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Cloning and Characterization of Mouse UBPy, a Deubiquitinating Enzyme That Interacts with the Ras Guanine Nucleotide Exchange Factor CDC25Mm/Ras-GRF1

Cloning and Characterization of Mouse UBPy, a Deubiquitinating Enzyme That Interacts with the Ras... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 42, Issue of October 19, pp. 39448 –39454, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Cloning and Characterization of Mouse UBPy, a Deubiquitinating Enzyme That Interacts with the Ras Guanine Nucleotide Exchange Mm Factor CDC25 /Ras-GRF1* Received for publication, April 18, 2001, and in revised form, July 31, 2001 Published, JBC Papers in Press, August 10, 2001, DOI 10.1074/jbc.M103454200 Nerina Gnesutta‡§, Michela Ceriani¶§, Metello Innocenti‡, Isabella Mauri‡, Renata Zippel‡, Emmapaola Sturani‡, Barbara Borgonovo, Giovanna Berruti, and Enzo Martegani¶** From the ‡Dipartimento di Fisiologia e Biochimica Generali, Universita ` di Milano, Via Celoria 26, Milano, Italy 20133, ¶Dipartimento di Biotecnologie e Bioscienze, Universita ` di Milano-Bicocca, Piazza della Scienza 2, Milano, Italy 20126, and Dipartimento di Biologia, Universita ` degli Studi di Milano, Milano, Italy 20133 Mm We used yeast “two-hybrid” screening to isolate brain-specific Ras-GEF, called CDC25 (1–2) or Ras-GRF1. Mm cDNA-encoding proteins interacting with the N-termi- CDC25 is a large protein (140 kDa) that contains a Ras- nal domain of the Ras nucleotide exchange factor exchange domain in the C-terminal region and several differ- Mm CDC25 . Three independent overlapping clones were ent domains in the large N-terminal region, namely two PH isolated from a mouse embryo cDNA library. The full- (pleckstrin homology) domains, one DH (Dbl homology) do- length cDNA was cloned by RACE-polymerase chain re- main, and an illimaquinone (IQ) domain (2–3). In addition a action. It encodes a large protein (1080 amino acids) coiled-coil region and a PEST sequence were identified (4). highly homologous to the human deubiquitinating en- Several evidences indicate that the large N-terminal region zyme hUBPy and contains a well conserved domain typ- Mm of CDC25 has a regulatory function and may interact with ical of ubiquitin isopeptidases. Therefore we called this other cellular components. We have previously shown that the new protein mouse UBPy (mUBPy). Northern blot anal- expression in mouse fibroblasts of a truncated form of ysis revealed a 4-kilobase mRNA present in several Mm CDC25 , lacking the Ras exchange domain, behaves as a mouse tissues and highly expressed in testis; a good dominant negative protein (5). In addition PH domains could level of expression was also found in brain, where Mm interact with phospholipids (6) and with the  subunits of CDC25 is exclusively expressed. Using a glutathione heterotrimeric G proteins (7), whereas the IQ domain binds S-transferase fusion protein, we demonstrated an “in calmodulin (3, 8) and is thought to be responsible for the vitro” interaction between mUBPy and the N-terminal Mm activation of GEF activity by calcium (3). Further evidence that half (amino acids 1– 625) of CDC25 . In addition “in Mm vivo” interaction was demonstrated after cotransfection the N-terminal region of CDC25 protein is involved in spe- Mm cific protein interaction(s) also comes from the work of Kiyono in mammalian cells. We also showed that CDC25 ,ex- Mm pressed in HEK293 cells, is ubiquitinated and that the et al. (9) in which it was shown that CDC25 /Ras-GRF1 was coexpression of mUBPy decreases its ubiquitination. In able to activate Rac1 and that for this activity a functional DH addition the half-life of CDC25Mm protein was consid- domain is required. Moreover the DH domain was required for erably increased in the presence of mUBPy. The specific homodimerization of Ras-GRF1 or for heterodimerization with function of the human homolog hUBPy is not defined, Ras-GRF2 (10) Mm although its expression was correlated with cell prolif- However, so far, only the specific interaction CDC25 /cal- eration. Our results suggest that mUBPy may play a role modulin has been demonstrated in vivo (3, 8). In an effort to Mm in controlling degradation of CDC25 , thus regulating identify mammalian proteins that could interact with the large the level of this Ras-guanine nucleotide exchange factor. Mm N-terminal region of CDC25 , we used a yeast “two-hybrid” system for the screening of mouse embryo cDNA libraries (11). 1 Here we report that the cloning and characterization of a cDNA Ras-guanine nucleotide exchange factors (GEFs) are pro- that was positive in this screening. This cDNA was found to teins that stimulate the exchange of guanine nucleotides (GDP/ encode a new deubiquitinating enzyme belonging to the ubiq- GTP) on Ras proteins. We have previously cloned a mouse uitin isopeptidase family (UBPs) and was highly homologous to This is an open access article under the CC BY license. the recently identified human enzyme hUBPy (12); therefore * This work was supported by C.E. Contract BIO4-CT98 – 0297, Con- we called this new cDNA mouse UBPy (mUBPy). siglio Nazionale delle Ricerche Target project on Biotechnology (to E. S.), and MURST 40%-Cofin 2000 (to R. Z.). The costs of publication of EXPERIMENTAL PROCEDURES this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance Two-hybrid Screening—The system developed by Vojtek et al. (11) with 18 U.S.C. Section 1734 solely to indicate this fact. was used for the two-hybrid screening. Two mouse embryo cDNA li- The nucleotide sequence(s) reported in this paper has been submitted braries (9.5 and 10.5 dpc) constructed in pVP16 vector (a gift of Stan TM to the GenBank /EBI Data Bank with accession number(s) AF057146. Hollenberg) were used. Three bait plasmids were prepared by cloning § These authors contributed equally to the work. Mm fragments of the N-terminal region of CDC25 in the pBTM116 vec- ** To whom correspondence should be addressed: Dipartimento di tor, which contained the Lex-A DNA binding domain (11). Biotecnologie e Bioscienze, Piazza della Scienza 2, 20126 Milano, Italy. Mm The first bait plasmid (pBTEE) contains 1877 bp of CDC25 IV Tel.: 39-02-64483533; Fax: 39-02-64483565; E-mail: enzo.martegani@ (2), which corresponds to the first 625 amino acids; this region includes unimib.it. the first PH domain, the IQ motif, the Dbl homology (DH) domain, and The abbreviations used are: Ras-GEF, guanine nucleotide exchange Mm the second PH domain. pBTES contains 1519 bp of CDC25 IV, factor for Ras; UBP, ubiquitin isopeptidase; DH, Dbl homolog; PH, corresponding to the first 506 amino acids, and lacks the second PH pleckstrin homolog; IQ, illimaquinone; bp, base pair(s); aa, amino ac- domain, whereas pBTEP contains 436 bp, corresponding to the first 145 id(s); GST, glutathione S-transferase; HA, hemagglutinin; RIPA, radio- immune precipitation; dpc, days post-coitus. amino acids, and contains only the first PH domain. 39448 This paper is available on line at http://www.jbc.org Mm Mouse UBPy Interacts with the Ras-GEF CDC25 39449 The yeast strain L40 (MATa, his3, trp1, leu2, ade2, 1:1000). Bound antibodies were detected with peroxidase-conjugated LYS2::(lexAop) -HIS3, URA3::(lexAop) -lacZ) was transformed with the goat anti-rabbit immunoglobulins and revealed by the ECL detection 4 8 bait vector (initially pBTEE) and with the mouse embryo cDNA librar- system (Amersham Pharmacia Biotech). ies. Selection was performed as described by Vojtek et al. (11) in selec- Cells cultures were harvested, washed with phosphate-buffered sa- tive minimal plates containing 5 mM 3-aminotriazole. Positive clones, line, and lysed in RIPA buffer (Tris-HCl, 50 mM; NaCl, 150 mM; sodium obtained only by the 10.5-dpc library, were also tested for -galactosid- deoxycholate, 0.5% w/v; SDS, 0.1% w/v; Triton X-100, 1% w/v) contain- ase activity. Plasmids were recovered from positive clones and used for ing protease inhibitors (leupeptin, 1 M; aprotinin, 0.1 M; sodium a new round of screening to test their positivity. Positive clones were vanadate, 0.1 mM; phenylmethylsulfonyl fluoride, 0.1 mM). Aliquots of sequenced and then tested with the other plasmids containing reduced cleared cell lysate were separated on SDS-polyacrylamide gels, trans- Mm regions of CDC25 (pBTES and pBTEP). ferred to nitrocellulose, and immunoblotted with anti-mUBPy RACE-PCR, Cloning, Plasmids, Sequencing, and Northern Blot Hy- antibodies. bridization—RACE reactions were done using Marathon-Ready cDNA Cell Culture and Transfection—NIH-3T3, COS7, and Hek-293 cells (CLONTECH) from 11.5-dpc mouse embryo and a mix of polymerases were cultured in Dulbecco’s modified Eagle’s medium supplemented (Advantage Klen-Taq Mix, CLONTECH) suitable for amplification of with 10% fetal bovine serum. Subconfluent cells were transfected using long regions of DNA with high fidelity. The PCR conditions were set and the LipofectAMINE (Life Technologies, Inc.) method according to the performed according to the protocols given by CLONTECH. Four oligo- protocol of the manufacturer. nucleotides were used, two for 3 RACE and two for 5 RACE using a Pull-down Experiments—48 h after transfection, COS7 cells were nested PCR procedure to obtain an increased specificity. For 3 RACE, harvested, washed with phosphate-buffered saline, and lysed in RIPA the external primer was GSS1 (5-GCTCAGCGAGAACCTTTGAC- buffer or in lysis buffer (HNGT) (Hepes, 25 mM; NaCl, 50 mM; glycerol, GAGAGC-3), and the internal one was GSS2 (5-CCTGAAATGGCTC- 10% w/v; Triton X-100, 1% w/v). The buffers contained protease CTCGTCTGCACC-3). For 5 RACE, the external primer was GSA1 inhibitors. (5-GGTGCAGACGAAGGAGCCATTTCAGG-3), and the internal one Aliquots of lysates (450 l) were then incubated with 50 l of gluta- was GSA2 (5-GCTCTCGTCAAAGGTTCTCGCTGAGC-3). thione-Sepharose resin loaded either with GST-mUBPy fusion 525– 660 Two oligonucleotides (i.e. GSS1 and GSA1) were also used in a protein or with GST alone for2hat4 °C. After incubation, the resin control reaction to verify the presence of the specific cDNA in the pool. was recovered by gentle centrifugation and washed five times with the Positivity was