRG-47, TGTP/Mg21, or LRG-47, neither GTP bind- ing nor had any other biochemical or physiological function been determined. To facilitate the study of IGTP, rabbit polyclonal antisera were generated that recognized the carboxyl-terminal 16 amino acids of IGTP (Fig. 5). This sequence was not shared with any of the three related proteins, and the antibody that was gener- ated immunoprecipitated in vitro translated IGTP but not in vitro translated LRG-47 protein (data not shown). From the [ S]methionine-labeled protein lysates of RAW 264.7 cells, the FIG.4. Mapping of the Igtp gene to the central region of mouse antibody immunoprecipitated a dominant band of about 48 chromosome 11 by interspecific backcross analysis. Igtp was kDa (Fig. 6) that probably represented IGTP. This band had placed on mouse chromosome 11 by interspecific backcross analysis. the same relative migration as that of in vitro translated IGTP The segregation pattern of Igtp and the flanking genes in 122 backcross animals that were typed for all loci is shown at the top of the figure. (data not shown) and was not precipitated by preimmune rab- Each column represents the chromosomes identified in the backcross bit serum (Fig. 6). In addition, the band was weak in cells that progeny that was inherited from the (C57BL/6J 3 M. spretus)F parent. had not been exposed to IFNg, and its intensity increased Shaded boxes represent the presence of the C57BL/6J allele, and white markedly following IFNg stimulation, reaching peak levels in boxes represent the presence of an M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of 3–4 h (Fig. 6). Two other bands of about 55–60 kDa were also each column. A partial chromosome 11 linkage map showing the loca- immunoprecipitated by the IGTP antibody but not by preim- tion of Igtp is shown at the bottom of the figure. Recombination dis- mune serum. These bands could have been proteins that asso- tances between loci in centimorgans are shown to the left. The map ciated with IGTP and were coprecipitated; however, because positions of loci in human, where known, are shown to the right. (References for the human map positions can be obtained from the the immunoprecipitation was done in the presence of SDS and Genome Data Base, a computerized data base of human linkage infor- because the intensities of the bands did not fluctuate with the mation maintained by the William H. Welch Medical Library, The intensity of the IGTP band, it seemed more likely that they Johns Hopkins University, Baltimore, MD.) were unrelated proteins that were also recognized by the anti- body. In Western blotting experiments using the same anti- and are shown in the segregation analysis (Fig. 4). Each locus body, the 48-kDa IGTP was not detectable in RAW cells that was analyzed in pairwise combinations for recombination fre- had not been exposed to IFNg but was easily detectable in cells quencies using the additional data. The ratios of the total that had been exposed to IFNg for4hor longer (Fig. 9 and data number of mice exhibiting recombinant chromosomes to the not shown). total number of mice analyzed for each pair of loci and the most To determine whether IGTP was able to hydrolyze GTP to likely gene order were as follows: centromere-II3-4/122-Igtp- GDP, as suggested by the presence of putative GTP binding 9/122-Trp53. The recombination frequencies (expressed as ge- sites, protein that had been immunoprecipitated from RAW netic distances in centimorgans (cM) 6 S.E.) were II3-3.3 6 cells was incubated with a-[ P]GTP for various times, and the 6.6-Igtp-7.4 6 2.4-Trp53. radiolabeled nucleotides were separated by thin layer chroma- Based on the high IGTP mRNA levels in spleen, a spleen tography (Fig. 7). Very little a-[ P]GTP was converted to GDP cDNA library was chosen for screening with the original 150- by protein immunoprecipitated by preimmune rabbit serum base IGTP cDNA fragment. Several positive clones were ob- from either cells incubated under control conditions or cells tained, one of which was 1927 bases and contained a 1419-base exposed to IFNg for 4 h (Fig. 7). Similarly, very little GTPase open reading frame between bases 204 and 1619 (GenBank activity was immunoprecipitated by the IGTP antibody from accession U53219); the clone contained no other open reading cells not exposed to IFNg; however, greater than four times frames longer than 200 bases. The first potential translation more GTPase activity was precipitated with the IGTP antibody initiation codon within the long open reading frame occurred at from cells that had been exposed to IFNg for 4 h (Fig. 7). The base 348, and translation from this codon produced a 424- increase in GTPase activity was probably due to the increased amino acid polypeptide with a calculated M of 48,507 and a pI of 7.79. The translated sequence showed substantial homology amounts of IGTP that are present in IFNg-stimulated RAW cells (Fig. 6), which suggested that IGTP was a GTPase. Alter- to the translated sequences of three previously cloned cDNAs: IRG-47 (11), TGTP (12)/Mg21 (13), and LRG-47 (14) (Fig. 5). natively, it was also possible that another IFNg-inducible fac- tor that had GTPase activity, or a factor that was able to These cDNAs were cloned as cDNAs whose expression was increased by IFNg in 70Z/3 pre-B cells (11), by T cell receptor catalyze the IGTP GTPase activity, was also present in the 1 1 cross-linking in CD4 8 thymocytes (12) and IFNg in perito- immune pellet from IFNg-stimulated cells. Similar experi- neal macrophages (13), or by IFNg in RAW 264.7 macrophages ments were performed using an antibody that immunoprecipi- IGTP, an IFNg-induced GTPase 20403 FIG.5. Alignment of the translated IGTP, LRG-47, IRG-47, and TGTP/Mg21 protein sequences. Alignments were done using the program pileup (Genetics Computer Group, Madison, WI). Amino acid positions are numbered at the right. Residues that were absolutely conserved or showed conservative substitutions in three of the four proteins are placed in boxes, and residues that are part of the GTP binding motifs are indicated with asterisks (*). blotting (Fig. 9). In some experiments, protein fractions were prepared by briefly incubating the cells in a hypotonic buffer, disrupting the cytosolic membranes by drawing the cells through a 25-gauge needle, and then separating nuclei and particulates from the cytosolic protein fraction by centrifuga- tion. Using this procedure, IGTP was readily detected in the nuclear/particulate protein fraction of IFNg-treated cells but not in the cytosolic protein fraction (Fig. 9). Conversely, when the cytosolic membranes were lysed by incubating the cells in an isotonic buffer containing 0.5% (v/v) Nonidet P-40, IGTP localized to the cytosolic protein fraction (Fig. 9). With both procedures, the nuclei appeared intact as determined by light microscopy (data not shown), and b-tubulin was detected only in the cytosolic protein fractions (Fig. 9). These data raised the possibility that IGTP may have been a cytosolic protein that was loosely associated with membranes in the nuclear/partic- FIG.6. Time course of IGTP accumulation in RAW 264.7 cells ulate fractions and that this association was disrupted by mild exposed to interferon g. Cells were exposed to [ S]methionine for 4 h and to 100 units/ml interferon g for the indicated times during the treatment with a nonionic detergent such as Nonidet P-40. labeling period. Cell lysates were prepared and used for immunopre- Cytosolic localization of IGTP was also supported by prelimi- cipitation with the IGTP antibody; the precipitated proteins were sep- nary results using the IGTP antibody