Identification of a Novel Ligand-Receptor Pair Constitutively Activated by ras Oncogenes
2000; Elsevier BV; Volume: 275; Issue: 32 Linguagem: Inglês
10.1074/jbc.m001958200
ISSN1083-351X
AutoresRong Zhang, Zhongjia Tan, Peng Liang,
Tópico(s)HER2/EGFR in Cancer Research
ResumoThe Ras signaling pathway is thought to control the expression of a subset of yet to be defined genes that are crucial for cell growth and differentiation. Here we have identified by differential display a novel oncogenic Ras target, mob-5, encoding a 23-kDa cytokine-like secreted protein. Mob-5 expression could be induced by oncogenic Ha-ras and Ki-ras, but not by normal ras activation. Inhibitors of both Ha-Ras and mitogen-activated protein kinase kinase completely abolished the mob-5 expression in ras transformed cells, with concomitant loss of the transformation phenotype. Using an alkaline phosphatase-tagged Mob-5 as ligand, a putative Mob-5 receptor was identified on the cell surface of oncogenic ras transformed cells. Thus, the Mob-5/Mob-5 receptor may represent a novel putative autocrine loop coordinately activated by rasoncogenes. The Ras signaling pathway is thought to control the expression of a subset of yet to be defined genes that are crucial for cell growth and differentiation. Here we have identified by differential display a novel oncogenic Ras target, mob-5, encoding a 23-kDa cytokine-like secreted protein. Mob-5 expression could be induced by oncogenic Ha-ras and Ki-ras, but not by normal ras activation. Inhibitors of both Ha-Ras and mitogen-activated protein kinase kinase completely abolished the mob-5 expression in ras transformed cells, with concomitant loss of the transformation phenotype. Using an alkaline phosphatase-tagged Mob-5 as ligand, a putative Mob-5 receptor was identified on the cell surface of oncogenic ras transformed cells. Thus, the Mob-5/Mob-5 receptor may represent a novel putative autocrine loop coordinately activated by rasoncogenes. farnesyltransferase inhibitor mitogen-activated protein rat intestinal epithelial polymerase chain reaction base pairs isopropyl-1-thio-β-d-galactopyranoside alkaline phosphatase interleukin 10 Ras proteins are among the most important molecular switch molecules that relay mitogenic or differentiation signals from the cell surface to the nucleus where selective gene expression takes place. Oncogenic mutations lock the Ras proteins into a permanent “on” position, leading to unregulated cell proliferation, which is the hallmark of cancer (1Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3778) Google Scholar, 2Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1124) Google Scholar). Mutations in ras oncogenes have been detected in over 90% of pancreatic cancers and 50% of colorectal cancer (3Kiaris H. Spandidos D.A. Int. J. Oncol. 1995; 7: 413-421PubMed Google Scholar). In contrast to the extensive body of knowledge related to the genetics of ras activation (4Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1761) Google Scholar), relatively little is known of the transcriptional events triggered by Ras. Of a fewras target genes previously identified, including transin/stromelysin-1 (5Matrisian L.M. Glaichenhaus N. Gesnel M-C. Breathnach R. EMBO J. 1985; 4: 1435-1440Crossref PubMed Scopus (227) Google Scholar), glucose transporter (6Flier J. Mueckler M.M. Usher P. Lodish H.F. Science. 1987; 235: 1492-1495Crossref PubMed Scopus (688) Google Scholar, 7Godwin A. Lieberman M. Oncogene. 1990; 5: 1231-1241PubMed Google Scholar), Pai-2 (8Cohen L.R. Niclas J. Lee W.M.F. Wun T.-C. Crowley C.W. Levinson A.D. Sadler J.E. Shuman M.A. J. Biol. Chem. 1989; 264: 8375-8383Abstract Full Text PDF PubMed Google Scholar), heparin binding epidermal growth factor (9McCarthy S.A. Samuels M.L. Pritchard C.A. Abraham J.A. McMahon M. Genes Dev. 1995; 9: 1953-1964Crossref PubMed Scopus (170) Google Scholar), and mob-1/IP10 (10Liang P. Averboukh L. Zhu W. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12515-12519Crossref PubMed Scopus (59) Google Scholar,11Zhang R. Zhang H. Zhu W. Coffey R. Pardee A.B. Liang P. Oncogene. 1997; 14: 1607-1610Crossref PubMed Scopus (27) Google Scholar), nearly all were shown to be inducible in normal cells by serum growth factors, likely through the activation of endogenousras (12Dobrowolski S. Harter M. Stacey D.W. Mol. Cell. Biol. 1994; 14: 5441-5449Crossref PubMed Scopus (99) Google Scholar, 13Durkin J.P. Whitfield J.F. Mol. Cell. Biol. 1986; 6: 1386-1392Crossref PubMed Scopus (32) Google Scholar, 14Taylor S.J. Shalloway D. Curr. Biol. 1996; 6: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). One long standing puzzle surroundingras has been that although the effects of Ras activation are very similar to those of serum growth factors,ras-transformed cells may be oncogenic, whereas the parental cells continuously exposed to serum growth factors are not. To search for additional ras target genes, especially those that may be oncogenic ras-specific, optimized differential display technology (15Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4700) Google Scholar, 16Liang P. Zhu W. Zhang X. Guo Z. O'Connell R. Averboukh L. Wang F. Pardee A.B. Nucleic Acids Res. 1994; 22: 5763-5764Crossref PubMed Scopus (309) Google Scholar) was employed to systematically search for such genes in comparative studies involving two carefully chosen paradigms. Since the development of the method, it has been realized that differential display, like other competitive methods such as DNA microarray, is mechanism-based, rather than function-based gene screening tool (17Liang P. Methods. 