Characterization of a Second Member of the Sentrin Family of Ubiquitin-like Proteins

Sentrin is a novel ubiquitin-like protein that can be conjugated to other proteins in a manner analogous to ubiquitination. Two additional cDNA sequences that encode proteins highly homologous to sentrin have been reported to GenBank™. It is not known whether these sentrin-like proteins could also function as protein modifiers. In this report, a second member of the sentrin family was characterized in detail. Sentrin-2 is a 95-amino acid polypeptide that is 46% identical and 66% homologous to sentrin-1. Northern blot analysis showed that the sentrin-2 message was expressed in all tissues, but was barely detectable in the liver and placenta. The ability of sentrin-2 to conjugate to other proteins was tested by expressing hemagglutinin epitope-tagged sentrin-2 in COS cells. Western blot analysis showed that sentrin-2 could be transferred to other proteins in a pattern similar to that of sentrin-1 conjugation and had similar C-terminal processing. We further showed that both sentrin-1 and sentrin-2 could covalently modify RanGAP1, a Ran GTPase-activating protein critically involved in nuclear transport. Immunocytochemical analysis showed that sentrin-2 derivatives were highly enriched in the nucleus. Taken together, our results demonstrate that sentrin-2 is another protein modifier for the sentrinization pathway. Sentrin is a novel ubiquitin-like protein that can be conjugated to other proteins in a manner analogous to ubiquitination. Two additional cDNA sequences that encode proteins highly homologous to sentrin have been reported to GenBank™. It is not known whether these sentrin-like proteins could also function as protein modifiers. In this report, a second member of the sentrin family was characterized in detail. Sentrin-2 is a 95-amino acid polypeptide that is 46% identical and 66% homologous to sentrin-1. Northern blot analysis showed that the sentrin-2 message was expressed in all tissues, but was barely detectable in the liver and placenta. The ability of sentrin-2 to conjugate to other proteins was tested by expressing hemagglutinin epitope-tagged sentrin-2 in COS cells. Western blot analysis showed that sentrin-2 could be transferred to other proteins in a pattern similar to that of sentrin-1 conjugation and had similar C-terminal processing. We further showed that both sentrin-1 and sentrin-2 could covalently modify RanGAP1, a Ran GTPase-activating protein critically involved in nuclear transport. Immunocytochemical analysis showed that sentrin-2 derivatives were highly enriched in the nucleus. Taken together, our results demonstrate that sentrin-2 is another protein modifier for the sentrinization pathway. Sentrin was isolated in our laboratory because of its ability to interact with the death domains of both Fas and tumor necrosis factor receptor-1 (1Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.F. Chang H.M. Yeh E.T.H. J. Immunol. 1996; 157: 4277-4281PubMed Google Scholar). Overexpression of sentrin in mammalian cells protects them against anti-Fas or tumor necrosis factor-induced cell death (1Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.F. Chang H.M. Yeh E.T.H. J. Immunol. 1996; 157: 4277-4281PubMed Google Scholar). Analysis of the amino acid sequence of sentrin revealed a ubiquitin-like domain (residues 22–97), which is 18% identical and 48% homologous to human ubiquitin. In contrast to ubiquitin, sentrin contains 21 additional amino acids at the N terminus and 4 more amino acids at the C terminus (see Fig. 1). Using a COS cell expression system, we showed that the C terminus of sentrin is efficiently processed to allow sentrin to conjugate to other proteins via the conserved Gly-97 residue (2Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). However, the conjugation patterns between hemagglutinin (HA) 1The abbreviations used are: HA, hemagglutinin; PBS, phosphate-buffered saline; UCRP, ubiquitin cross-reactive protein; mAb, monoclonal antibody. epitope-tagged sentrin versus ubiquitin are entirely distinct (2Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Furthermore, most of the sentrinized proteins appear to be localized in the nucleus, in contrast to the uniform expression of