to immunostain IFNg- arated on a 9% SDS-polyacrylamide gel. The positions of selected mo- treated RAW cells (data not shown). lecular mass markers are shown at the left. Other details are described in the text. DISCUSSION We report here the identification of a 48-kDa GTPase, IGTP, tated the LRG-47 protein, and, again, increased GTPase activ- whose expression was rapidly and markedly increased by IFNg ity was detected in immune pellets from IFNg-treated cells, in macrophages and fibroblasts. Its expression pattern sug- relative to control cells (data not shown). Taken together, these gested that IGTP could potentially mediate some IFNg-induced data suggest that IGTP, LRG-47, and possibly the other related responses in macrophages and fibroblasts, and its high expres- proteins are GTPases. sion in mouse thymus and spleen suggested that it may also In similar experiments, IGTP was expressed in bacteria as a function in other immune cell populations. glutathione S-transferase (GST) fusion protein and, following We mapped the Igtp gene to a location on mouse chromosome partial purification by glutathione affinity chromatography 11 between Il3 and Trp53. We compared our interspecific map (data not shown), was used in a-[ P]GTP hydrolysis assays of chromosome 11 with a composite mouse linkage map that (Fig. 8). The GST-IGTP fusion protein converted about six reports the location of many uncloned mouse mutations ; Igtp times more GTP to GDP than was converted by an equivalent mapped in a region of the composite map that lacks mutations amount of GST protein (Fig. 8). This result suggested that the with a phenotype that might be expected for an alteration at IGTP GTPase activity was inherent to the protein and did not this locus. require other mammalian accessory proteins for basal activity. The central region of mouse chromosome 11 shares homology To determine the subcellular distribution of IGTP, RAW cells to regions of human chromosomes 5q and 17p, and thus, the were exposed to IFNg for4hto stimulate synthesis of the protein and then cellular protein was separated into cytosolic Compiled by M. T. Davisson, T. H. Roderick, A. L. Hillyard, D. P. and nuclear/particulate fractions; the presence of IGTP and the Doolittle and provided by GBASE, a computerized data base main- cytosolic protein b-tubulin (31) was determined by Western tained at the Jackson Laboratory, Bar Harbor, ME. 20404 IGTP, an IFNg-induced GTPase FIG.7. GTPase activity of immunoprecipitated IGTP. RAW 264.7 cells were exposed to control conditions or to 100 units/ml inter- feron g (IFNg) for 4 h, and the cells were then used for immunoprecipi- FIG.8. GTPase activity of a GST-IGTP fusion protein. Bacteri- tation with preimmune serum or anti-IGTP serum. a-[ P]GTP was ally expressed GST and GST-IGTP fusion proteins were partially puri- incubated with the immunoprecipitated proteins or with buffer at 37 °C fied using glutathione affinity chromatography. a-[ P]GTP was incu- for the indicated times, and the nucleotide products were separated by bated with 10 mg of the purified proteins or bovine serum albumin polyethyleneimine thin layer chromatography. A, the chromatogram (BSA) at 37 °C for the indicated times, and the nucleotide products were was used for autoradiography, and the positions of GTP and GDP were separated by polyethyleneimine thin layer chromatography. A, the determined using the fluorescent indicator in the PEI-cellulose plates. chromatogram was used for autoradiography, and the positions of GTP B, the amounts of radioactive GTP and GDP were determined with a and GDP were determined using the fluorescent indicator in the PEI- phosphorimager, and the ratio of GDP to GTP was plotted as a function cellulose plates. B, the amounts of radioactive GTP and GDP were of time. Other details are described in the text. M, buffer; L, control, determined with a phosphorimager, and the ratio of GDP to GTP was preimmune; E, control, anti-IGTP; ‚, IFN, preimmune; µ, IFN, plotted as a function of time. Other details are described in the text. M, anti-IGTP. BSA; L, GST; Q, GST-IGTP. human IGTP gene is likely to reside on 5q or 17p. Interestingly, monosomy for chromosome 5 or 5q deletions are frequently associated with myelodysplastic disorders, myeloproliferative syndromes, and acute myeloid leukemia (32, 33). Because Igtp is expressed in myeloid cells, it will be of interest to determine if the human IGTP gene maps to the region on chromosome 5 thought to contain this important myeloid tumor suppressor gene (32). The IGTP protein contained three GTP-binding motifs, GXXXXGKS/T, DXXG, and NKXD (29), and the protein had inherent GTPase activity. Although many other GTP-binding proteins have been identified, they have diverse cellular func- tions, and the presence of this biochemical activity in itself does not suggest a cellular function for IGTP. However, the exten- sive information gained in the biochemical studies of Ras and other GTP-binding proteins may facilitate study of IGTP. For instance, the x-ray crystal structure of Ras (34, 35) has shown that the first and second GTP-binding motifs both contact the phosphates of the GTP molecule and a Mg cofactor, whereas FIG.9. Subcellular localization of IGTP in RAW 264.7 cells. the third motif contacts the purine ring. Mutation of serine 17 Cellular protein was fractionated into nuclear/particulate (N/P) and in the first GTP-binding motif to asparagine blocks the ability cytosolic (Cy) fractions by lysis of the cells by incubation in a hypotonic buffer followed by passage through a 25-gauge needle (A) or by lysis of of Ras to hydrolyze GTP, and expression of this Ras mutant in the cells by incubation in a buffer containing 0.5% Nonidet P-40 (B). cells blocks the function of wild-type Ras (36). The analogous Fractionated protein was used for Western blotting with anti-IGTP and mutation in dynamin, another GTP-binding protein, also has a anti-b-tubulin antibodies. The positions of selected molecular mass dominant-negative effect (37, 38), and this may well be true of markers are indicated at the left. Other details are described in the text. IGTP, an IFNg-induced GTPase 20405 Acknowledgments—We thank Marilyn Powers for automated se- IGTP. Such a dominant negative mutant would be useful in quencing, Terry Copeland for generation of the rabbit polyclonal anti- determining the cellular function of IGTP. bodies, Debbie Gilbert for technical assistance, and Marianne Oskars- The IGTP amino acid sequence was very similar to the trans- son and Kenji Fukasawa for helpful discussions. lated sequences of three previously cloned cDNAs, IRG-47 (11), REFERENCES TGTP (12)/Mg21 (13), and LRG-47(14), each of which contained 1. Farrar, M. A., and Screiber, R. D. (1993) Annu. Rev. Immunol. 11, 571–611 three GTP-binding motifs and had molecular masses of about 2. Vilcek, J., Gray, P. W., Rinderknect, E., and Sevastopoulos, C. G. (1985) 47 kDa. Moreover, the increases in IRG-47, TGTP/Mg21, and Lymphokines 11, 1–32 3. Handa, K., Suzuki, R., Matsui, H., Shimizu, Y., and Kumagai, K. (1983) LRG-47 mRNA levels in IFNg-treated RAW macrophages, and J. Immunol. 130, 988–992 their expression patterns in mouse tissues, paralleled those of 4. King, D. P., and Jones, P. P. (1983) J. Immunol. 131, 315–318 5. Warren, M. K., and Vogel, S. N. (1985) J. Immunol. 134, 2462–2469 IGTP mRNA. Therefore, it seems likely that IGTP is represent- 6. Liew, F. Y., Li, Y., and Millott, S. (1990) J. Immunol. 145, 4306–4310 ative of a new family of proteins whose expression is regulated 7. Ding, A. H., Nathan, C. F., and Stuehr, D. J. (1988) J. Immunol. 