revealed by a 176-bp fragment. incubation buffer (RIPA or HGNT). At the end, the resin was treated The amplification products of 3 and 5 RACE were cloned in pMOS- with 30 lof2 SDS sample buffer and boiled for 10 min, and the Blue (Amersham Pharmacia Biotech) and sequenced. Three clones were recovered proteins were used for Western blot analysis. sequenced both for 3 and 5 amplification products. Coimmunoprecipitation and Immunoblotting—48 h after transfec- Sequencing was performed on both strands using the Taq Dye Ter- tion, cells (COS7 or Hek-293) were harvested and lysed in 500 l/plate minator Cycle Sequencing Kit (Applied Biosystems) with universal and (100 mm) of an ice-cold HNGT buffer as described (18). Immunoprecipi- walking primers and using an automated DNA sequence analyzer (Ap- tation from cleared lysates was performed with monoclonal anti-HA-11 plied Biosystems 373A). The full-length cDNA was obtained by using antibody (BAbCO) or with anti-mUBPy polyclonal antibodies for1hat the unique NdeI site present in the overlapping region and was sub- 4 °C. Immunocomplexes were recovered with protein G-Sepharose or cloned in pCDNA3 vector (Invitrogen). with protein A-Sepharose (Sigma) for1hat4 °C and then resuspended pCDNA3-mUBPy-HA vector was prepared by ligation of a cDNA in SDS sample buffer and analyzed by immunoblot analysis. Briefly ⁄50 fragment of 2.5 kilobase pairs comprising the coding sequence of aa of cleared cell lysates and the immunoprecipitates were resolved by 10% 542–1080 of mUBPy in pBSKSII-HA vector (Stratagene), and then a SDS-PAGE and transferred to nitrocellulose membranes. Membranes SphI-EcoRI fragment coding for a fusion between HA tag and the aa were probed with anti Ras-GRF1 (C-20, Santa Cruz, CA) or with poly- 542–1080 of mUBPy was subcloned in pCDNA3 plasmid. HA-ubiquitin clonal anti-mUBPy raised in our laboratory. Immunocomplexes were vector and pCDNA3-hUBPy plasmid were obtained by G. Draetta (12). detected by enhanced chemiluminescence (Amersham Pharmacia Bio- For Northern blots, polyadenylated RNAs from mouse tissues (total tech) using goat anti-rabbit antiserum coupled to horseradish peroxi- mouse RNA, Ambion) were separated on agarose-formaldehyde gels (5 dase as secondary antibodies (Cappel). g/lane), blotted to a Hybond-N nylon membrane (Amersham Pharma- Deubiquitination Assays—We used a mouse testis extract as a source cia Biotech), and hybridized with a digoxygenin-labeled riboprobe. Sig- of ubiquitinated protein as described in Ref. 19 with minor modifica- nals were detected with the Nucleic Acid Detection kit (Roche Molecular tions. The cleared extracts were dialyzed against Tris-HCl 50 mM, Biochemicals). The probe was prepared by subcloning a fragment of MgCl 5mM, and dithiothreitol 2 mM. The sample was then centrifuged mUBPy cDNA in a pGEM3z vector (Promega) and in vitro transcription and washed several times in a Centricon-3 concentrator (Amicon) to with T7 RNA polymerase and digoxygenin-labeled UTP (Roche Molec- remove residual N-ethylmaleimide (12). Aliquots (50 g) of the proteins ular Biochemicals) according to the manufacturer’s instructions. were then incubated with immunoprecipitates (anti-HA antibodies) Sequence Analysis—The searches for open reading frames and their from COS7 cells transfected with pCDNA3-mUBPy-HA vector at 37 °C conceptual translations were performed with DM software (13). The and processed as described in Ref. 19 using anti-ubiquitin antibodies homology search was done at the BLAST (14) server at NCBI (www. (Sigma). Mm ncbi.nlm.nih.gov/BLAST/). Protein alignments were done with MACAW In Vivo Ubiquitination of CDC25 —Hek-293 cells transfected with (15), while the search for motifs in protein was done with Profilescan at the different plasmids (as reported in the figure legends) were lysed in the Expasy server (www.expasy.ch/). RIPA buffer. Immunoprecipitation from the same amount of total pro- Recombinant Protein Production and Preparation of Polyclonal An- tein was performed with antibodies against Ras-GRF1 or anti-HA tibodies—The cDNA contained in one of the positive clones (6/12) was (BAbCO). Immunocomplexes were recovered with protein G-Sepharose subcloned in the SmaI site of the Escherichia coli expression vector (for anti-HA monoclonal antibodies) or protein A-Sepharose (for Ras- pGEX-2T (Amersham Pharmacia Biotech) to obtain a fusion protein GRF1 antibodies) and analyzed in Western blotting with the different between GST and 119 amino acids of mUBPy corresponding to position primary antibodies and then revealed with the corresponding second- 542 to 660 of the whole protein. E. coli DH5 cells bearing the vector for ary antibody and the ECL system. fusion protein were grown in LB broth, induced with 0.1 mM isopropyl- Determination of Stability of CDC25Mm Protein—Hek-293 cells were Mm 1-thio--D-galactopyranoside at 28 °C for 3 h, and collected for protein transiently transfected with 1 g of pCDNA3-CDC25 alone and then extraction. Fusion protein was purified with glutathione-Sepharose cotransfected with 1.5 g of pCDNA3-mUBPy or with 1.5 gof resin as described (16). About 1 mg of purified fusion protein (GST- pCDNA3-human UBPy antisense (12). After 24 h, cells were washed mUBPy ) was used to immunize New Zealand rabbits. Polyclonal two times with minimum Eagle’s medium without methionine. Cells 542– 660 antibodies were then affinity-purified using a GST-coupled Affi-Gel-10 were then incubated for 2 h with minimum Eagle’s medium without column (Bio-Rad) as described (17). For pull-down experiments, the methionine with [ S]methionine (100 C/60-mm dish), Hepes 25 mM, GST-mUBPy fusion protein was used, coupled to glutathione- and fetal calf serum 0.1%. After the incubation, cells were washed two 542– 660 Sepharose beads. times with Dulbecco’s modified Eagle’s medium and further incubated Western Blotting—Tissues from adult CD1 mice (Charles River, with Dulbecco’s modified Eagle’s medium supplemented with fetal bo- Calco (LC), Italy) were mechanically homogenized with 4 volumes of vine serum 10%. Tris (0.125 M, pH 6.8) and SDS (7% w/v). The homogenate was further At the appropriate time, cells were rinsed in phosphate-buffered sonicated in ice and centrifuged (20,000  g for 10 min); the solubilized saline and lysed in RIPA buffer, and the lysates were clarified. Immu- proteins were separated on SDS-polyacrylamide gel electrophoresis noprecipitation from lysates was performed in the presence of Ras- (SDS-PAGE) and transferred to nitrocellulose. Membranes were probed GRF1 antibody (Sigma) for2hat4 °C,andthe immunocomplexes were with affinity-purified anti-mUBPy polyclonal antibodies (diluted recovered with protein A (Sigma) for1hat4 °C with gentle rotation. Mm 39450 Mouse UBPy Interacts with the Ras-GEF CDC25 FIG.1. Amino acid sequence of mUBPy predicted open reading frame and comparison with hUBPy. The sequence in bold is that of the 6/12 clone isolated from two-hybrid screening. The underlined region identifies Cys and His boxes typical of deubiquitinating en- zymes (21, 22); the dotted underlined re- gion (aa 198 –311) represents the rho- danese/cdc25 fold homology (23). The TM sequence was deposited in GenBank , accession number AF057146. The align- ment between hUBPy (12) and mUBPy was done with Macaw (15). Equal numbers of trichloroacetic acid-precipitable counts from cell ex- (not shown). On the basis of these results, we can conclude that tracts were immunoprecipitated. Mm the region of CDC25 between aa 145 and aa 506, containing The immunoprecipitates were washed three times with RIPA buffer the IQ motif and the DH domain, is required for this in vivo solution and then resuspended in 50 l of SDS-sample buffer and interaction. The sequence of the 6/12 clone revealed an open separated by SDS-PAGE. The dry gels were exposed to x-ray films (Amersham Pharmacia Biotech), and the intensity of specific bands was reading frame (3-5 open) encoding a short proline-rich analyzed by densitometry with NIH-Image software. polypeptide (119 aa) homologous to a small central region of a human gene product called hUBPy, originally reported to RESULTS encode a putative deubiquitinating enzyme (20) and recently Screening for Proteins Interacting with the N-terminal demonstrated to be actually a deubiquitinase (12). Mm Region of CDC25 , Identification and Cloning of Mouse The full-length mouse cDNA was cloned by RACE-PCR using UBPy—We used the N-terminal region (first 625 aa) of a mouse embryo-cDNA (Marathon-Ready cDNA, CLONTECH) Mm CDC25 as a bait for a yeast two-hybrid screening. This starting from this central region. Several clones were obtained fragment comprises all the identified motifs except the cata- both for the 3 region and the 5 region and were completely lytic one, i.e. the first PH domain, the IQ domain, and the sequenced. The 3970-bp cDNA contains a complete open read- DH-PH module, and it is the same region that, when expressed ing frame of 3240 nucleotides encoding a 1080 aa protein, 18 bp in mammalian cells, behaves as a dominant negative protein upstream the putative AUG start codon and 713 bp down- (5). For the screening, we used two different mouse embryo stream the stop codon (Fig. 1). Because the open reading frame cDNA libraries in pVP16 vector given by S. Hollenberg (11) shared a great homology (higher than 90%) with the hUBPy, After two rounds of selection, we identified three “true” pos- we called this new mouse protein mouse UBPy (mUBPy). The itive clones, all derived from the 10.5-dpc cDNA library. These homology is very high in the C-terminal region (97% identity clones contained small cDNA inserts (about 400 bp), which between aa 591–1080) that contains the typical hallmarks of shared a common overlapping region of 300 bp. The largest the UBP family of deubiquitinating enzymes, i.e. a well con- clone (called 6/12) was further characterized for its ability to Mm served UCH-2 domain (aa 739 –1072, Profilescan) with the interact in yeast with different shorter regions of CDC25 .A characteristic cysteine and histidine boxes (21, 22). The N- positive interaction was obtained with pBTES plasmid contain- Mm terminal part of mUBPy also contains a highly homologous ing the first 506 aa of CDC25 and thus lacking the second region (88% identity aa 1–346) followed by a region of lower PH domain, whereas no interaction was observed with pBTEP Mm plasmid that contains only the first PH domain of CDC25 homology (68% identity aa 346 –590). A search for motifs in this Mm Mouse UBPy Interacts with the Ras-GEF CDC25 39451 FIG.3. mUBPy (aa 542–1080), expressed in COS7 cells, can remove ubiquitin from endogenous ubiquitinated proteins. Mouse testis extracts were prepared by homogenizing testes in the presence of 20 mM N-ethylmaleimide to inactivate endogenous deubiq- uitinating enzymes (19). Soluble fractions were dialyzed against 50 mM Tris (pH 8), 5 mM MgCl ,and2mM dithiothreitol and then centrifuged and washed four times in a Centricon-3 concentrator to remove residual N-ethylmaleimide. Aliquots containing 50 g of total proteins were incubated for 15 or 30 min with an HA-immunoprecipitate obtained from COS7 cells transfected with pCDNA3-mUBPy-HA vector (4 g/ 100-mm plate) expressing mUBPy tagged with HA epitope. The 542–1080 immunoprecipitates of cleared lysate obtained from two dishes were FIG.2. Expression of mUBPy in mouse tissues and cell lines. A, washed three times in HNGT buffer, and the equal aliquots (about ⁄4 of Northern blot analysis. 