ubiquitin in both the nuclear and cytosolic compartments (2Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Using sentrin as bait in the yeast two-hybrid system, we have identified Ubc9 as a preferred conjugating enzyme for the sentrinization pathway (3Gong L. Kamitani T. Fujise K. Caskey L. Yeh E.T.H. J. Biol. Chem. 1997; 272: 28198-28201Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar).In vitro translated sentrin could be precipitated by a glutathione S-transferase-Ubc9 fusion protein, but not by glutathione S-transferase. A β-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay. Substitution of the conserved Cys residue of Ubc9 by Ser abolished the formation of the Ubc9-sentrin conjugate. Thus, sentrin was able to form a thiol ester linkage with Ubc9 via the conserved Cys-93 residue. Recently, two other laboratories independently identified a novel ubiquitin-like protein called SUMO-1 or GMP-1 that could covalently modify RanGAP1, a Ran GTPase-activating protein critically involved in nuclear transport (4Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1010) Google Scholar, 5Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (961) Google Scholar). Remarkably, the amino acid sequences of SUMO-1 and GMP-1 are identical to that of sentrin (1Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.F. Chang H.M. Yeh E.T.H. J. Immunol. 1996; 157: 4277-4281PubMed Google Scholar). RanGAP1 exists as a 70-kDa monomer in the cytosol and is converted to a 90-kDa protein following sentrin modification. Thus, sentrinization of RanGAP1 is essential for the translocation of RanGAP1 from the cytosol to the nuclear pore complex to participate in nuclear protein import (4Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1010) Google Scholar, 5Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (961) Google Scholar). Taken together, sentrinization is a unique pathway for protein modification that is distinct from ubiquitination. Data base research revealed the presence of sentrin homologues inSaccharomyces cerevisiae (GenBank™ accession number U27233; 50% identity and 74% similarity), Caenorhabditis elegans(X99600; 66% identity and 83% similarity), Arabidopsis thaliana (X99609; 49% identity and 64% similarity), andOryza sativa (X99608; 48% identity and 66% similarity). The presence of sentrin homologues from A. thaliana toHomo sapiens suggests that sentrin is an evolutionary conserved protein that may have a specialized function in cellular metabolism. The yeast homologue of sentrin is termed Smt3, which is a high copy suppressor of the conditional lethal mif2mutation, which, at nonpermissive temperature, shows increased mitotic chromosome instability, sensitivity to anti-microtubule drugs, and formation of aberrant spindles that break in half during anaphase (6Meluh P.B. Koshland D. Mol. Biol. Cell. 1995; 6: 793-807Crossref PubMed Scopus (354) Google Scholar,7Brown M.T. Goetsch L. Hartwell L.H. J. Cell Biol. 1993; 123: 387-403Crossref PubMed Scopus (96) Google Scholar). We have preliminary results demonstrating that human sentrin could also suppress the yeast mif2mutation, 2L. Gong, L. Caskey, and E. T. H. Yeh, unpublished results. suggesting that human sentrin and yeast Smt3 are involved in a evolutionary conserved pathway of protein modification. Further analysis of the data base, however, revealed two additional human cDNA sequences (X99584 andX99585) that are highly homologous to sentrin. This is of interest because there is only one sentrin homologue reported for all of the nonmammalian genera and species. It is not known whether any of these human cDNAs could encode functional proteins that also participate in the sentrinization pathway. In this report, we analyze a second member of the sentrin family, demonstrating the remarkably conserved modification patterns between these two sentrins. COS-M6 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics. 12CA5 (Boehringer Mannheim) and 16B12 (BAbCo, Richmond, CA) are mouse monoclonal antibodies to the peptide sequence YPYDVPDYA of influenza HA. Mouse anti-RH (specific for the amino acid sequence RGSHHHH) monoclonal antibody was purchased from QIAGEN Inc. (Santa Clara, CA). Rabbit anti-RanGAP1 antiserum was kindly provided by Frauke Melchior (The