141, similarly and whose biochemical functions are similar. The 2407–2412 8. Beutler, B., Tkacenko, V., Milsark, I., Krochin, N., and Cerami, A. (1986) J. levels at which protein expression and biochemical function Exp. Med. 164, 1791–1796 differ will be the subject of future studies. 9. Schreiber, R. D., Hicks, L. J., Celada, A., Buchmeier, N. A., and Gray, P. W. (1985) J. Immunol. 134, 1609–1618 At least two other groups of interferon-induced GTP-binding 10. Munro, J. M., Pober, J. S., and Cotran, R. S. (1989) Am. J. Pathol. 135, proteins exist. The first of these is comprised of the Mx pro- 121–131 teins, a group of 72-kDa proteins involved in the interferon- 11. Gilly, M., and Wall, R. (1992) J. Immunol. 148, 3275–3281 12. Carlow, D. A., Marth, J., Clark-Lewis, I., and Teh, H.-S. (1995) J. Immunol. induced inhibition of viral proliferation (39). The second group 154, 1724–1734 includes several 65–67-kDa proteins that bind to GTP-agarose 13. LaFuse, W. P., Brown, D., Castle, L., and Zwilling, B. S. (1995) J. Leuk. Biol. 57, 477–483 but whose function is unknown (40, 41). However, because 14. Sorace, J. M., Johnson, R. J., Howard, D. L., and Drysdale, B. E. (1995) J. little sequence similarity exists between IGTP and either of Leuk. Biol. 58, 477–484 these groups of proteins, other than that found in the GTP- 15. Rong, S., Oskarsson, M., Faletto, D. L., Tsarfaty, I., Resau, J., Nakamura, T., Rosen, E., Hopkins, R., and Vande Woude, G. F. (1993) Cell Growth & binding motifs, no functional comparisons can be drawn. Differ. 4, 563–569 Future experiments will focus on determining the physiolog- 16. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156–159 17. Taylor, G. A., and Blackshear, P. J. (1995) J. Cell. Physiol. 162, 378–387 ical function of IGTP. Among the many possible functions of the 18. Stumpo, D. J., Graff, J. M., Albert, K. A., Greengard, P., and Blackshear, P. J. protein are mediation of IFNg signal transduction at some level (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4012–4016 and regulation of some of the multiple IFNg-induced pheno- 19. Tso, J. Y., Sun, X. H., Kao, T.-h., Reece, K. S., and Wu, R. (1985) Nucleic Acids Res. 13, 2485–2502 typic responses. It seems unlikely that IGTP is involved in any 20. Guan, K., and Dixon, J. E. (1991) Anal. Biochem. 192, 262–267 of the immediate signal transduction events stimulated by 21. Smith, D. B. (1991) Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and IFNg, given that its expression is not detectable until 1 h Struhl, K., eds) pp. 16.7.1–16.7.8, John Wiley and Sons, New York following stimulation. In addition, it seems unlikely that IGTP 22. Copeland, N. G., and Jenkins, N. A. (1991) Trends Genet. 7, 113–118 could have any direct effects on gene expression, as it is prob- 23. Jenkins, N. A., Copeland, N. G., Taylor, B. A., and Lee, B. K. (1982) J. Virol. 43, 26–36 ably not located in the cell nucleus. However, it is tempting to 24. Buchberg, A. M., Bedigan, H. G., Taylor, B. A., Brownwell, E., Ihle, J. N., speculate that because of the large increases in protein proc- Nagata, S., Jenkins, N. A., and Copeland, N. G. (1988) Oncogene Res. 2, 149–165 essing associated with cytokine secretion and cell surface pro- 25. Green, E. L. (1981) Genetics and Probability in Animal Breeding Experiments, tein expression in IFNg-treated cells (1), IGTP might be in- pp. 77–113, Oxford University Press, New York volved in protein processing. One GTP-binding protein that is 26. Vater, C. A., Raymond, C. K., Ekena, K., Howald-Stevenson, I., and Stevens, T. H. (1992) J. Cell Biol. 119, 773–786 involved in protein processing is SRP54, the 54-kDa component 27. Herschman, H. R. (1991) Annu. Rev. Biochem. 60, 281–319 of the signal recognition particle (SRP) that targets ribosomes 28. Luster, A. D., Unkeless, J. C., and Ravetch, J. V. (1985) Nature 315, 672–676 29. Dever, T. E., Glynias, M. J., and Merrick, W. C. (1987) Proc. Natl. Acad. Sci. synthesizing proteins with a signal sequence to the endoplas- U. S. A. 84, 1814–1818 mic reticulum (for review see Ref. 42). One model of SRP54 30. Obar, R. A., Collins, C. A., Hammerback, J. A., Shpetner, H. S., and Vallee R. function (43) suggests that while in the GTP-bound state, the B. (1990) Nature 347, 256–261 31. Kirschner, M. W. (1979) Int. Rev. Cytol. 54, 1–71 SRP54 component of SRP recognizes a ribosome from which the 32. LeBeau, M. M., Espinosa, R., Newman, W. L., Stock, W., Roulston, D., Larson, nascent signal sequence of a presecretory protein has just R. A., Keinaren, M., and Westbrook, C. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5484–5488 emerged, then binds this complex, and targets it to the signal 33. Boultwood, J., Fidler, C., Lewis, S., Kelly, S., Sheridan, H., Littlewood, T. J., recognition receptor complex on the surface of the endoplasmic Buckle, V. J., and Wainscoat, J. S. (1994) Genomics 19, 425–432 reticulum. Once transfer to the endoplasmic reticulum is com- 34. Pai, E. F., Krengel, U., Petsko, G. A., Goody, R. S., Kabsch, W., and Witting- hofer, A. (1990) EMBO J. 9, 2351–2359 plete, the GTP is somehow hydrolyzed catalyzing transfer of 35. Pai, E. F., Kabsch, W., Krengel, U., Holmes, K. C., John, J., and Wittinghofer, the signal sequence to the translocon and release of the SRP A. (1989) Nature 341, 209–214 36. Sigal, I. S., Gibbs, J. B., D’Alonzo, J. S., Temeles, G. L., Wolanski, B. S., Socher, with the SRP54 component in the GDP-bound state (43). Be- S. H., and Scolnick, E. M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 952–956 cause IGTP shows some similarities to mouse SRP54 (44), it is 37. Herskovits, J. S., Burgess, C. C., Obar, R. A., and Vallee, R. B. (1993) J. Cell possible that IGTP could have a similar function. At the amino Biol. 122, 565–578 38. van der Bliek, A. M., Redelmeier, T. E., Damle, H., Tisdale, E. J., Meyerwitz, acid level, the two proteins are about 15% identical and 43% E. M., and Schmid, S. L. (1993) J. Cell Biol. 122, 553–563 similar; this includes residues in the GTP-binding motifs and 39. 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High, S., and Dobberstein, B. (1991) J. Cell Biol. 113, 229–234 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Identification of a Novel GTPase, the Inducibly Expressed GTPase, That Accumulates in Response to Interferon γ

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 34, Issue of August 23, pp. 20399–20405, 1996 Printed in U.S.A. Identification of a Novel GTPase, the Inducibly Expressed GTPase, That Accumulates in Response to Interferon g* (Received for publication, April 16, 1996, and in revised form, May 31, 1996) Gregory A. Taylor, Michael Jeffers, David A. Largaespada‡, Nancy A. Jenkins, Neal G. Copeland, and George F. Vande Woude§ From the ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 factor a, markedly increases expression of major histocompat- Interferon g is a pleiotropic cytokine that regulates many immune functions. We have identified a novel pro- ibility class I molecules and the cell adhesion molecules, tein, inducibly expressed GTPase (IGTP), whose expres- ICAM-1 and ELAM-1, thereby promoting recruitment of im- sion was regulated by interferon g in macrophages. In mune cells to areas of inflammation (10). These pleiotropic mouse RAW 264.7 macrophages, IGTP mRNA levels were responses are initiated by binding of IFNg to its receptor, almost undetectable but increased within1hof expo- followed by receptor dimerization and a cascade of primary and sure to interferon g, peaked at very high levels within secondary events (1). However, these molecular events have 3 h, and remained at high levels to at least 48 h; pretreat- not been