5 g of total RNA extracted from various mouse the total) were used for each assay. As a control, an immunoprecipitate tissues were separated on agarose-formaldehyde gels, blotted to a nylon was washed in the presence of 20 mM N-ethylmaleimide ( NEM). At membrane, and hybridized with a digoxygenin-labeled mUBPy ribo- the indicated times, samples were quenched with SDS-sample buffer probe. Signals were detected with the Nucleic Acid Detection Kit (Roche and analyzed by immunoblotting with anti-ubiquitin antibodies. Molecular Biochemicals). Lane 1, liver; lane 2, lung; lane 3, thymus; lane 4, heart; lane 5, 11.5-dpc embryo; lane 6, spleen; lane 7, testis; lane 8, ovary; lane 9, kidney; lane 10, brain. The amount of RNA loaded in of a strong specific immunoreactive band of 120 kDa (in agree- each lane was comparable, as indicated by staining of the gel with ethidium bromide (not shown). B, total proteins extracted from adult ment with the expected molecular size of the mUBPy open mouse tissues, separated on SDS-PAGE 7% gels, blotted to nitrocellu- reading frame: 122,579 Da) in mouse brain and testis homoge- lose, and probed with anti-mUBPy antibodies. A strong 120-kDa band nates (Fig. 2B). A lower signal was observed in pancreas, lung, was observed in brain and testis. About 50 g of protein were loaded in small intestine, and placenta homogenates, whereas a barely each lane. Lane 1, brain; lane 2, lung; lane 3, heart; lane 4, liver; lane 5, detectable signal was present in liver, spleen, kidney, and spleen; lane 6, gut; lane 7, kidney; lane 8, pancreas; lane 9, ovary; lane 10, placenta; lane 11, testis. C, proteins extracted from cultured cells, heart (Fig. 2B). These results are generally in agreement with separated on SDS-PAGE gels, blotted to nitrocellulose, and probed with the results obtained with Northern blot experiments. Therefore anti-mUBPy antibodies. Lane 1, growing NIH-3T3; lane 2, confluent the mUBPy protein is expressed at a very low level in most of NIH-3T3; lane 3, human neuroblastoma cells SK-N-BE; lane 4, human the adult mouse tissues with a strong expression in brain and Hek-297 cells. About 50 g of protein were loaded in each lane. testis only. Moreover, mUBPy is not an easily soluble protein N-terminal region revealed only a highly conserved Rhodanese/ because to detect it in most tissues, we needed strong detergent cdc25 fold (aa 198 –311) (23). Interestingly the Rhodanese/ conditions (i.e. 7% SDS). When a mild extraction procedure (1% cdc25 fold has been found in the noncatalytic region of several Triton X-100) was used, mUBPy could be detected only in brain yeast ubiquitin hydrolases (yeast Ubp4, 5 and 7), in the non- and testis (not shown). Since the human homolog hUBPy is catalytic domain of dual specificity MAPK-phosphatases, and expressed in several cell lines (12), we also looked for the in the yeast phosphotyrosine phosphate-phosphatase (23). expression of the mUBPy protein in mouse fibroblasts; more- Other interesting features of mUBPy protein are the presence over we also probed two human cell lines with our antibodies. of two long proline-rich regions (aa 300 – 450 and aa 560 –740, A 120-kDa band was detected in the mouse NIH-3T3 fibro- respectively), which contain several PEST-like motifs, and two blasts extract (Fig. 2C, lanes 1 and 2); however, no difference coiled-coil regions (aa 95–140 and 460 –500). was observed between proliferating and density-arrested NIH- Expression of mUBPy in Mouse Tissues—The human homo- 3T3 fibroblasts. In addition a sharp 130-kDa band was detected log hUBPy was initially identified as the predicted product of a in both Hek-293 cells and SN-K-BE neuroblastoma cells, indi- cDNA found in myeloblasts (20). Successively, Naviglio et al. cating that our antibodies also recognize the human hUBPy (12) found the protein in several human cell lines (human protein. This was not unexpected because the protein region we fibroblasts WI-38, osteosarcoma U2OS, HeLa cells, etc.). used for the preparation of anti-mUBPy polyclonal antibodies Northern blot analysis performed on RNA extracted from contains a large amino acid sequence (aa 592– 657 of mUBPy, several mouse tissues evidenced the presence of a 4-kilobase see Fig. 1), which is identical in the two proteins. mUBPy mRNA in most of the probed tissues (Fig. 2A). As mUBPy Is a Deubiquitinating Enzyme—Since mUBPy has a expected, this mRNA was also present in mouse embryo and in high homology with hUBPy that has been demonstrated to adult mouse brain; however, mUBPy mRNA was found to be have a UBP (ubiquitin-isopeptidase) activity (12), we expected particularly abundant in testis. that also mUBPy has an UBP activity. To verify this, we used Rabbit polyclonal antibodies were raised against a GST fu- a mouse testis protein extract, pretreated with N-ethylmaleim- sion protein (containing 119 aa of mUBPy) expressed in E. coli. ide, as a source of ubiquitinated proteins as reported by Lin et The antiserum was treated with immobilized GST to remove al. (19). As shown in Fig. 3, the addition of HA-immunoprecipi- the anti-GST component and then used for Western blot tates from COS7 cells transfected with HA-tagged mUBPy (aa experiments. 542–1080) expressing vector greatly reduced the amount of An immunoscreening of mouse tissues revealed the presence ubiquitinated protein recognized by anti-ubiquitin antibodies, Mm 39452 Mouse UBPy Interacts with the Ras-GEF CDC25 FIG.4. In vitro interaction of mUBPy (aa 542– 660) with the Mm N-terminal domain of CDC25 (aa 1– 625). COS7 cells transfected Mm with pHC21 vector, expressing the N-terminal region of CDC25 (aa 1– 625) (5),(5 g/100-mm plate), or untransfected COS7 cells were lysed with HGNT buffer. Lysates were incubated with glutathione-Sepharose resin loaded with either GST-mUBPy or GST alone as described 542– 660 (see “Experimental Procedures”). Protein bound to the resin was recov- ered and processed for Western blot analysis with anti Ras-GRF1 antibodies. A, Western blot analysis of protein bound to glutathione- Sepharose beads coupled to GST-mUBPy (lanes 1 and 3)orGST 542– 660 alone (lanes 2 and 4). Lanes 1 and 2, control COS7 cells; lanes 3 and 4, Mm extracts of COS7 cells expressing CDC25 . B, immunodetection 1– 625 Mm of CDC25 on extracts of COS7 cells. Lane 1, total cell protein 1– 625 (lysis in RIPA buffer) of control COS7 cells; lane 2, total cell protein of Mm COS7 cells expressing CDC25 ; lane 3, lysis buffer (HGNT) ex- 1– 625 Mm tract of COS7 cells expressing CDC25 . The arrows indicate the 1– 625 Mm 70-kDa band expected for CDC25 . IP, immunoprecipitate. 1– 625 Mm FIG.5. In vivo interaction between CDC25 and mUBPy and hUBPy. A, COS7 cells were transfected with 4 g/100 mm plate of indicating that mUBPy can deubiquitinate ubiquitin from high Mm the indicated vectors (GRF1, pCDNA3-CDC25 expressing the full- molecular weight ubiquitinated proteins. Mm length CDC25 protein; mUBPy, pCDNA3-mUBPy-HA expressing Mm Interaction of mUBPy with CDC25 —We have previously mUBPy tagged with HA epitope) using pCDNA3 when required 542–1080 identified mUBPy in a two-hybrid screening for protein inter- to keep the total DNA to 8 g/plate. After 48 h, cells were harvested and Mm lysed in HGNT buffer, and lysates were immunoprecipitated with an- action with the Ras-GEF CDC25 . It was, however, impor- ti-HA antibodies. Immunoprecipitated proteins (IPP) and equal ali- tant to confirm this interaction directly in vitro and succes- quots of cleared total lysates (30 l) were separated by SDS-PAGE, sively in an in vivo assay using mammalian cells. blotted, and probed with either anti-GRF1 antibodies or anti-mUBPy A GST fusion protein containing the 119 aa of the clone 6/12 antibodies. B, Hek-293 cells were transfected with 4 g/100-mm plate of Mm found to be positive in the yeast two-hybrid screening was the indicated vectors (GRF1, pCDNA3-CDC25 expressing the full- Mm length CDC25 protein; hUBPy, pCDNA3-hUBPy expressing hUBPy tested for in vitro interaction with the N-terminal region of Mm (12)) using pCDNA3 when required to keep the total DNA to 8 g/plate. CDC25 by pull-down experiments. As shown in Fig. 4,a After 48 h, cells were harvested and lysed in HGNT buffer, and lysates Mm specific interaction with the CDC25 N-terminal fragment were immunoprecipitated with anti-mUBPy antibodies. Immunopre- can be revealed. This result is in agreement with that obtained cipitated proteins were separated by SDS-PAGE, blotted, and probed with either anti-GRF1 antibodies or anti-mUBPy antibodies. by the yeast two-hybrid system. To validate the above results, we tested the in vivo interac- Mm tion between CDC25 and mUBPy by co-expressing the two pCDN3-RasGRF1 and pCDNA3-hUBPy, and then the cell ly- Mm proteins in mammalian cells. CDC25 and a HA-tagged sates were immunoprecipitated with anti-mUPBy antibodies mUBPy (aa 542–1080) were transfected in COS7 cells either and immunoblotted with anti-GRF1 antibodies. As shown in Mm separately or together. 