Scripps Research Institute). To express HA-tagged proteins in COS-M6 cells, two vectors for amino-terminal tagging (pcDNA3/HA-N) and carboxyl-terminal tagging (pcDNA3/HA-C) were constructed as described previously (2Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). HA adaptor duplexes were inserted into pcDNA3 (Invitrogen, San Diego, CA), and then the cDNAs of ubiquitin, sentrin-1, Rad51, and sentrin-2 mutants were isolated by polymerase chain reaction using appropriate primers, followed by ligation into pcDNA3/HA-N or pcDNA3/HA-C. To express RGS(H)6-tagged RanGAP1 (RH-RanGAP1) in COS-M6 cells, the amino-terminal tagging vector (pcDNA3/RH-N) was constructed in the same way as pcDNA3/HA-N. The cDNA of RanGAP1 was isolated by polymerase chain reaction from a cDNA library of human testis and inserted into pcDNA3/RH-N to make pcDNA3/RH-RanGAP1. The sequence of each insert was confirmed by automated DNA sequencing. Plasmids were transfected into COS-M6 cells using LipofectAMINE (Life Technologies, Inc.). The transfected cells were harvested for Western blotting or immunostaining 16 h after transfection. A full-length cDNA fragment of human sentrin-1 or sentrin-2 from plasmid pcDNA3/sentrin-1-HA or pcDNA3/sentrin-2-HA, respectively, was labeled with [α-32P]dCTP with a Megaprime labeling kit (Amersham Pharmacia Biotech). The radioactive probe was hybridized with human multiple tissue Northern blots purchased fromCLONTECH (Palo Alto, CA). Cells (1 × 106) were harvested, washed twice with cold PBS, and centrifuged. To prevent protein degradation, the cell pellet was immediately transferred into liquid nitrogen, and then the frozen pellet was treated at 45 °C for 1 h in 300 μl of 2% SDS treating solution containing 5% β-mercaptoethanol. DNA in the sample was sheared with a 25-gauge needle. After SDS-polyacrylamide gel electrophoresis, using 3 μl of the sample (equivalent to 1 × 104 cells) per lane, Western blotting was performed following the protocol of the ECL detection system (Amersham Pharmacia Biotech.). As a secondary antibody, horseradish peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) was used. To investigate the conjugation of sentrin-2 to RanGAP1, RH-RanGAP1 was coexpressed with HA-Rad51 (negative control), HA-ubiquitin, HA-sentrin-1 (positive control), or HA-sentrin-2 in COS-M6 cells by the cotransfection method. Since the sequence of the RH tag is RGSHHHHHH, RH-RanGAP1 can be purified by nickel-charged beads (8Kamitani T. Katsumi K. Nguyen R. Yeh E.T.H. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). The total cell lysate of the transfectants expressing RH-RanGAP1 and HA-tagged protein was prepared in lysis buffer (6 m guanidine hydrochloride, 20 mm sodium phosphate, and 500 mm sodium chloride (pH 7.8)). DNA in the sample was sheared with a 22-gauge needle, and then the lysate was centrifuged at 100,000 × g for 30 min at 15 °C. The supernatant was incubated with nickel-charged agarose resin beads (Invitrogen) for 1 h at room temperature. The beads were washed twice with washing buffer (8 m urea, 20 mm sodium phosphate, and 500 mm sodium chloride) (pH 7.8), followed by washing twice with washing buffer (pH 6.0). Finally, the beads were washed twice with PBS and treated in 2% SDS treating solution for SDS-polyacrylamide gel electrophoresis. The solubilized proteins were analyzed by Western blotting using anti-HA antibody (16B12) to detect RH-RanGAP1 conjugated to HA-tagged proteins and also using anti-RH antibody to detect every derivative of RH-RanGAP1. Immunohistochemical staining was performed by the avidin-biotin-horseradish peroxidase complex method (9Kamitani T. Suzuki H. Yano S. Clin. Immunol. Immunopathol. 1991; 58: 217-235Crossref PubMed Scopus (8) Google Scholar) using the VECTASTAIN ABC kit system (Vector Laboratories, Inc., Burlingame, CA). Transfected COS-M6 cells on coverslips were fixed in 3.7% paraformaldehyde solution for 20 min and permeabilized in 0.1% Triton X-100 for 10 min at room temperature. After washing with PBS, the fixed cells were incubated with PBS containing 0.1% H2O2 for 10 min to quench endogenous peroxidase activity and then washed with PBS. The cells were incubated for 10 min with PBS containing 5% horse serum for blocking, followed by further incubation with anti-HA antibody (16B12) for 30 min at 37 °C. After rinsing with PBS, the cells were incubated with biotinylated anti-mouse IgG for 30 min at 37 °C, washed with PBS, and then treated with the avidin-biotin-horseradish peroxidase complex for 30 min at 37 °C. Finally, the enzymatic disclosing procedure was performed as reported previously (9Kamitani T. Suzuki H. Yano S. Clin. Immunol. Immunopathol. 