completely defined, and identification of proteins ment of the cells with cycloheximide blocked the major- whose expression is regulated by IFNg is the subject of active ity of mRNA accumulation. In the mouse, the mRNA was investigation. highly expressed in thymus, spleen, lung, and small in- We report here the identification of a 48-kDa protein, desig- testine. Using interspecific backcross analysis, the Igtp nated IGTP, whose expression was rapidly and dramatically gene was mapped to mouse chromosome 11. The IGTP increased by IFNg in macrophages and fibroblasts. The protein cDNA encoded a putative polypeptide of M 48,507 and was first recognized as the product of a differentially displayed pI 7.79 that contained three consensus GTP binding mo- cDNA in hepatocyte growth factor (HGF)-treated C127 mouse tifs, GXXXXGK(S/T), DXXG, and NTKXD. Both IGTP that fibroblasts. In these cells, the IGTP mRNA levels showed a had been immunoprecipitated from RAW cells and a slight transient increase following HGF exposure; in addition, glutathione S-transferase IGTP fusion protein were able mRNA levels were constitutively elevated in C127 and NIH/ to convert GTP to GDP in vitro. Subcellular protein 3T3 cells that were transformed by coexpression of HGF and its fractionation and Western blotting localized IGTP to the receptor Met. These observations and their significance will be cytosol of RAW cells. In addition, the protein was homol- ogous to proteins encoded by three previously cloned discussed elsewhere. In the present work, we characterize the cDNAs, IRG-47, TGTP/Mg21, and LRG-47, and thus may IFNg-induced IGTP gene expression, and we demonstrate that be representative of a new family of interferon g-regu- the protein product is a GTPase. Because of its expression lated GTPases. pattern and its biochemical activity, the protein has been des- ignated inducibly-expressed GTPase (IGTP). The protein had high sequence homology with the proteins encoded by three Interferon g (IFNg) is a 20-kDa protein that regulates a other cDNAs, IRG-47 (11), TGTP(12)/Mg21(13), and LRG-47(14), wide variety of immunological and inflammatory processes (for for which no biochemical or physiological function had been de- review see Ref. 1). While production of IFNg is limited to CD8 scribed previously, but which also contained putative GTP binding T cells, some CD4 T cell subsets (2), and natural killer cells sequences. IGTP, therefore, may be representative of a new family (3), its receptor is found on almost all cell types where it elicits of GTPases that could potentially mediate the effects of IFNg in many diverse physiological responses. In macrophages for ex- macrophages and other cells. ample, IFNg induces major histocompatibility class II expres- EXPERIMENTAL PROCEDURES sion (4), increases F receptor-mediated phagocytosis (5), and Cells and Culture—C127 cells (ATCC CRL-1616, American Type mediates removal of neoplastic cells and virally and parasiti- Culture Collection), Met-transfected C127 cells (C127/Met), and RAW cally infected cells by initiating secretion of cytosidal com- 264.7 cells (ATCC TIB-71) were maintained in Dulbecco’s modified pounds and tumor necrosis factor a (6–9). In endothelial cells Eagle’s medium (Life Technologies, Inc.) supplemented with 10% (v/v) on the other hand, IFNg, in combination with tumor necrosis fetal bovine serum (Life Technologies, Inc.), 0.292 mg/ml L-glutamine (Life Technologies, Inc.), 100 units/ml penicillin (Life Technologies, Inc.), and 100 units/ml streptomycin (Life Technologies, Inc.). Hepato- * This research is sponsored by the National Cancer Institute, cyte growth factor was purified from the supernatant of transformed DHHS, under contract with ABL. The costs of publication of this article NIH/3T3 cells that overproduce the factor (15); it was added directly to were defrayed in part by the payment of page charges. This article must the growth medium of C127 cells at 200 units/ml. Recombinant mouse therefore be hereby marked “advertisement” in accordance with 18 interferon g (Boehringer Mannheim) and lipopolysaccharide (serotype U.S.C. Section 1734 solely to indicate this fact. 055:B5, Sigma) were added directly to the culture media of C127 and ‡ Leukemia Society of America Fellow. RAW 264.7 cells at the concentrations indicated in the text. § To whom correspondence should be addressed: ABL-Basic Research Northern Blotting—Total RNA was prepared from cultured cells (16) Program, NCI-Frederick Cancer Research & Development Center, P. O. and from mouse tissues (17) using standard acidic phenol/chloroform Box B, Frederick, MD 21702. Tel.: 301-846-1584; Fax: 301-846-5038. extraction protocols. 15 mg of RNA samples were separated on 1.2% The abbreviations used are: IFNg, interferon g; HGF, hepatocyte agarose/formaldehyde gels and used for Northern blot analysis as de- growth factor; GST, glutathione S-transferase; IGTP, inducibly ex- pressed GTPase; PBS, phosphate-buffered saline; PEI, polyethylenei- mine; kb, kilobase pair(s); SRP, signal recognition particle; RFLPs, restriction fragment length polymorphisms; CHAPSO, 3-[(3-cholamido- M. Jeffers, S. Rong, M. Oskarsson, M. Anver, and G. F. Vande propyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid. Woude, submitted for publication. This is an open access article under the CC BY license. 20399 20400 IGTP, an IFNg-induced GTPase scribed previously (18). The blots were probed with a P-labeled human rotator at 4 °C for 1 h, and with 6 mg of protein A-Sepharose (Pharma- glyceraldehyde-phosphate dehydrogenase probe isolated as a 1.2-kb cia, Uppsala, Sweden) for an additional hour at 4 °C. Following a 2-min PstI fragment of pHcGAP (19), a mouse IP10 probe isolated as a 0.5-kb centrifugation at 500 3 g to pellet the protein A-Sepharose, the super- EcoRI fragment of the mouse C7–1 (ATCC 63135), a human b-actin natant was transfered to a new microcentrifuge tube and incubated for cDNA probe (Clontech; supplied with product 7760–1), or an IGTP 3–4 h with anti-IGTP polyclonal rabbit serum that had been generated probe isolated as a 0.28-kb EcoRI fragment of the IGTP cDNA (bases against the peptide CSLRKALKDSVLPPEIH-OH corresponding to the 1645–1927, GenBank accession U53219). The IGTP probe contained 16 carboxyl-terminal amino acids of IGTP. Next, 6 mg of protein A- entirely 39-untranslated sequences that were not conserved with the Sepharose were added, and the slurry was incubated for an additional related cDNAs referred to in the text; thus the probe was specific for 1 h at 4 °C. Finally, the protein A-Sepharose was washed four times IGTP. with ice-cold lysis buffer and 0.1 ml of SDS buffer (24% (w/v) sucrose, Differential Display Screening and Library Screening—C127/Met 0.36 M dithiothreitol, 0.048 M EDTA, 4.8% (w/v) sodium dodecyl sulfate, cells were exposed to 200 units/ml hepatocyte growth factor for various 0.05% (w/v) bromphenol blue) that had been diluted 1:3 with PBS was times, and total RNA was isolated and used for differential display added; the suspension was mixed vigorously and boiled for 5 min, and screening with an RNAimage kit 1 (GenHunter Corp., Brookline, MA) the resin was pelleted by centrifugation for 5 min at 15,000 3 g. The according to the protocols supplied by the manufacturer. One 150-base proteins in the SDS buffer were separated by 9% SDS-polyacrylamide cDNA fragment, designated A2c, was isolated using primers H-T A gel electrophoresis. and H-AP2; levels of A2c were slightly increased in cDNA generated GTP Hydrolysis Assays—Protein was immunoprecipitated from cell from C127/Met cells exposed to HGF for 3–8 h. The cDNA fragment was