48 h after transfection, lysates were Fig. 5B, CDC25 was coimmunoprecipitated only if hUBPy prepared and processed for immunoprecipitation with anti-HA was expressed; this indicates that the human homolog is also Mm antibodies. The recovered immunocomplexes were separated able to interact with CDC25 . When the same filter was by SDS-PAGE and blotted. The blots were probed with anti- probed with anti-mUBPy antibodies, a strong band was de- Ras-GRF1 antibodies and with polyclonal antibodies raised tected in extracts of cells transfected with hUBPy, whereas a Mm against mUBPy. Fig. 5A shows that CDC25 was coimmuno- weak immunoreactive band was also detected in cells not precipitated only from extracts obtained by cells expressing treated with pCDNA3-hUBPy, which is likely due to endoge- Mm both CDC25 and HA-mUBPy (lane 3) but not from extracts nous hUBPy protein. Mm Mm of cells transfected with CDC25 alone or with HA-mUBPy CDC25 Is Ubiquitinated in Vivo and mUBPy Decreases Its alone (lanes 1–2). Aliquots of the same total cell lysates were Level of Ubiquitination—As a general rule, regulatory proteins Mm loaded in the same gel to check the expression of CDC25 are subjected to a fast turnover, and this is expected to be the (lane 4), HA-mUBPy (lane 5), and both proteins in cotrans- case also for the Ras exchange factors. At our knowledge, no Mm fected cells (lane 6). This experiment clearly demonstrates an data regarding the turnover of CDC25 /RasGRF1 have been Mm in vivo interaction between CDC25 and mUBPy. Interest- so far reported in the literature, although RasGRF1 is known to Mm ingly the amount of total CDC25 protein found in COS7 cells contain a PEST motif (4). However, it has been recently shown lysates increased when the mUBPy was co-expressed, suggest- that RasGRF2, a protein closely related to RasGRF1, is ubiq- ing a potential ability of mUBPy to regulate the levels of uitinated and is likely degraded by the 26 S proteasome (24); in Mm CDC25 . addition the ubiquitous mammalian Ras GEF, hSos2, is also To test if the human homolog hUBPy is also able to interact rapidly degraded through an ubiquitination step (25). Since we Mm Mm in vivo with CDC25 , we performed similar cotransfection found an interaction between CDC25 and a deubiquitinat- experiments. Briefly Hek-293 cells were cotransfected with ing enzyme (mUBPy), it is reasonable to suppose that Mm Mouse UBPy Interacts with the Ras-GEF CDC25 39453 Mm FIG.7. Stability of CDC25 protein expressed in Hek-293 Mm cells. Hek-293 cells were transfected with pCDNA3-CDC25 (Con- Mm trol) or cotransfected with pCDNA3-CDC25 and pCDNA3-mUBPy Mm (mUBPy) or pCDNA3-CDC25 and pCDNA3-hUBPy-Antisense Mm (AS-hUBPy). For transfection, we used 1 g of pCDNA3-CDC25 and 1.5 g of the other plasmids/60-mm dish with the empty pCDNA3 vector if required. After 24 h, cells were labeled for 2 h with [ S]me- thionine (100 C/dish) and then chased with nonradiolabeled methio- nine for the number of hours indicated, immunoprecipitated with anti- RasGRF1, and processed for SDS-PAGE. The gels were autoradiographed, and the intensity of the specific bands was analyzed Mm by densitometry. A, relative amount of labeled CDC25 immunopre- cipitated after the chase. Open bar, control; black bar, mUBPy; gray bar,  AS-hUBPy. B, autoradiography of the immunoprecipitated la- Mm beled CDC25 after 0, 3, 6, and9hof chase. Mm Mm mUBPy Stabilizes the CDC25 Protein Expressed in Hek- FIG.6. In vivo ubiquitination of CDC25 . Hek-293 cells Mm were cotransfected with the different plasmids (RasGRF1, 293 Cells—The finding that Ras-GRF1/CDC25 is ubiquiti- Mm pCDNA3-CDC25 expressing the full-length CDC25Mm; HA-Ubi, nated in vivo strongly suggests that this protein may be rapidly a pCDNA3 vector expressing HA-tagged ubiquitin; mUBPy, degraded. To measure the stability of the protein, Hek-293 cells pCDNA3-mUBPy expressing the full-length mUPBy). 2 g of each Mm transfected with a vector expressing CDC25 were metabol- plasmid were used, keeping a total of 6 g of DNA/60-mm dish with the empty pCDNA3 vector if required. Cells were lysed in RIPA buffer after ically labeled and subjected to a pulse-chase analysis (Fig. 7). Mm 48 h. A, equal amounts of protein lysates immunoprecipitated with anti The apparent half-life of CDC25 was about 5– 6 h, thus GRF1 antibodies, separated by SDS-PAGE, blotted, and probed with confirming that this protein is unstable, at least in our exper- anti-GRF1 or anti-Ha antibodies. B, panel 1, equal amounts of total imental conditions. proteins (30 g) separated by SDS-PAGE, blotted, and probed with Mm In parallel experiments, we cotransfected Hek-293 cells with anti-GRF1 antibodies. The arrow indicates the 140-kDa CDC25 pro- Mm tein. Panel 2, equal amounts of the same lysates used for panel 1 plasmids expressing CDC25 and mUBPy or with a vector immunoprecipitated with anti-HA antibodies and probed with anti- expressing the antisense of hUBPy (12). The latter was used GRF1 antibodies. A diffuse band corresponding to ubiquitinated Mm because Naviglio et al. (12) showed that the cDNA of hUBPy CDC25 species with an apparent molecular size of 150 –160 kDa is cloned in the antisense orientation increased protein ubiquiti- evident in the second lane and barely detectable in the third lane. nation, likely by interfering with the expression of endogenous hUBPy that it is present in most human cell lines and also in Mm CDC25 could be ubiquitinated and then rapidly degraded. Hek-293 (Fig. 2). Mm Mm To assay in vivo the ubiquitination of CDC25 , we cotrans- Clearly the expression of mUBPy stabilized the CDC25 fected Hek-293 cells with pCDNA3-RasGRF1 and HA-ubiq- protein (Fig. 7), the half-life increases to more than 9 h, uitin vector (HA-Ub) (26). The epitope-tagged ubiquitin can be whereas the expression of antisense of hUBPy further destabi- correctly conjugated in vivo to cellular proteins, which then lized the protein (Fig. 7). These data strongly support the become targets for proteolytic cleavage (26). The HA-ubiquitin hypothesis that mUBPy (and its homolog hUBPy) can regulate Mm allows a good quantitative and sensitive detection of ubiquiti- the turnover of CDC25 . nation of proteins either by immunoblotting or immunoprecipi- DISCUSSION tation with anti-HA monoclonal antibodies (12, 26). As shown in Fig. 6A, immunoprecipitation with anti-Ras- We started this work with the aim to isolate and identify GRF1 antibodies and immunoblotting with anti-HA allows the regulatory proteins able to interact with the large N-terminal Mm detection of high molecular weight HA-immunoreactive spe- part of the brain-specific Ras exchange factor CDC25 (Ras- Mm cies, indicating that CDC25 /Ras-GRF1 is ubiquitinated un- GRF1). At the moment our results partially fulfill this purpose Mm der our experimental conditions. because we have identified a CDC25 interacting protein, a In other experiments, we cotransfected Hek-293 cells with deubiquitinating enzyme called mUBPy, highly homologous to pCDNA3-RasGRF, HA-Ub, and also with a plasmid expressing the human hUBPy as described by Naviglio et al. (12). Mm mUBPy (Fig. 6B). The amount of CDC25 present in total At first glance, a deubiquitinating enzyme should not be extracts clearly decreased in the presence of HA-Ub, suggesting directly involved in signal transduction mechanisms, but actu- that an increased availability of ubiquitin might induce a faster ally there is increasing evidence in the literature that deubiq- Mm turnover of CDC25 protein, whereas a partial recovery was uitinases may play relevant roles in several pathways control- observed in cotransfection with mUBPy (Fig. 6B, panel 1). ling growth and/or differentiation. In fact the deubiquitinating Mm More interestingly, the amount of ubiquitinated CDC25 enzymes are thought to act also through the stabilization of forms detected with anti-RasGRF1 antibodies on HA-immuno- some key components of the pathway, counterbalancing the precipitates clearly decreased in the presence of mUBPy (Fig. activity of specific ubiquitinating factor(s) (27). It is worthwhile 6B, panel 2, third lane; compare third lane with second lane). to mention that the faf gene product, involved in eye develop- Mm 39454 Mouse UBPy Interacts with the Ras-GEF CDC25 ment in Drosophila (28). In yeast the UBP3 and UBP4 (named pate in the deubiquitination processes of proteasome sub- also DOA4) gene products, are involved in transcriptional reg- strates (12), whereas in the tissues where mUBPy is strongly ulation (21, 29), whereas DUB1 protein is required for cell cycle expressed (brain and testis), it could exert a more specialized progression in mouse lymphocytes (30), and UNP deubiquiti- function through its interaction with specific substrates. One of nase causes tumors in nude mice (31) and is overexpressed in these substrates can reasonably be the brain-specific Ras ex- Mm human small cell lung primary carcinoma (32). These few ex- change factor CDC25 /Ras-GRF1. amples indicate that deubiquitinating enzymes could interact Acknowledgments—We thank Stan Hollenberg for the gift of mouse in a specific way with a limited variety of substrates. Indeed, embryo cDNA libraries and pBTM116 vector and Giulio Draetta for more than 90 deubiquitinating enzymes have been so far iden- HA-ubiquitin vector and for pCDNA3-hUBPy. tified (27), and this may explain the marked heterogeneity in their sequence, apart from the catalytic domain. This peculiar- REFERENCES ity is present also in mUBPy; in fact its large N-terminal region 1. Martegani, E., Vanoni, M., Zippel, R., Coccetti, P., Brambilla, R., Ferrari, C., Sturani, E., and Alberghina, L. (1992) EMBO J. 11, 2151–2157 (aa 1–700) shows no homology with any other known deubiq- 2. Cen, H., Papageorge, A. C., Zippel, R., Lowy, D. R., and Zhang, K. (1992) uitinase with the exception of hUBPy, clearly a human homo- EMBO J. 11, 4007– 4015 3. Farnsworth, C. L., Freshney, N. W., Rosen, L. B., Ghosh, A., Greenberg, M. E., log of the mouse protein. and Feig, L. A. (1995) Nature 376, 524 –527 Our results indicate that mUBPy interacts both in vitro and 4. 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(1999) Proc. Natl. Acad. Sci. U. S. A. by coexpression of mUBPy. 96, 4826 – 4831 10. Anborgh, P. H., Qian, X. L., Papageorge, A. G., Vass, W. C., DeClue, J. E., and Furthermore the expression of mUBPy stabilizes the Mm Lowy, D. R. (1999) Mol. Cell. Biol. 19, 4611– 4622 CDC25 protein expressed in Hek-293 increasing its half-life. 11. Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993) Cell 74, 205–214 Therefore we could hypothesize that mUBPy can take part in 12. Naviglio, S., Matteucci, C., Matoskova, B., Nagase, T., Nomura, N., Di Fiore, Mm P. P., and Draetta, G. (1998) EMBO J. 17, 3241–3250 the regulation of CDC25 turnover in neurons or alterna- 13. Mount, D. W., and Conrad, B. (1984) Nucleic Acids Res. 12, 811– 818 tively play a more general role in the control of proteasome 14. Altschul, S. F., Madden, T. L., Scha ¨ ffer, A. A., Zhang, J., Zhang, Z., Miller, W., function as previously suggested (12). Further work is needed and Lipman, D. J. 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Cloning and Characterization of Mouse UBPy, a Deubiquitinating Enzyme That Interacts with the Ras Guanine Nucleotide Exchange Factor CDC25Mm/Ras-GRF1