1991; 58: 217-235Crossref PubMed Scopus (8) Google Scholar). Sentrin is a 101-amino acid polypeptide that is 18% identical and 48% homologous to ubiquitin (1Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.F. Chang H.M. Yeh E.T.H. J. Immunol. 1996; 157: 4277-4281PubMed Google Scholar). A data base search revealed two human cDNA sequences (GenBank™ accession numbersX99584 and X99585) that are more closely related to sentrin than to ubiquitin. One of these sequences (X99585) encodes a 95-amino acid polypeptide that is 46% identical and 66% similar to sentrin. An identical polypeptide sequence is also encoded in a gene insertion in the p125 region of a cytopathic bovine viral diarrhea virus genome (U89439). Because this novel polypeptide shares several biochemical characteristics with sentrin (see below), we have renamed the original sentrin as sentrin-1 and the new protein as sentrin-2. Sentrin-1 was named UBL1 (10Shen Z. Pardington-Purtymun P.E. Comeaux J.C. Moyzis R.K. Chen D.J. Genomics. 1996; 36: 271-279Crossref PubMed Scopus (183) Google Scholar), PIC-1 (11Boddy M.N. Howe K. Etkin L.D. Solomon E. Freemont P.S. Oncogene. 1996; 13: 971-982PubMed Google Scholar), GMP-1 (5Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (961) Google Scholar), SUMO-1 (4Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1010) Google Scholar), and SMT3C (12Lapenta V. Chiurazzi P. van der Spek P. Pizzuti A. Hanaoka F. Brahe C. Genomics. 1997; 40: 362-366Crossref PubMed Scopus (103) Google Scholar) by other laboratories. Sentrin-2 was named SMT3B or GMP-related protein in these reports. Fig. 1 shows the amino acid alignment of human sentrin-1, sentrin-2, and ubiquitin. As shown, sentrin-2 contains a ubiquitin domain, an N-terminal extension, and the conserved Gly-Gly residues in the C terminus. The amino acids proximal to the C-terminal Gly-Gly residues are QEQT or QQQT for sentrin-1 or sentrin-2, respectively, in contrast to LRLR in the ubiquitin molecule. Furthermore, the amino acid residue equivalent to Lys-48 in ubiquitin has been substituted with Gln in both sentrin-1 and sentrin-2. To determine the expression of sentrin-2 in human tissues, Northern blot analysis was performed using 32P-labeled human sentrin-1 or sentrin-2 cDNA as a probe. As shown in Fig. 2, the pattern of expression is similar for both sentrin-1 and sentrin-2, with a of all the and in the and Because sentrin-2 is highly homologous to sentrin-1, we to determine whether sentrin-2 could also be conjugated to other proteins in a manner analogous to sentrin-1. this HA epitope-tagged sentrin-2 was expressed in COS cells as described previously (2Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). HA epitope-tagged sentrin-1 or ubiquitin was used as a and total cell prepared from transfected COS cells were analyzed by Western blot analysis with anti-HA antibody. As shown in Fig. both sentrin-1 and sentrin-2 were In sentrin-2 a number of similar to of sentrin-1. Two anti-HA monoclonal antibodies were used in Western blot 12CA5 was more than 16B12 and could detect a in both the and The expression of the sentrin-2 monomer and conjugated proteins is more than the expression of sentrin-1. However, the conjugation patterns of sentrin-1 and sentrin-2 are suggesting that could modify proteins (see The C terminus of sentrin-2 contains amino and to the conserved and sentrin-2 conjugation to this C-terminal be As shown in Fig. the HA tag was at the C terminus of sentrin-2, was efficiently in transfected COS cells Substitution of for the C terminus of the sentrin-2 monomer more to and Thus, only was have been with the of the C terminus of sentrin-1 (2Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). of the C-terminal residues of sentrin-2 abolished the conjugation activity of sentrin-2 Thus, the of the C terminus of sentrin-2 is identical to that of sentrin-1. However, is not known whether the same enzyme is As shown in 3 the conjugation patterns of sentrin-1 and sentrin-2 are Furthermore, a 90-kDa was in both and Since sentrin-1 has been shown to conjugate to RanGAP1 to form a 90-kDa protein (4Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1010) Google Scholar, 5Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (961) Google Scholar, T. Katsumi K. Nguyen R. Yeh E.T.H. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar), was of interest to determine whether sentrin-2 could also be conjugated to To this the N terminus of RanGAP1 was with the amino acid sequence RGSHHHHHH, termed RH which for of the RanGAP1 fusion protein with resin Plasmids HA epitope-tagged sentrin-1, sentrin-2, ubiquitin, and were with a plasmid RH-RanGAP1 into COS cells as described cell prepared from the transfectants were precipitated with resin followed by The were analyzed by Western blot analysis using anti-HA antibody. As shown in Fig. A RanGAP1 could be modified by sentrin-1 or sentrin-2, but not by ubiquitin or The purified RH-RanGAP1 derivatives were in a Western blot analysis anti-RH monoclonal antibody. As shown in both RanGAP1 and sentrinized RanGAP1 could be in all It be that in was from RanGAP1 modified by sentrin in COS cells. This was further by the of a in the and and The is most RanGAP1, and is most RanGAP1 and In Fig. we that sentrin-1 or sentrin-2 mutants in which the second conserved residue was could not form any mutants were used to further that the formation of a linkage between RanGAP1 and sentrin-1 or sentrin-2. Plasmids HA epitope-tagged mutants were with a plasmid RH-RanGAP1 into COS cells, and the were prepared as described As shown in Fig. the formation of sentrin and RanGAP1 the presence of the conserved residue in the C terminus of the sentrin molecule. To the of of sentrin-2 into endogenous versus RanGAP1, HA-sentrin-2 RH-RanGAP1 was expressed in COS cells. The of the were with antibodies to detect both endogenous and RanGAP1, anti-RH to detect RanGAP1, and anti-HA to detect As shown in Fig. all endogenous RanGAP1 proteins were modified by endogenous 1 and of HA-tagged sentrin-2 not the of modified RanGAP1 of RanGAP1 with HA-tagged sentrin-2 increased the total of modified RanGAP1 However, of the RanGAP1 proteins were not conjugated and This was not to the presence of a of sentrin-2 because of sentrin-2 were in the sample and Thus, there to be of the total of sentrinized RanGAP1 in COS cells. It is that the of sentrinized RanGAP1 proteins is by binding at the nuclear RanGAP1 to conjugate to sentrin-2 more efficiently than other proteins results were HA-tagged sentrin-1 was coexpressed with RanGAP1 not We have previously shown that proteins are localized in the (2Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The of proteins was as described in our (2Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). COS cells were also transfected with HA epitope-tagged sentrin-1, sentrin-2, or ubiquitin and with anti-HA antibody (16B12) as described previously (2Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). HA epitope-tagged sentrin-2 is expressed in the nucleus, with cytosolic expression not In HA epitope-tagged ubiquitin is in both the and cytosol not Thus, the of both sentrin-1 and sentrin-2 to be modification by ubiquitin has been in the A. A. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). has been shown to a in cell and Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, C. L. K. R. L. D. Cell. Full Text PDF PubMed Scopus Google Scholar, A. 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Full Text PDF PubMed Google however, the of proteins have not been The of sentrin-1 and the of its ability to modify RanGAP1 further to this of (2Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1010) Google Scholar, 5Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (961) Google Scholar). laboratory has characterized another ubiquitin-like protein, which could also form protein in a manner similar to protein (8Kamitani T. Katsumi K. Nguyen R. Yeh E.T.H. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). The results shown in this sentrin-2 as the after UCRP, sentrin-1, and of the ubiquitin-like protein modifiers.