lysates as described above, except that the cells were lysed in a lysis used to screen a mouse spleen cDNA library (Stratagene, La Jolla, CA), buffer of 10 mM CHAPSO, 50 mM Tris, pH 8.0, and 0.15 M NaCl; from which a 1927-base cDNA clone was isolated and later designated immunoprecipitation using this buffer results in slightly higher as the IGTP cDNA. The clone was sequenced on both strands using the GTPase activities for some GTP binding proteins (26). a-[ P]GTP hy- Applied Biosystems (Foster City, CA) Prism DyeDeoxy Sequencing drolysis and separation of the nucleotide products by polyethyleneimine System. One end of this cDNA corresponded to the 150-base A2c clone. (PEI) chromatography were carried out as described previously (26). Based on a partial poly(A) tail present in A2c, the IGTP cDNA was Subcellular Fractionation and Western Blotting—Cells on 60-mm oriented, and a 1419-base open reading frame was identified. The DNA plates were washed three times with ice-cold PBS, gently scraped into sequence and translated protein were analyzed using Genetics Com- 0.5 ml of PBS, and pelleted by centrifugation at 500 3 g for 5 min at puter Group (Madison, WI) software. 4 °C. Next, cellular protein was fractionated by either of two methods. Plasmid Subcloning and Preparation of GST Fusion Proteins—The In one method, cells were gently resuspended in a hypotonic buffer (10 plasmid encoding GST-IGTP, pGEX/IGTP, was created by ligating a mM Tris, pH 7.5, 10 mM NaCl, 1.5 mM MgCl , 0.06 mg/ml aprotinin, 0.5 1.6-kb NcoI-XhoI fragment of the IGTP CDNA into the NcoI-XhoI site of mg/ml leupeptin, and 0.4 mM Pefabloc (Boehringer Mannheim)) and pGEX-KG (20); this produced an in-frame fusion of the GST gene and recentrifuged at 500 3 g for 5 min at 4 °C. Then the cells were resus- the IGTP cDNA. pGEX-KG and pGEX/IGTP were transformed into the pended in 0.5 ml of hypotonic buffer and incubated on ice for 5 min. The Escherichia coli strain BL21/DE3, and the GST fusion proteins were cells were lysed by passing through a 25-gauge needle attached to a purified using glutathione-Sepharose (Pharmacia) as described previ- syringe 15–20 times. The broken cell suspension was centrifuged at ously (21). 3000 3 g for 5 min at 4 °C, producing a nuclear/particulate pellet and a Interspecific Backcross Mapping—Interspecific backcross progeny cytosolic supernatant. The pellet was resuspended in 1 ml of hypotonic were generated by mating (C57BL/6J 3 Mus spretus)F females and buffer, recentrifuged at 500 3 g for 5 min, and finally resuspended in C57BL/6J males as described previously (22). A total of 205 N mice 0.5 ml of hypotonic buffer; 0.25 volume of an SDS buffer (24% (w/v) were used to map the Igtp locus (see the text for details). DNA isolation, sucrose, 0.36 M dithiothreitol, 4.8% (w/v) sodium dodecyl sulfate, 0.048 restriction enzyme digestion, agarose gel electrophoresis, and Southern M EDTA, and 0.05% (w/v) bromphenol blue) was added to the suspen- blotting were performed as described previously (23). All blots were sion. The cytosolic fraction was centrifuged at 15,000 3 g to remove prepared with Hybond-N nylon membranes (Amersham Corp.). The residual nuclei and particulates; an aliquot was removed for protein probe, a 0.28-kb EcoRI fragment of the 39-untranslated region of the quantification using the Bio-Rad assay (Hercules, CA), and 0.25 volume IGTP cDNA, was labeled with an a-[ P]dCTP random primer labeling of SDS buffer was added to the remainder. system (number 300385, Stratagene, La Jolla, CA); washing was done Alternatively, cellular protein was fractionated by gently resuspend- to a final stringency of 0.25 3 SSCP, 0.1% (w/v) SDS, 65 °C. A fragment ing the pelleted cells in a Nonidet P-40 buffer (0.5% (v/v) Nonidet P-40, of 2.5-kb was detected in SacI-digested C57BL/6J DNA, and a 3.0- or 0.06 mg/ml aprotinin, 0.5 mg/ml leupeptin, and 0.4 mM Pefabloc in PBS) 4.5-kb fragment was detected in SacI-digested M. spretus DNA. The and incubating them on ice for 5 min. The nuclear/particulate and presence or absence of the 3.0- or 4.5-kb SacI M. spretus-specific frag- cytosolic protein fractions were then separated by centrifugation as in ment was followed in backcross mice. the first fractionation method. With both methods, lysis of the cells and A description of the probes and restriction fragment length polymor- integrity of the nuclei were monitored by light microscopy. phisms (RFLPs) for Il3 and Trp53, two of the loci linked to Igtp, has 25 mg of cytosolic protein and the equivalent volume of the nuclear/ been published (24). Recombination distances were calculated using the particulate protein fraction were separated on a 9% SDS- polyacryl- computer program SPRETUS MADNESS (25). Gene order was deter- amide gel; the gel was transfered to an Immobilon-P membrane (Mil- mined by minimizing the number of recombination events required to lipore, Bedford, MA) with a TEX50 wet transfer apparatus (Hoefer, San explain the allele distribution patterns. Francisco, CA) using 25 mM Tris, 480 mM glycine, 0.1%(w/v) SDS, and Immunoprecipitation—Cells were plated on 60-mm plates and grown 50%(v/v) methanol buffer. Western blotting and detection were carried to confluence. In some experiments, the cells were labeled by washing out using the ECL Detection System (Amersham Int., Buckingham- them three times with Dulbecco’s modified Eagle’s medium without shire, UK) according to the manufacturer’s protocols. The rabbit poly- methionine (Life Technologies, Inc.) and then incubating them in the clonal anti-IGTP antiserum and the mouse monoclonal anti-b-tubulin same medium supplemented with 10% fetal calf serum (v/v) and 0.5 antibody (Sigma) were used at a 1:1000 dilutions; the peroxidase- mCi of [ S]methionine (DuPont NEN) for 4 h. Cells were exposed to 100 conjugated goat anti-mouse IgG and anti-rabbit IgG secondary antibod- units/ml mouse interferon g (Boehringer Mannheim) for various times ies (Boehringer Mannheim) were used at 1:10,000 dilutions. during the labeling period. RESULTS Total cellular protein lysates were prepared by washing the cells three times with ice-cold phosphate-buffered saline and then scraping The IGTP cDNA was first identified as a 150-base cDNA them into 0.5-ml ice-cold lysis buffer (1%(v/v) Triton X-100, 0.1%(w/v) fragment that was differentially displayed in hepatocyte sodium dodecyl sulfate, 50 mM Tris, pH 7.5, 0.15 M NaCl, 5 mM EDTA) growth factor-treated C127 mouse fibroblasts (data not shown). with plastic cell scrapers. Following sonication for 15 s in a cup-horn However, it was observed during these experiments that IGTP sonicator (Sonifier 450, Branson, Danbury, CT), the lysates were cleared by centrifugation in microcentrifuge tubes in a refrigerated mRNA levels were dramatically increased by 100 units/ml microcentrifuge (MTX-150, Tomy Seiko, Tokyo) for 10 min at 15,000 3 IFNg, in both this cell line (data not shown) and in RAW 264.7 g. The samples were matched for trichloroacetic acid-precipitated ra- mouse macrophages (Fig. 1). While basal IGTP mRNA levels dioactive counts and then incubated with normal rabbit serum on a were almost undetectable, they were easily detectable within 1hofIFNg exposure, peaking at very high levels at 3 h, and remaining at high levels to at least 48 h (Fig. 1). Longer Program manual for the Wisconsin Package, Version 8, September, 1994, Genetics Computer Group, 575 Science Dr., Madison, WI 53711. exposures of the blot showed that trace amounts of IGTP IGTP, an IFNg-induced GTPase 20401 FIG.1. Effects of interferon g and lipopolysaccharide on IGTP mRNA accumulation in RAW 264.7 cells. Cells were exposed to 100 units/ml interferon g or 1 mg/ml lipopolysaccharide for the indicated times. The cells were then used for preparation of total cellular RNA and Northern blotting with an IGTP probe or a glyceraldehyde-3-phos- phate dehydrogenase probe. The positions of the major ribosomal RNA species, as determined by the stained gel, are indicated. Other details are described in the text. FIG.2. Effect of cycloheximide on interferon g-induced accu- mulation of IGTP mRNA in RAW 264.7 cells. Cells were exposed to mRNA were present before exposure to IFNg (data not shown). 