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 42, Issue of October 19, pp. 39448 –39454, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Cloning and Characterization of Mouse UBPy, a Deubiquitinating Enzyme That Interacts with the Ras Guanine Nucleotide Exchange Mm Factor CDC25 /Ras-GRF1* Received for publication, April 18, 2001, and in revised form, July 31, 2001 Published, JBC Papers in Press, August 10, 2001, DOI 10.1074/jbc.M103454200 Nerina Gnesutta‡§, Michela Ceriani¶§, Metello Innocenti‡, Isabella Mauri‡, Renata Zippel‡, Emmapaola Sturani‡, Barbara Borgonovo, Giovanna Berruti, and Enzo Martegani¶** From the ‡Dipartimento di Fisiologia e Biochimica Generali, Universita ` di Milano, Via Celoria 26, Milano, Italy 20133, ¶Dipartimento di Biotecnologie e Bioscienze, Universita ` di Milano-Bicocca, Piazza della Scienza 2, Milano, Italy 20126, and Dipartimento di Biologia, Universita ` degli Studi di Milano, Milano, Italy 20133 Mm We used yeast “two-hybrid” screening to isolate brain-specific Ras-GEF, called CDC25 (1–2) or Ras-GRF1. Mm cDNA-encoding proteins interacting with the N-termi- CDC25 is a large protein (140 kDa) that contains a Ras- nal domain of the Ras nucleotide exchange factor exchange domain in the C-terminal region and several differ- Mm CDC25 . Three independent overlapping clones were ent domains in the large N-terminal region, namely two PH isolated from a mouse embryo cDNA library. The full- (pleckstrin homology) domains, one DH (Dbl homology) do- length cDNA was cloned by RACE-polymerase chain re- main, and an illimaquinone (IQ) domain (2–3). In addition a action. It encodes a large protein (1080 amino acids) coiled-coil region and a PEST sequence were identified (4). highly homologous to the human deubiquitinating en- Several evidences indicate that the large N-terminal region zyme hUBPy and contains a well conserved domain typ- Mm of CDC25 has a regulatory function and may interact with ical of ubiquitin isopeptidases. Therefore we called this other cellular components. We have previously shown that the new protein mouse UBPy (mUBPy). Northern blot anal- expression in mouse fibroblasts of a truncated form of ysis revealed a 4-kilobase mRNA present in several Mm CDC25 , lacking the Ras exchange domain, behaves as a mouse tissues and highly expressed in testis; a good dominant negative protein (5). In addition PH domains could level of expression was also found in brain, where Mm interact with phospholipids (6) and with the  subunits of CDC25 is exclusively expressed. Using a glutathione heterotrimeric G proteins (7), whereas the IQ domain binds S-transferase fusion protein, we demonstrated an “in calmodulin (3, 8) and is thought to be responsible for the vitro” interaction between mUBPy and the N-terminal Mm activation of GEF activity by calcium (3). Further evidence that half (amino acids 1– 625) of CDC25 . In addition “in Mm vivo” interaction was demonstrated after cotransfection the N-terminal region of CDC25 protein is involved in spe- Mm cific protein interaction(s) also comes from the work of Kiyono in mammalian cells. We also showed that CDC25 ,ex- Mm pressed in HEK293 cells, is ubiquitinated and that the et al. (9) in which it was shown that CDC25 /Ras-GRF1 was coexpression of mUBPy decreases its ubiquitination. In able to activate Rac1 and that for this activity a functional DH addition the half-life of CDC25Mm protein was consid- domain is required. Moreover the DH domain was required for erably increased in the presence of mUBPy. The specific homodimerization of Ras-GRF1 or for heterodimerization with function of the human homolog hUBPy is not defined, Ras-GRF2 (10) Mm although its expression was correlated with cell prolif- However, so far, only the specific interaction CDC25 /cal- eration. Our results suggest that mUBPy may play a role modulin has been demonstrated in vivo (3, 8). In an effort to Mm in controlling degradation of CDC25 , thus regulating identify mammalian proteins that could interact with the large the level of this Ras-guanine nucleotide exchange factor. Mm N-terminal region of CDC25 , we used a yeast “two-hybrid” system for the screening of mouse embryo cDNA libraries (11). 1 Here we report that the cloning and characterization of a cDNA Ras-guanine nucleotide exchange factors (GEFs) are pro- that was positive in this screening. This cDNA was found to teins that stimulate the exchange of guanine nucleotides (GDP/ encode a new deubiquitinating enzyme belonging to the ubiq- GTP) on Ras proteins. We have previously cloned a mouse uitin isopeptidase family (UBPs) and was highly homologous to This is an open access article under the CC BY license. the recently identified human enzyme hUBPy (12); therefore * This work was supported by C.E. Contract BIO4-CT98 – 0297, Con- we called this new cDNA mouse UBPy (mUBPy). siglio Nazionale delle Ricerche Target project on Biotechnology (to E. S.), and MURST 40%-Cofin 2000 (to R. Z.). The costs of publication of EXPERIMENTAL PROCEDURES this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance Two-hybrid Screening—The system developed by Vojtek et al. (11) with 18 U.S.C. Section 1734 solely to indicate this fact. was used for the two-hybrid screening. Two mouse embryo cDNA li- The nucleotide sequence(s) reported in this paper has been submitted braries (9.5 and 10.5 dpc) constructed in pVP16 vector (a gift of Stan TM to the GenBank /EBI Data Bank with accession number(s) AF057146. Hollenberg) were used. Three bait plasmids were prepared by cloning § These authors contributed equally to the work. Mm fragments of the N-terminal region of CDC25 in the pBTM116 vec- ** To whom correspondence should be addressed: Dipartimento di tor, which contained the Lex-A DNA binding domain (11). Biotecnologie e Bioscienze, Piazza della Scienza 2, 20126 Milano, Italy. Mm The first bait plasmid (pBTEE) contains 1877 bp of CDC25 IV Tel.: 39-02-64483533; Fax: 39-02-64483565; E-mail: enzo.martegani@ (2), which corresponds to the first 625 amino acids; this region includes unimib.it. the first PH domain, the IQ motif, the Dbl homology (DH) domain, and The abbreviations used are: Ras-GEF, guanine nucleotide exchange Mm the second PH domain. pBTES contains 1519 bp of CDC25 IV, factor for Ras; UBP, ubiquitin isopeptidase; DH, Dbl homolog; PH, corresponding to the first 506 amino acids, and lacks the second PH pleckstrin homolog; IQ, illimaquinone; bp, base pair(s); aa, amino ac- domain, whereas pBTEP contains 436 bp, corresponding to the first 145 id(s); GST, glutathione S-transferase; HA, hemagglutinin; RIPA, radio- immune precipitation; dpc, days post-coitus. amino acids, and contains only the first PH domain. 39448 This paper is available on line at http://www.jbc.org Mm Mouse UBPy Interacts with the Ras-GEF CDC25 39449 The yeast strain L40 (MATa, his3, trp1, leu2, ade2, 1:1000). Bound antibodies were detected with peroxidase-conjugated LYS2::(lexAop) -HIS3, URA3::(lexAop) -lacZ) was transformed with the goat anti-rabbit immunoglobulins and revealed by the ECL detection 4 8 bait vector (initially pBTEE) and with the mouse embryo cDNA librar- system (Amersham Pharmacia Biotech). ies. Selection was performed as described by Vojtek et al. (11) in selec- Cells cultures were harvested, washed with phosphate-buffered sa- tive minimal plates containing 5 mM 3-aminotriazole. Positive clones, line, and lysed in RIPA buffer (Tris-HCl, 50 mM; NaCl, 150 mM; sodium obtained only by the 10.5-dpc library, were also tested for -galactosid- deoxycholate, 0.5% w/v; SDS, 0.1% w/v; Triton X-100, 1% w/v) contain- ase activity. Plasmids were recovered from positive clones and used for ing protease inhibitors (leupeptin, 1 M; aprotinin, 0.1 M; sodium a new round of screening to test their positivity. Positive clones were vanadate, 0.1 mM; phenylmethylsulfonyl fluoride, 0.1 mM). Aliquots of sequenced and then tested with the other plasmids containing reduced cleared cell lysate were separated on SDS-polyacrylamide gels, trans- Mm regions of CDC25 (pBTES and pBTEP). ferred to nitrocellulose, and immunoblotted with anti-mUBPy RACE-PCR, Cloning, Plasmids, Sequencing, and Northern Blot Hy- antibodies. bridization—RACE reactions were done using Marathon-Ready cDNA Cell Culture and Transfection—NIH-3T3, COS7, and Hek-293 cells (CLONTECH) from 11.5-dpc mouse embryo and a mix of polymerases were cultured in Dulbecco’s modified Eagle’s medium supplemented (Advantage Klen-Taq Mix, CLONTECH) suitable for amplification of with 10% fetal bovine serum. Subconfluent cells were transfected using long regions of DNA with high fidelity. The PCR conditions were set and the LipofectAMINE (Life Technologies, Inc.) method according to the performed according to the protocols given by CLONTECH. Four oligo- protocol of the manufacturer. nucleotides were used, two for 3 RACE and two for 5 RACE using a Pull-down Experiments—48 h after transfection, COS7 cells were nested PCR procedure to obtain an increased specificity. For 3 RACE, harvested, washed with phosphate-buffered