0.1 mM cycloheximide for the indicated times, to 100 units/ml interferon In RAW cells, IGTP mRNA levels were also induced by 1 mg/ml g for the indicated times, or to 0.1 mM cycloheximide for 30 min and lipopolysaccharide with similar kinetics but to a lesser extent then to 100 units/ml interferon g for the indicated times. The cells were (Fig. 1). With increased lipopolysaccharide exposure times, the then used for preparation of total cellular RNA and Northern blotting with an IGTP probe, an IP-10 probe, or a glyceraldehyde-3-phosphate mRNA showed a slightly increased mobility which could have dehydrogenase probe. The positions of the major ribosomal mRNA resulted from shortening of the poly(A) tail; however, this pos- species, as determined by the stained gel, are indicated. sibility was not addressed experimentally. The half-life of the IGTP mRNA was determined by exposing RAW cells to 100 units/ml IFNg for 3 h and then to the tran- scriptional inhibitor actinomycin D (4 mM) for various times; the decay of the accumulated mRNA was then followed by North- ern blotting. Under these conditions, the mRNA decayed with a half-life of about 4.5 h (data not shown). Inducibly expressed genes are often classified as primary response genes if induction requires only previously translated transcription factors or as secondary response genes if newly translated factors are required (for review see Ref. 27). To determine if IGTP accumulation required protein synthesis, RAW cells were exposed to 0.1 mM cycloheximide for 30 min and then to 100 units/ml IFNg; mRNA accumulation was fol- lowed by Northern blotting (Fig. 2). Inhibition of protein syn- thesis in this manner blocked the majority of the IGTP mRNA FIG.3. Expression of IGTP mRNA in various mouse tissues. accumulation, although a small amount of mRNA did accumu- Total cellular RNA was prepared from a 3-month-old male C57BL/6 late (Fig. 2). Conversely, under the same conditions, cyclohex- mouse. 15-mg samples were subjected to Northern blotting with an IGTP cDNA probe or a b-actin cDNA probe. The positions of the major imide did not affect the accumulation of IP10 mRNA, which is ribosomal mRNA species, as determined by the stained gel, are a primary IFNg response gene in macrophages (28) (Fig. 2). indicated. Therefore, because IGTP mRNA levels did not show detectable increases until 1 h following IFNg exposure, and because full induction required protein synthesis, IGTP accumulation may This interspecific backcross mapping panel has been typed for be classified as a secondary IFNg response. over 2100 loci that are well distributed among the autosomes, Hybridization of the IGTP cDNA to a total RNA blot contain- as well as the X chromosome (22). C57BL/6J and M. spretus ing RNA from several mouse tissues revealed very high expres- DNAs were digested with several enzymes and analyzed by sion in the thymus and slightly lower expression in the spleen, Southern blot hybridization for informative restriction map lung, and small intestine; expression in brain, heart, kidney, polymorphisms (RFLPs) using a fragment from the 39-untrans- liver, skeletal muscle, and testes was very low or undetectable lated region of the IGTP cDNA as probe. The 3.0- or 4.5-kb SacI (Fig. 3). The tissue expression pattern suggested that IGTP M. spretus RFLP (see “Experimental Procedures”) was used to may be highly expressed in immune cell populations. follow the segregation of the Igtp locus in backcross mice. The The mouse chromosomal location of Igtp was determined by mapping results indicated that Igtp was located in the central interspecific backcross analysis using progeny derived from region of mouse chromosome 11 linked to Il3 and Trp53. One matings of ((C57BL/6J 3 Mus spretus)F and C57BL/6J) mice. hundred and twenty-two mice were analyzed for every marker 1 20402 IGTP, an IFNg-induced GTPase (14), respectively. In IFNg-treated RAW cells, levels of the three mRNAs increased markedly with kinetics similar to the increases in IGTP mRNA levels (data not shown). In addition, each mRNA was highly expressed in mouse thymus, spleen, lung, and small intestine (data not shown). Thus, expression of IGTP and these three related cDNAs was nearly identical in several cell types and tissues. Comparison of the amino acid sequences showed that in at least three of the four proteins 36% of the amino acids were absolutely conserved or were replaced with conservative substitutions (Fig. 5). Sequence homology was found throughout the proteins and was particularly high in the regions of three conserved GTP binding motifs, GXXXXGKS/T, DXXG, and N/TKXD (29); these motifs are found in many functionally diverse GTP-binding proteins in- cluding Ras (29) and dynamin (30). In the case of the proteins encoded by IRG-47, TGTP/Mg21, or LRG-47, neither GTP bind- ing nor had any other biochemical or physiological function been determined. To facilitate the study of IGTP, rabbit polyclonal antisera were generated that recognized the carboxyl-terminal 16 amino acids of IGTP (Fig. 5). This sequence was not shared with any of the three related proteins, and the antibody that was gener- ated immunoprecipitated in vitro translated IGTP but not in vitro translated LRG-47 protein (data not shown). From the [ S]methionine-labeled protein lysates of RAW 264.7 cells, the FIG.4. Mapping of the Igtp gene to the central region of mouse antibody immunoprecipitated a dominant band of about 48 chromosome 11 by interspecific backcross analysis. Igtp was kDa (Fig. 6) that probably represented IGTP. This band had placed on mouse chromosome 11 by interspecific backcross analysis. the same relative migration as that of in vitro translated IGTP The segregation pattern of Igtp and the flanking genes in 122 backcross animals that were typed for all loci is shown at the top of the figure. (data not shown) and was not precipitated by preimmune rab- Each column represents the chromosomes identified in the backcross bit serum (Fig. 6). In addition, the band was weak in cells that progeny that was inherited from the (C57BL/6J 3 M. spretus)F parent. had not been exposed to IFNg, and its intensity increased Shaded boxes represent the presence of the C57BL/6J allele, and white markedly following IFNg stimulation, reaching peak levels in boxes represent the presence of an M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of 3–4 h (Fig. 6). Two other bands of about 55–60 kDa were also each column. A partial chromosome 11 linkage map showing the loca- immunoprecipitated by the IGTP antibody but not by preim- tion of Igtp is shown at the bottom of the figure. Recombination dis- mune serum. These bands could have been proteins that asso- tances between loci in centimorgans are shown to the left. The map ciated with IGTP and were coprecipitated; however, because positions of loci in human, where known, are shown to the right. (References for the human map positions can be obtained from the the immunoprecipitation was done in the presence of SDS and Genome Data Base, a computerized data base of human linkage infor- because the