saline, and lysed in RIPA the external primer was GSS1 (5-GCTCAGCGAGAACCTTTGAC- buffer or in lysis buffer (HNGT) (Hepes, 25 mM; NaCl, 50 mM; glycerol, GAGAGC-3), and the internal one was GSS2 (5-CCTGAAATGGCTC- 10% w/v; Triton X-100, 1% w/v). The buffers contained protease CTCGTCTGCACC-3). For 5 RACE, the external primer was GSA1 inhibitors. (5-GGTGCAGACGAAGGAGCCATTTCAGG-3), and the internal one Aliquots of lysates (450 l) were then incubated with 50 l of gluta- was GSA2 (5-GCTCTCGTCAAAGGTTCTCGCTGAGC-3). thione-Sepharose resin loaded either with GST-mUBPy fusion 525– 660 Two oligonucleotides (i.e. GSS1 and GSA1) were also used in a protein or with GST alone for2hat4 °C. After incubation, the resin control reaction to verify the presence of the specific cDNA in the pool. was recovered by gentle centrifugation and washed five times with the Positivity was revealed by a 176-bp fragment. incubation buffer (RIPA or HGNT). At the end, the resin was treated The amplification products of 3 and 5 RACE were cloned in pMOS- with 30 lof2 SDS sample buffer and boiled for 10 min, and the Blue (Amersham Pharmacia Biotech) and sequenced. Three clones were recovered proteins were used for Western blot analysis. sequenced both for 3 and 5 amplification products. Coimmunoprecipitation and Immunoblotting—48 h after transfec- Sequencing was performed on both strands using the Taq Dye Ter- tion, cells (COS7 or Hek-293) were harvested and lysed in 500 l/plate minator Cycle Sequencing Kit (Applied Biosystems) with universal and (100 mm) of an ice-cold HNGT buffer as described (18). Immunoprecipi- walking primers and using an automated DNA sequence analyzer (Ap- tation from cleared lysates was performed with monoclonal anti-HA-11 plied Biosystems 373A). The full-length cDNA was obtained by using antibody (BAbCO) or with anti-mUBPy polyclonal antibodies for1hat the unique NdeI site present in the overlapping region and was sub- 4 °C. Immunocomplexes were recovered with protein G-Sepharose or cloned in pCDNA3 vector (Invitrogen). with protein A-Sepharose (Sigma) for1hat4 °C and then resuspended pCDNA3-mUBPy-HA vector was prepared by ligation of a cDNA in SDS sample buffer and analyzed by immunoblot analysis. Briefly ⁄50 fragment of 2.5 kilobase pairs comprising the coding sequence of aa of cleared cell lysates and the immunoprecipitates were resolved by 10% 542–1080 of mUBPy in pBSKSII-HA vector (Stratagene), and then a SDS-PAGE and transferred to nitrocellulose membranes. Membranes SphI-EcoRI fragment coding for a fusion between HA tag and the aa were probed with anti Ras-GRF1 (C-20, Santa Cruz, CA) or with poly- 542–1080 of mUBPy was subcloned in pCDNA3 plasmid. HA-ubiquitin clonal anti-mUBPy raised in our laboratory. Immunocomplexes were vector and pCDNA3-hUBPy plasmid were obtained by G. Draetta (12). detected by enhanced chemiluminescence (Amersham Pharmacia Bio- For Northern blots, polyadenylated RNAs from mouse tissues (total tech) using goat anti-rabbit antiserum coupled to horseradish peroxi- mouse RNA, Ambion) were separated on agarose-formaldehyde gels (5 dase as secondary antibodies (Cappel). g/lane), blotted to a Hybond-N nylon membrane (Amersham Pharma- Deubiquitination Assays—We used a mouse testis extract as a source cia Biotech), and hybridized with a digoxygenin-labeled riboprobe. Sig- of ubiquitinated protein as described in Ref. 19 with minor modifica- nals were detected with the Nucleic Acid Detection kit (Roche Molecular tions. The cleared extracts were dialyzed against Tris-HCl 50 mM, Biochemicals). The probe was prepared by subcloning a fragment of MgCl 5mM, and dithiothreitol 2 mM. The sample was then centrifuged mUBPy cDNA in a pGEM3z vector (Promega) and in vitro transcription and washed several times in a Centricon-3 concentrator (Amicon) to with T7 RNA polymerase and digoxygenin-labeled UTP (Roche Molec- remove residual N-ethylmaleimide (12). Aliquots (50 g) of the proteins ular Biochemicals) according to the manufacturer’s instructions. were then incubated with immunoprecipitates (anti-HA antibodies) Sequence Analysis—The searches for open reading frames and their from COS7 cells transfected with pCDNA3-mUBPy-HA vector at 37 °C conceptual translations were performed with DM software (13). The and processed as described in Ref. 19 using anti-ubiquitin antibodies homology search was done at the BLAST (14) server at NCBI (www. (Sigma). Mm ncbi.nlm.nih.gov/BLAST/). Protein alignments were done with MACAW In Vivo Ubiquitination of CDC25 —Hek-293 cells transfected with (15), while the search for motifs in protein was done with Profilescan at the different plasmids (as reported in the figure legends) were lysed in the Expasy server (www.expasy.ch/). RIPA buffer. Immunoprecipitation from the same amount of total pro- Recombinant Protein Production and Preparation of Polyclonal An- tein was performed with antibodies against Ras-GRF1 or anti-HA tibodies—The cDNA contained in one of the positive clones (6/12) was (BAbCO). Immunocomplexes were recovered with protein G-Sepharose subcloned in the SmaI site of the Escherichia coli expression vector (for anti-HA monoclonal antibodies) or protein A-Sepharose (for Ras- pGEX-2T (Amersham Pharmacia Biotech) to obtain a fusion protein GRF1 antibodies) and analyzed in Western blotting with the different between GST and 119 amino acids of mUBPy corresponding to position primary antibodies and then revealed with the corresponding second- 542 to 660 of the whole protein. E. coli DH5 cells bearing the vector for ary antibody and the ECL system. fusion protein were grown in LB broth, induced with 0.1 mM isopropyl- Determination of Stability of CDC25Mm Protein—Hek-293 cells were Mm 1-thio--D-galactopyranoside at 28 °C for 3 h, and collected for protein transiently transfected with 1 g of pCDNA3-CDC25 alone and then extraction. Fusion protein was purified with glutathione-Sepharose cotransfected with 1.5 g of pCDNA3-mUBPy or with 1.5 gof resin as described (16). About 1 mg of purified fusion protein (GST- pCDNA3-human UBPy antisense (12). After 24 h, cells were washed mUBPy ) was used to immunize New Zealand rabbits. Polyclonal two times with minimum Eagle’s medium without methionine. Cells 542– 660 antibodies were then affinity-purified using a GST-coupled Affi-Gel-10 were then incubated for 2 h with minimum Eagle’s medium without column (Bio-Rad) as described (17). For pull-down experiments, the methionine with [ S]methionine (100 C/60-mm dish), Hepes 25 mM, GST-mUBPy fusion protein was used, coupled to glutathione- and fetal calf serum 0.1%. After the incubation, cells were washed two 542– 660 Sepharose beads. times with Dulbecco’s modified Eagle’s medium and further incubated Western Blotting—Tissues from adult CD1 mice (Charles River, with Dulbecco’s modified Eagle’s medium supplemented with fetal bo- Calco (LC), Italy) were mechanically homogenized with 4 volumes of vine serum 10%. Tris (0.125 M, pH 6.8) and SDS (7% w/v). The homogenate was further At the appropriate time, cells were rinsed in phosphate-buffered sonicated in ice and centrifuged (20,000  g for 10 min); the solubilized saline and lysed in RIPA buffer, and the lysates were clarified. Immu- proteins were separated on SDS-polyacrylamide gel electrophoresis noprecipitation from lysates was performed in the presence of Ras- (SDS-PAGE) and transferred to nitrocellulose. Membranes were probed GRF1 antibody (Sigma) for2hat4 °C,andthe immunocomplexes were with affinity-purified anti-mUBPy polyclonal antibodies (diluted recovered with protein A (Sigma) for1hat4 °C with gentle rotation. Mm 39450 Mouse UBPy Interacts with the Ras-GEF CDC25 FIG.1. Amino acid sequence of mUBPy predicted open reading frame and comparison with hUBPy. The sequence in bold is that of the 6/12 clone isolated from two-hybrid screening. The underlined region identifies Cys and His boxes typical of deubiquitinating en- zymes (21, 22); the dotted underlined re- gion (aa 198 –311) represents the rho- danese/cdc25 fold homology (23). The TM sequence was deposited in GenBank , accession number AF057146. The align- ment between hUBPy (12) and mUBPy was done with Macaw (15). Equal numbers of trichloroacetic acid-precipitable counts from cell ex- (not shown). On the basis of these results, we can conclude that tracts were immunoprecipitated. Mm the region of CDC25 between aa 145 and aa 506, containing The immunoprecipitates were washed three times with RIPA buffer the IQ motif and the DH domain, is required for this in vivo solution and then resuspended in 50 l of SDS-sample buffer and interaction. The sequence of the 6/12 clone revealed an open separated by SDS-PAGE. The dry gels were exposed to x-ray films (Amersham Pharmacia Biotech), and the intensity of specific bands was reading frame (3-5 open) encoding a short proline-rich analyzed by densitometry with NIH-Image software. polypeptide (119 aa) homologous to a small central region of a human gene product called hUBPy, originally reported to RESULTS encode a putative deubiquitinating enzyme (20) and recently Screening for Proteins Interacting with the N-terminal demonstrated to be actually a deubiquitinase (12). Mm Region of CDC25 , Identification and Cloning of Mouse The full-length mouse cDNA was cloned by RACE-PCR using UBPy—We used the N-terminal region (first 625 aa) of a mouse embryo-cDNA (Marathon-Ready cDNA, CLONTECH) Mm CDC25 as a bait for a yeast two-hybrid screening. This starting from this central region. Several clones were obtained fragment comprises all the identified motifs except the cata- both for the 3 region and the 5 region and were completely lytic one, i.e. the first PH domain, the IQ domain, and the sequenced. The 3970-bp cDNA contains a complete open read- DH-PH module, and it is the same region that, when expressed ing frame of 3240 nucleotides encoding a 1080 aa protein, 18 bp in mammalian cells, behaves as a dominant negative protein upstream the putative AUG start codon and 713 bp down- (5). For the screening, we used two different mouse embryo stream the stop codon (Fig. 1). Because the open reading frame cDNA libraries in pVP16 vector given by S. Hollenberg (11) shared a great homology (higher than 90%) with the hUBPy, After two rounds of selection, we identified three “true” pos- we called this new mouse protein mouse UBPy (mUBPy). The itive clones, all derived from the 10.5-dpc cDNA library. These homology is very high in the C-terminal region (97% identity clones contained small cDNA inserts (about 400 bp), which between aa 591–1080) that contains the typical hallmarks of shared a common overlapping region of 300 bp. The largest the UBP family of deubiquitinating enzymes, i.e. a well con- clone (called 6/12) was further characterized for its ability to Mm served UCH-2 domain (aa 739 –1072, Profilescan) with the interact in yeast with different shorter regions of CDC25 .A characteristic cysteine and histidine boxes (21, 22). The N- positive interaction was obtained with pBTES plasmid contain- Mm terminal part of mUBPy also contains a highly homologous ing the first 506 aa of CDC25 and thus lacking the second region (88% identity aa 1–346) followed by a region of lower PH domain, whereas no interaction was observed with pBTEP Mm plasmid that contains only the first PH domain of CDC25 homology (68% identity aa 346 –590). A search for motifs in this Mm Mouse UBPy Interacts with the Ras-GEF CDC25 39451 FIG.3. mUBPy (aa 542–1080), expressed in COS7 cells, can remove ubiquitin from endogenous ubiquitinated proteins. Mouse testis extracts were prepared by homogenizing testes in the presence of 20 mM N-ethylmaleimide to inactivate endogenous deubiq- uitinating enzymes (19). Soluble fractions were dialyzed against 50 mM Tris (pH 8), 5 mM MgCl ,and2mM dithiothreitol and then centrifuged and washed four times in a Centricon-3 concentrator to remove residual N-ethylmaleimide. Aliquots containing 50 g of total proteins were incubated for 15 or 30 min with an HA-immunoprecipitate obtained from COS7 cells transfected with pCDNA3-mUBPy-HA vector (4 g/ 100-mm plate) expressing mUBPy tagged with HA epitope. The 542–1080 immunoprecipitates of cleared lysate obtained from two dishes were FIG.2. Expression of mUBPy in mouse tissues and cell lines. A, washed three times in HNGT buffer, and the equal aliquots (about ⁄4 of Northern blot analysis. 5 g of total RNA extracted from various mouse the total) were used for each assay. As a control, an immunoprecipitate tissues were separated on agarose-formaldehyde gels, blotted to a nylon was washed in the presence of 20 mM N-ethylmaleimide ( NEM). At membrane, and hybridized with a digoxygenin-labeled mUBPy ribo- the indicated times, samples were quenched with SDS-sample buffer probe. Signals were detected with the Nucleic Acid Detection Kit (Roche and analyzed by immunoblotting with anti-ubiquitin antibodies. Molecular Biochemicals). Lane 1, liver; lane 2, lung; lane 3, thymus; lane 4, heart; lane 5, 11.5-dpc embryo; lane 6, spleen; lane 7, testis; lane 8, ovary; lane 9, kidney; lane 10, brain. The amount of RNA loaded in of a strong specific immunoreactive band of 120 kDa (in agree- each lane was comparable, as indicated by staining of the gel with ethidium bromide (not shown). B, total proteins extracted from adult ment with the expected molecular size of the mUBPy open mouse tissues, separated on SDS-PAGE 7% gels, blotted to nitrocellu- reading frame: 122,579 Da) in mouse brain and testis homoge- lose, and probed with anti-mUBPy antibodies. A strong 120-kDa band nates (Fig. 2B). A lower signal was observed in pancreas, lung, was observed in brain and testis. About 50 g of protein were loaded in small intestine, and placenta homogenates, whereas a barely each lane. Lane 1, brain; lane 2, lung; lane 3, heart; lane 4, liver; lane 5, detectable signal was present in liver, spleen, kidney, and spleen; lane 6, gut; lane 7, kidney; lane 8, pancreas; lane 9, ovary; lane 10, placenta; lane 11, testis. C, proteins extracted from cultured cells, heart (Fig. 2B). These results are generally in agreement with separated on SDS-PAGE gels, blotted to nitrocellulose, and probed with the results obtained with Northern blot experiments. Therefore anti-mUBPy antibodies. Lane 1, growing NIH-3T3; lane 2, confluent the mUBPy protein is expressed at a very low level in most of NIH-3T3; lane 3, human neuroblastoma cells SK-N-BE; lane 4, human the adult mouse tissues with a strong expression in brain and Hek-297 cells. About 50 g of protein were loaded in each lane. testis only. Moreover, mUBPy is not an easily soluble protein N-terminal region revealed only a highly conserved Rhodanese/ because to detect it in most tissues, we needed strong detergent cdc25 fold (aa 198 –311) (23). Interestingly the Rhodanese/ conditions (i.e. 7% SDS). When a mild extraction procedure (1% cdc25 fold has been found in the noncatalytic region of several Triton X-100) was used, mUBPy could be detected only in brain yeast ubiquitin hydrolases (yeast Ubp4, 5 and 7), in the non- and testis (not shown). Since the human homolog hUBPy is catalytic domain of dual specificity MAPK-phosphatases, and expressed in several cell lines (12), we also looked for the in the yeast phosphotyrosine phosphate-phosphatase (23). expression of the mUBPy protein in mouse fibroblasts; more- Other interesting features of mUBPy protein are the presence over we also probed two human cell lines with our antibodies. of two long proline-rich regions (aa 300 – 450 and aa 560 –740, A 120-kDa band was detected in the mouse NIH-3T3 fibro- respectively), which contain several PEST-like motifs, and two blasts extract (Fig. 2C, lanes 1 and 2); however, no difference coiled-coil regions (aa 95–140 and 460 –500). was observed between proliferating and density-arrested NIH- Expression of mUBPy in Mouse Tissues—The human homo- 3T3 fibroblasts. In addition a sharp 130-kDa band was detected log hUBPy was initially identified as the predicted product of a in both Hek-293 cells and SN-K-BE neuroblastoma cells, indi- cDNA found in myeloblasts (20). Successively, Naviglio et al. cating that our antibodies also recognize the human hUBPy (12) found the protein in several human cell lines (human protein. This was not unexpected because the protein region we fibroblasts WI-38, osteosarcoma U2OS, HeLa cells, etc.). used for the preparation of anti-mUBPy polyclonal antibodies Northern blot analysis performed on RNA extracted from contains a large amino acid sequence (aa 592– 657 of mUBPy, several mouse tissues evidenced the presence of a 4-kilobase see Fig. 1), which is identical in the two proteins. mUBPy mRNA in most of the probed tissues (Fig. 2A). As mUBPy Is a Deubiquitinating Enzyme—Since mUBPy has a expected, this mRNA was also present in mouse embryo and in high homology with hUBPy that has been demonstrated to adult mouse brain; however, mUBPy mRNA was found to be have a UBP (ubiquitin-isopeptidase) activity (12), we expected particularly abundant in testis. that also mUBPy has an UBP activity. To verify this, we used Rabbit polyclonal antibodies were raised against a GST fu- a mouse testis protein extract, pretreated with N-ethylmaleim- sion protein (containing 119 aa of mUBPy) expressed in E. coli. ide, as a source of ubiquitinated proteins as reported by Lin et The antiserum was treated with immobilized GST to remove al. (19). As shown in Fig. 3, the addition of HA-immunoprecipi- the anti-GST component and then used for Western blot tates from COS7 cells transfected with HA-tagged mUBPy (aa experiments. 542–1080) expressing vector greatly reduced the amount of An immunoscreening of mouse tissues revealed the presence ubiquitinated protein recognized by anti-ubiquitin antibodies, Mm 39452 Mouse UBPy Interacts with the Ras-GEF CDC25 FIG.4. In vitro interaction of mUBPy (aa 542– 660) with the Mm N-terminal domain of CDC25 (aa 1– 625). COS7 cells transfected Mm with pHC21 vector, expressing the N-terminal region of CDC25 (aa 1– 625) (5),(5 g/100-mm plate), or untransfected COS7 cells were lysed with HGNT buffer. Lysates were incubated with glutathione-Sepharose resin loaded with either GST-mUBPy or GST alone as described 542– 660 (see “Experimental Procedures”). Protein bound to the resin was recov- ered and processed for Western blot analysis with anti Ras-GRF1 antibodies. A, Western blot analysis of protein bound to glutathione- Sepharose beads coupled to GST-mUBPy (lanes 1 and 3)orGST 542– 660 alone (lanes 2 and 4). Lanes 1 and 2, control COS7 cells; lanes 3 and 4, Mm extracts of COS7 cells expressing CDC25 . B, immunodetection 1– 625 Mm of CDC25 on extracts of COS7 cells. Lane 1, total cell protein 1– 625 (lysis in RIPA buffer) of control COS7 cells; lane 2, total cell protein of Mm COS7 cells expressing CDC25 ; lane 3, lysis buffer (HGNT) ex- 1– 625 Mm tract of COS7 cells expressing CDC25 . The arrows indicate the 1– 625 Mm 70-kDa band expected for CDC25 . IP, immunoprecipitate. 1– 625 Mm FIG.5. In vivo interaction between CDC25 and mUBPy and hUBPy. A, COS7 cells were transfected with 4 g/100 mm plate of indicating that mUBPy can deubiquitinate ubiquitin from high Mm the indicated vectors (GRF1, pCDNA3-CDC25 expressing the full- molecular weight ubiquitinated proteins. Mm length CDC25 protein; mUBPy, pCDNA3-mUBPy-HA expressing Mm Interaction of mUBPy with CDC25 —We have previously mUBPy tagged with HA epitope) using pCDNA3 when required 542–1080 identified mUBPy in a two-hybrid screening for protein inter- to keep the total DNA to 8 g/plate. After 48 h, cells were harvested and Mm lysed in HGNT buffer, and lysates were immunoprecipitated with an- action with the Ras-GEF CDC25 . It was, however, impor- ti-HA antibodies. Immunoprecipitated proteins (IPP) and equal ali- tant to confirm this interaction directly in vitro and succes- quots of cleared total lysates (30 l) were separated by SDS-PAGE, sively in an in vivo assay using mammalian cells. blotted, and probed with either anti-GRF1 antibodies or anti-mUBPy A GST fusion protein containing the 119 aa of the clone 6/12 antibodies. B, Hek-293 cells were transfected with 4 g/100-mm plate of Mm found to be positive in the yeast two-hybrid screening was the indicated vectors (GRF1, pCDNA3-CDC25 expressing the full- Mm length CDC25 protein; hUBPy, pCDNA3-hUBPy expressing hUBPy tested for in vitro interaction with the N-terminal region of Mm (12)) using pCDNA3 when required to keep the total DNA to 8 g/plate. CDC25 by pull-down experiments. As shown in Fig. 4,a After 48 h, cells were harvested and lysed in HGNT buffer, and lysates Mm specific interaction with the CDC25 N-terminal fragment were immunoprecipitated with anti-mUBPy antibodies. Immunopre- can be revealed. This result is in agreement with that obtained cipitated proteins were separated by SDS-PAGE, blotted, and probed with either anti-GRF1 antibodies or anti-mUBPy antibodies. by the yeast two-hybrid system. To validate the above results, we tested the in vivo interac- Mm tion between CDC25 and mUBPy by co-expressing the two pCDN3-RasGRF1 and pCDNA3-hUBPy, and then the cell ly- Mm proteins in mammalian cells. CDC25 and a HA-tagged sates were immunoprecipitated with anti-mUPBy antibodies mUBPy (aa 542–1080) were transfected in COS7 cells either and immunoblotted with anti-GRF1 antibodies. As shown in Mm separately or together. 