intensities of the bands did not fluctuate with the mation maintained by the William H. Welch Medical Library, The intensity of the IGTP band, it seemed more likely that they Johns Hopkins University, Baltimore, MD.) were unrelated proteins that were also recognized by the anti- body. In Western blotting experiments using the same anti- and are shown in the segregation analysis (Fig. 4). Each locus body, the 48-kDa IGTP was not detectable in RAW cells that was analyzed in pairwise combinations for recombination fre- had not been exposed to IFNg but was easily detectable in cells quencies using the additional data. The ratios of the total that had been exposed to IFNg for4hor longer (Fig. 9 and data number of mice exhibiting recombinant chromosomes to the not shown). total number of mice analyzed for each pair of loci and the most To determine whether IGTP was able to hydrolyze GTP to likely gene order were as follows: centromere-II3-4/122-Igtp- GDP, as suggested by the presence of putative GTP binding 9/122-Trp53. The recombination frequencies (expressed as ge- sites, protein that had been immunoprecipitated from RAW netic distances in centimorgans (cM) 6 S.E.) were II3-3.3 6 cells was incubated with a-[ P]GTP for various times, and the 6.6-Igtp-7.4 6 2.4-Trp53. radiolabeled nucleotides were separated by thin layer chroma- Based on the high IGTP mRNA levels in spleen, a spleen tography (Fig. 7). Very little a-[ P]GTP was converted to GDP cDNA library was chosen for screening with the original 150- by protein immunoprecipitated by preimmune rabbit serum base IGTP cDNA fragment. Several positive clones were ob- from either cells incubated under control conditions or cells tained, one of which was 1927 bases and contained a 1419-base exposed to IFNg for 4 h (Fig. 7). Similarly, very little GTPase open reading frame between bases 204 and 1619 (GenBank activity was immunoprecipitated by the IGTP antibody from accession U53219); the clone contained no other open reading cells not exposed to IFNg; however, greater than four times frames longer than 200 bases. The first potential translation more GTPase activity was precipitated with the IGTP antibody initiation codon within the long open reading frame occurred at from cells that had been exposed to IFNg for 4 h (Fig. 7). The base 348, and translation from this codon produced a 424- increase in GTPase activity was probably due to the increased amino acid polypeptide with a calculated M of 48,507 and a pI of 7.79. The translated sequence showed substantial homology amounts of IGTP that are present in IFNg-stimulated RAW cells (Fig. 6), which suggested that IGTP was a GTPase. Alter- to the translated sequences of three previously cloned cDNAs: IRG-47 (11), TGTP (12)/Mg21 (13), and LRG-47 (14) (Fig. 5). natively, it was also possible that another IFNg-inducible fac- tor that had GTPase activity, or a factor that was able to These cDNAs were cloned as cDNAs whose expression was increased by IFNg in 70Z/3 pre-B cells (11), by T cell receptor catalyze the IGTP GTPase activity, was also present in the 1 1 cross-linking in CD4 8 thymocytes (12) and IFNg in perito- immune pellet from IFNg-stimulated cells. Similar experi- neal macrophages (13), or by IFNg in RAW 264.7 macrophages ments were performed using an antibody that immunoprecipi- IGTP, an IFNg-induced GTPase 20403 FIG.5. Alignment of the translated IGTP, LRG-47, IRG-47, and TGTP/Mg21 protein sequences. Alignments were done using the program pileup (Genetics Computer Group, Madison, WI). Amino acid positions are numbered at the right. Residues that were absolutely conserved or showed conservative substitutions in three of the four proteins are placed in boxes, and residues that are part of the GTP binding motifs are indicated with asterisks (*). blotting (Fig. 9). In some experiments, protein fractions were prepared by briefly incubating the cells in a hypotonic buffer, disrupting the cytosolic membranes by drawing the cells through a 25-gauge needle, and then separating nuclei and particulates from the cytosolic protein fraction by centrifuga- tion. Using this procedure, IGTP was readily detected in the nuclear/particulate protein fraction of IFNg-treated cells but not in the cytosolic protein fraction (Fig. 9). Conversely, when the cytosolic membranes were lysed by incubating the cells in an isotonic buffer containing 0.5% (v/v) Nonidet P-40, IGTP localized to the cytosolic protein fraction (Fig. 9). With both procedures, the nuclei appeared intact as determined by light microscopy (data not shown), and b-tubulin was detected only in the cytosolic protein fractions (Fig. 9). These data raised the possibility that IGTP may have been a cytosolic protein that was loosely associated with membranes in the nuclear/partic- FIG.6. Time course of IGTP accumulation in RAW 264.7 cells ulate fractions and that this association was disrupted by mild exposed to interferon g. Cells were exposed to [ S]methionine for 4 h and to 100 units/ml interferon g for the indicated times during the treatment with a nonionic detergent such as Nonidet P-40. labeling period. Cell lysates were prepared and used for immunopre- Cytosolic localization of IGTP was also supported by prelimi- cipitation with the IGTP antibody; the precipitated proteins were sep- nary results using the IGTP antibody to immunostain IFNg- arated on a 9% SDS-polyacrylamide gel. The positions of selected mo- treated RAW cells (data not shown). lecular mass markers are shown at the left. Other details are described in the text. DISCUSSION We report here the identification of a 48-kDa GTPase, IGTP, tated the LRG-47 protein, and, again, increased GTPase activ- whose expression was rapidly and markedly increased by IFNg ity was detected in immune pellets from IFNg-treated cells, in macrophages and fibroblasts. Its expression pattern sug- relative to control cells (data not shown). Taken together, these gested that IGTP could potentially mediate some IFNg-induced data suggest that IGTP, LRG-47, and possibly the other related responses in macrophages and fibroblasts, and its high expres- proteins are GTPases. sion in mouse thymus and spleen suggested that it may also In similar experiments, IGTP was expressed in bacteria as a function in other immune cell populations. glutathione S-transferase (GST) fusion protein and, following We mapped the Igtp gene to a location on mouse chromosome partial purification by glutathione affinity chromatography 11 between Il3 and Trp53. We compared our interspecific map (data not shown), was used in a-[ P]GTP hydrolysis assays of chromosome 11 with a composite mouse linkage map that (Fig. 8). The GST-IGTP fusion protein converted about six reports the location of many uncloned mouse mutations ; Igtp times more GTP to GDP than was converted by an equivalent mapped in a region of the composite map that lacks mutations amount of GST protein (Fig. 8). This result suggested that the with a phenotype that might be expected for an alteration at IGTP GTPase activity was inherent to the protein and did not this locus. require other mammalian accessory proteins for basal activity. The central region of mouse chromosome 11 shares homology To determine the subcellular distribution of IGTP, RAW cells to regions of human chromosomes 5q and 17p, and thus, the were exposed to IFNg for4hto stimulate synthesis of the protein and then cellular protein was separated into cytosolic Compiled by M. T. Davisson, T. H. Roderick, A. L. Hillyard, D. P. and nuclear/particulate fractions; the presence of IGTP and the Doolittle and provided by GBASE, a computerized data base main- cytosolic protein