48 h after transfection, lysates were Fig. 5B, CDC25 was coimmunoprecipitated only if hUBPy prepared and processed for immunoprecipitation with anti-HA was expressed; this indicates that the human homolog is also Mm antibodies. The recovered immunocomplexes were separated able to interact with CDC25 . When the same filter was by SDS-PAGE and blotted. The blots were probed with anti- probed with anti-mUBPy antibodies, a strong band was de- Ras-GRF1 antibodies and with polyclonal antibodies raised tected in extracts of cells transfected with hUBPy, whereas a Mm against mUBPy. Fig. 5A shows that CDC25 was coimmuno- weak immunoreactive band was also detected in cells not precipitated only from extracts obtained by cells expressing treated with pCDNA3-hUBPy, which is likely due to endoge- Mm both CDC25 and HA-mUBPy (lane 3) but not from extracts nous hUBPy protein. Mm Mm of cells transfected with CDC25 alone or with HA-mUBPy CDC25 Is Ubiquitinated in Vivo and mUBPy Decreases Its alone (lanes 1–2). Aliquots of the same total cell lysates were Level of Ubiquitination—As a general rule, regulatory proteins Mm loaded in the same gel to check the expression of CDC25 are subjected to a fast turnover, and this is expected to be the (lane 4), HA-mUBPy (lane 5), and both proteins in cotrans- case also for the Ras exchange factors. At our knowledge, no Mm fected cells (lane 6). This experiment clearly demonstrates an data regarding the turnover of CDC25 /RasGRF1 have been Mm in vivo interaction between CDC25 and mUBPy. Interest- so far reported in the literature, although RasGRF1 is known to Mm ingly the amount of total CDC25 protein found in COS7 cells contain a PEST motif (4). However, it has been recently shown lysates increased when the mUBPy was co-expressed, suggest- that RasGRF2, a protein closely related to RasGRF1, is ubiq- ing a potential ability of mUBPy to regulate the levels of uitinated and is likely degraded by the 26 S proteasome (24); in Mm CDC25 . addition the ubiquitous mammalian Ras GEF, hSos2, is also To test if the human homolog hUBPy is also able to interact rapidly degraded through an ubiquitination step (25). Since we Mm Mm in vivo with CDC25 , we performed similar cotransfection found an interaction between CDC25 and a deubiquitinat- experiments. Briefly Hek-293 cells were cotransfected with ing enzyme (mUBPy), it is reasonable to suppose that Mm Mouse UBPy Interacts with the Ras-GEF CDC25 39453 Mm FIG.7. Stability of CDC25 protein expressed in Hek-293 Mm cells. Hek-293 cells were transfected with pCDNA3-CDC25 (Con- Mm trol) or cotransfected with pCDNA3-CDC25 and pCDNA3-mUBPy Mm (mUBPy) or pCDNA3-CDC25 and pCDNA3-hUBPy-Antisense Mm (AS-hUBPy). For transfection, we used 1 g of pCDNA3-CDC25 and 1.5 g of the other plasmids/60-mm dish with the empty pCDNA3 vector if required. After 24 h, cells were labeled for 2 h with [ S]me- thionine (100 C/dish) and then chased with nonradiolabeled methio- nine for the number of hours indicated, immunoprecipitated with anti- RasGRF1, and processed for SDS-PAGE. The gels were autoradiographed, and the intensity of the specific bands was analyzed Mm by densitometry. A, relative amount of labeled CDC25 immunopre- cipitated after the chase. Open bar, control; black bar, mUBPy; gray bar,  AS-hUBPy. B, autoradiography of the immunoprecipitated la- Mm beled CDC25 after 0, 3, 6, and9hof chase. Mm Mm mUBPy Stabilizes the CDC25 Protein Expressed in Hek- FIG.6. In vivo ubiquitination of CDC25 . Hek-293 cells Mm were cotransfected with the different plasmids (RasGRF1, 293 Cells—The finding that Ras-GRF1/CDC25 is ubiquiti- Mm pCDNA3-CDC25 expressing the full-length CDC25Mm; HA-Ubi, nated in vivo strongly suggests that this protein may be rapidly a pCDNA3 vector expressing HA-tagged ubiquitin; mUBPy, degraded. To measure the stability of the protein, Hek-293 cells pCDNA3-mUBPy expressing the full-length mUPBy). 2 g of each Mm transfected with a vector expressing CDC25 were metabol- plasmid were used, keeping a total of 6 g of DNA/60-mm dish with the empty pCDNA3 vector if required. Cells were lysed in RIPA buffer after ically labeled and subjected to a pulse-chase analysis (Fig. 7). Mm 48 h. A, equal amounts of protein lysates immunoprecipitated with anti The apparent half-life of CDC25 was about 5– 6 h, thus GRF1 antibodies, separated by SDS-PAGE, blotted, and probed with confirming that this protein is unstable, at least in our exper- anti-GRF1 or anti-Ha antibodies. B, panel 1, equal amounts of total imental conditions. proteins (30 g) separated by SDS-PAGE, blotted, and probed with Mm In parallel experiments, we cotransfected Hek-293 cells with anti-GRF1 antibodies. The arrow indicates the 140-kDa CDC25 pro- Mm tein. Panel 2, equal amounts of the same lysates used for panel 1 plasmids expressing CDC25 and mUBPy or with a vector immunoprecipitated with anti-HA antibodies and probed with anti- expressing the antisense of hUBPy (12). The latter was used GRF1 antibodies. A diffuse band corresponding to ubiquitinated Mm because Naviglio et al. (12) showed that the cDNA of hUBPy CDC25 species with an apparent molecular size of 150 –160 kDa is cloned in the antisense orientation increased protein ubiquiti- evident in the second lane and barely detectable in the third lane. nation, likely by interfering with the expression of endogenous hUBPy that it is present in most human cell lines and also in Mm CDC25 could be ubiquitinated and then rapidly degraded. Hek-293 (Fig. 2). Mm Mm To assay in vivo the ubiquitination of CDC25 , we cotrans- Clearly the expression of mUBPy stabilized the CDC25 fected Hek-293 cells with pCDNA3-RasGRF1 and HA-ubiq- protein (Fig. 7), the half-life increases to more than 9 h, uitin vector (HA-Ub) (26). The epitope-tagged ubiquitin can be whereas the expression of antisense of hUBPy further destabi- correctly conjugated in vivo to cellular proteins, which then lized the protein (Fig. 7). These data strongly support the become targets for proteolytic cleavage (26). The HA-ubiquitin hypothesis that mUBPy (and its homolog hUBPy) can regulate Mm allows a good quantitative and sensitive detection of ubiquiti- the turnover of CDC25 . nation of proteins either by immunoblotting or immunoprecipi- DISCUSSION tation with anti-HA monoclonal antibodies (12, 26). As shown in Fig. 6A, immunoprecipitation with anti-Ras- We started this work with the aim to isolate and identify GRF1 antibodies and immunoblotting with anti-HA allows the regulatory proteins able to interact with the large N-terminal Mm detection of high molecular weight HA-immunoreactive spe- part of the brain-specific Ras exchange factor CDC25 (Ras- Mm cies, indicating that CDC25 /Ras-GRF1 is ubiquitinated un- GRF1). At the moment our results partially fulfill this purpose Mm der our experimental conditions. because we have identified a CDC25 interacting protein, a In other experiments, we cotransfected Hek-293 cells with deubiquitinating enzyme called mUBPy, highly homologous to pCDNA3-RasGRF, HA-Ub, and also with a plasmid expressing the human hUBPy as described by Naviglio et al. (12). Mm mUBPy (Fig. 6B). The amount of CDC25 present in total At first glance, a deubiquitinating enzyme should not be extracts clearly decreased in the presence of HA-Ub, suggesting directly involved in signal transduction mechanisms, but actu- that an increased availability of ubiquitin might induce a faster ally there is increasing evidence in the literature that deubiq- Mm turnover of CDC25 protein, whereas a partial recovery was uitinases may play relevant roles in several pathways control- observed in cotransfection with mUBPy (Fig. 6B, panel 1). ling growth and/or differentiation. In fact the deubiquitinating Mm More interestingly, the amount of ubiquitinated CDC25 enzymes are thought to act also through the stabilization of forms detected with anti-RasGRF1 antibodies on HA-immuno- some key components of the pathway, counterbalancing the precipitates clearly decreased in the presence of mUBPy (Fig. activity of specific ubiquitinating factor(s) (27). It is worthwhile 6B, panel 2, third lane; compare third lane with second lane). to mention that the faf gene product, involved in eye develop- Mm 39454 Mouse UBPy Interacts with the Ras-GEF CDC25 ment in Drosophila (28). In yeast the UBP3 and UBP4 (named pate in the deubiquitination processes of proteasome sub- also DOA4) gene products, are involved in transcriptional reg- strates (12), whereas in the tissues where mUBPy is strongly ulation (21, 29), whereas DUB1 protein is required for cell cycle expressed (brain and testis), it could exert a more specialized progression in mouse lymphocytes (30), and UNP deubiquiti- function through its interaction with specific substrates. One of nase causes tumors in nude mice (31) and is overexpressed in these substrates can reasonably be the brain-specific Ras ex- Mm human small cell lung primary carcinoma (32). These few ex- change factor CDC25 /Ras-GRF1. amples indicate that deubiquitinating enzymes could interact Acknowledgments—We thank Stan Hollenberg for the gift of mouse in a specific way with a limited variety of substrates. 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Published: Oct 1, 2001

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