b-tubulin (31) was determined by Western tained at the Jackson Laboratory, Bar Harbor, ME. 20404 IGTP, an IFNg-induced GTPase FIG.7. GTPase activity of immunoprecipitated IGTP. RAW 264.7 cells were exposed to control conditions or to 100 units/ml inter- feron g (IFNg) for 4 h, and the cells were then used for immunoprecipi- FIG.8. GTPase activity of a GST-IGTP fusion protein. Bacteri- tation with preimmune serum or anti-IGTP serum. a-[ P]GTP was ally expressed GST and GST-IGTP fusion proteins were partially puri- incubated with the immunoprecipitated proteins or with buffer at 37 °C fied using glutathione affinity chromatography. a-[ P]GTP was incu- for the indicated times, and the nucleotide products were separated by bated with 10 mg of the purified proteins or bovine serum albumin polyethyleneimine thin layer chromatography. A, the chromatogram (BSA) at 37 °C for the indicated times, and the nucleotide products were was used for autoradiography, and the positions of GTP and GDP were separated by polyethyleneimine thin layer chromatography. A, the determined using the fluorescent indicator in the PEI-cellulose plates. chromatogram was used for autoradiography, and the positions of GTP B, the amounts of radioactive GTP and GDP were determined with a and GDP were determined using the fluorescent indicator in the PEI- phosphorimager, and the ratio of GDP to GTP was plotted as a function cellulose plates. B, the amounts of radioactive GTP and GDP were of time. Other details are described in the text. M, buffer; L, control, determined with a phosphorimager, and the ratio of GDP to GTP was preimmune; E, control, anti-IGTP; ‚, IFN, preimmune; µ, IFN, plotted as a function of time. Other details are described in the text. M, anti-IGTP. BSA; L, GST; Q, GST-IGTP. human IGTP gene is likely to reside on 5q or 17p. Interestingly, monosomy for chromosome 5 or 5q deletions are frequently associated with myelodysplastic disorders, myeloproliferative syndromes, and acute myeloid leukemia (32, 33). Because Igtp is expressed in myeloid cells, it will be of interest to determine if the human IGTP gene maps to the region on chromosome 5 thought to contain this important myeloid tumor suppressor gene (32). The IGTP protein contained three GTP-binding motifs, GXXXXGKS/T, DXXG, and NKXD (29), and the protein had inherent GTPase activity. Although many other GTP-binding proteins have been identified, they have diverse cellular func- tions, and the presence of this biochemical activity in itself does not suggest a cellular function for IGTP. However, the exten- sive information gained in the biochemical studies of Ras and other GTP-binding proteins may facilitate study of IGTP. For instance, the x-ray crystal structure of Ras (34, 35) has shown that the first and second GTP-binding motifs both contact the phosphates of the GTP molecule and a Mg cofactor, whereas FIG.9. Subcellular localization of IGTP in RAW 264.7 cells. the third motif contacts the purine ring. Mutation of serine 17 Cellular protein was fractionated into nuclear/particulate (N/P) and in the first GTP-binding motif to asparagine blocks the ability cytosolic (Cy) fractions by lysis of the cells by incubation in a hypotonic buffer followed by passage through a 25-gauge needle (A) or by lysis of of Ras to hydrolyze GTP, and expression of this Ras mutant in the cells by incubation in a buffer containing 0.5% Nonidet P-40 (B). cells blocks the function of wild-type Ras (36). The analogous Fractionated protein was used for Western blotting with anti-IGTP and mutation in dynamin, another GTP-binding protein, also has a anti-b-tubulin antibodies. The positions of selected molecular mass dominant-negative effect (37, 38), and this may well be true of markers are indicated at the left. Other details are described in the text. IGTP, an IFNg-induced GTPase 20405 Acknowledgments—We thank Marilyn Powers for automated se- IGTP. Such a dominant negative mutant would be useful in quencing, Terry Copeland for generation of the rabbit polyclonal anti- determining the cellular function of IGTP. bodies, Debbie Gilbert for technical assistance, and Marianne Oskars- The IGTP amino acid sequence was very similar to the trans- son and Kenji Fukasawa for helpful discussions. lated sequences of three previously cloned cDNAs, IRG-47 (11), REFERENCES TGTP (12)/Mg21 (13), and LRG-47(14), each of which contained 1. Farrar, M. A., and Screiber, R. D. (1993) Annu. Rev. Immunol. 11, 571–611 three GTP-binding motifs and had molecular masses of about 2. Vilcek, J., Gray, P. W., Rinderknect, E., and Sevastopoulos, C. G. (1985) 47 kDa. Moreover, the increases in IRG-47, TGTP/Mg21, and Lymphokines 11, 1–32 3. Handa, K., Suzuki, R., Matsui, H., Shimizu, Y., and Kumagai, K. (1983) LRG-47 mRNA levels in IFNg-treated RAW macrophages, and J. Immunol. 130, 988–992 their expression patterns in mouse tissues, paralleled those of 4. King, D. P., and Jones, P. P. (1983) J. Immunol. 131, 315–318 5. Warren, M. K., and Vogel, S. N. (1985) J. Immunol. 134, 2462–2469 IGTP mRNA. Therefore, it seems likely that IGTP is represent- 6. Liew, F. Y., Li, Y., and Millott, S. (1990) J. Immunol. 145, 4306–4310 ative of a new family of proteins whose expression is regulated 7. Ding, A. H., Nathan, C. F., and Stuehr, D. J. (1988) J. Immunol. 141, similarly and whose biochemical functions are similar. The 2407–2412 8. Beutler, B., Tkacenko, V., Milsark, I., Krochin, N., and Cerami, A. (1986) J. levels at which protein expression and biochemical function Exp. Med. 164, 1791–1796 differ will be the subject of future studies. 9. Schreiber, R. D., Hicks, L. J., Celada, A., Buchmeier, N. A., and Gray, P. W. (1985) J. Immunol. 134, 1609–1618 At least two other groups of interferon-induced GTP-binding 10. Munro, J. M., Pober, J. S., and Cotran, R. S. (1989) Am. J. Pathol. 135, proteins exist. The first of these is comprised of the Mx pro- 121–131 teins, a group of 72-kDa proteins involved in the interferon- 11. Gilly, M., and Wall, R. (1992) J. Immunol. 148, 3275–3281 12. Carlow, D. A., Marth, J., Clark-Lewis, I., and Teh, H.-S. (1995) J. Immunol. induced inhibition of viral proliferation (39). The second group 154, 1724–1734 includes several 65–67-kDa proteins that bind to GTP-agarose 13. LaFuse, W. P., Brown, D., Castle, L., and Zwilling, B. S. (1995) J. Leuk. Biol. 57, 477–483 but whose function is unknown (40, 41). However, because 14. Sorace, J. M., Johnson, R. J., Howard, D. L., and Drysdale, B. E. (1995) J. little sequence similarity exists between IGTP and either of Leuk. Biol. 58, 477–484 these groups of proteins, other than that found in the GTP- 15. Rong, S., Oskarsson, M., Faletto, D. L., Tsarfaty, I., Resau, J., Nakamura, T., Rosen, E., Hopkins, R., and Vande Woude, G. F. (1993) Cell Growth & binding motifs, no functional comparisons can be drawn. Differ. 4, 563–569 Future experiments will focus on determining the physiolog- 16. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156–159 17. Taylor, G. A., and Blackshear, P. J. (1995) J. Cell. Physiol. 162, 378–387 ical function of IGTP. Among the many possible functions of the 18. Stumpo, D. J., Graff, J. M., Albert, K. A., Greengard, P., and Blackshear, P. J. protein are mediation of IFNg signal transduction at some level (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4012–4016 and regulation of some of the multiple IFNg-induced pheno- 19. Tso, J. Y., Sun, X. H., Kao, T.-h., Reece, K. S., and Wu, R. (1985) Nucleic Acids Res. 13, 2485–2502 typic responses. 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Published: Aug 1, 1996

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