1998; 16: 361-364Crossref PubMed Scopus (26) Google Scholar). Therefore, much effort should be made to establish the comparative systems based on as direct and simple a mechanism as possible to identify the relevant genes. To this end, alteration in gene expression in Rat-1 embryo fibroblasts upon conditional expression of oncogenic Ha-ras was analyzed to identify the early ras inducible genes. In the other complementary screening, changes in gene expression was followed in the oncogenic Ha-ras transformed Rat-1 cells upon the treatment with inhibitors of either Ras farnesyltransferase (FTI)1 (18Kohl N.E. Mosser S.D. deSolms S.J. Giuliani E.A. Pompiano D.L. Graham S.L. Smith R.L. Scolnick E.M. Oliff A. Gibbs J.B. Science. 1993; 260: 1934-1937Crossref PubMed Scopus (619) Google Scholar) or MAP kinase kinase (19Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar), which is one of the major Ras downstream kinases required for cell transformation. Here we describe the identification and biochemical characterization of mob-5, the only oncogenicras-specific gene identified in both paradigms. We demonstrate that mob-5 encodes a cytokine-like secreted protein, which binds specifically to its putative cell surface receptor (Mob-5R) expressed by the ras-transformed cells, but not by their normal parental controls. We propose that the coordinate activation of this novel ligand/receptor loop may play an important role inras oncogene-mediated neoplasia. All cell lines including Rat1, Rat1(ras), Rat-1:iRas (10Liang P. Averboukh L. Zhu W. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12515-12519Crossref PubMed Scopus (59) Google Scholar, 35Zhang R. Averboukh L. Zhu W. Zhang H. Jo H. Dempsey P.J. Coffey R., A. Pardee A.B. Liang P. Mol. Cell. Biol. 1998; 18: 6131-6141Crossref PubMed Scopus (131) Google Scholar), rat intestinal epithelial (RIE), RIE(Ha-ras), and RIE(Ki-ras) (Ko, 1995) were routinely grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% bovine calf serum (HyClone, Logan, UT) and 1% penicillin-streptomycin (Life Technologies, Inc.) at 37 °C with 10% CO2. 293T cells and their derivatives were maintained under the same condition as above except 10% fetal bovine serum (HyClone, Logan, UT) was used in place of 10% bovine calf serum. Serum starvation and restimulation of the Rat-1 cells in the absence and presence of cyclohexamide (Sigma) was carried out essentially as described previously (11Zhang R. Zhang H. Zhu W. Coffey R. Pardee A.B. Liang P. Oncogene. 1997; 14: 1607-1610Crossref PubMed Scopus (27) Google Scholar). Total RNA from cells were purified using the RNApure reagent following the manufacture's instruction (GenHunter Corp., Nashville, TN). DNase I treatment of RNA prior to differential display was carried out using the MessageClean kit (GenHunter Corp., Nashville, TN). Differential display PCR reactions were setup in 96-well PCR plates (Perkin-Elmer) by the Beckman Biomek 2000 automated liquid dispensing workstation using one-base anchored oligo(dT) primers and rationally designed arbitrary 13-mers (16Liang P. Zhu W. Zhang X. Guo Z. O'Connell R. Averboukh L. Wang F. Pardee A.B. Nucleic Acids Res. 1994; 22: 5763-5764Crossref PubMed Scopus (309) Google Scholar) from the RNAimage kits (GenHunter Corp., Nashville, TN). The cDNAs were amplified in the presence of [α-33P]dATP (NEN Life Science Products) using Taq DNA polymerase (Qiagene) and separated on 6% denaturing polyacrylamide gels (National Diagnostic, Atlanta, GA). Total RNA was isolated from Ha-Ras-transformed rat embryo fibroblasts as described (16Liang P. Zhu W. Zhang X. Guo Z. O'Connell R. Averboukh L. Wang F. Pardee A.B. Nucleic Acids Res. 1994; 22: 5763-5764Crossref PubMed Scopus (309) Google Scholar) and then further purified by poly(A) selection using a poly(A)Tract mRNA isolation system (Promega, Madison, WI). The lambda ZAP II Vector/Gigapack cloning kit was used to construct the cDNA library following the instructions provided by the manufacturer. 500,000 plaques were screened for full-length mob-5 cDNA using α 32P-labeled 394-bp mob-5 cDNA probe from differential display. Positive plaques were excised as phagemids and sequenced by the molecular biology core facility of the Dana-Farber Cancer Institute, Boston, MA. The entire mda-7 coding region was amplified from RNA isolated from human colon cancer tissues by reverse transcriptase-PCR based on the published sequence (24Jiang H. Lin J.J. Su Z. Goldstein N.I. Fisher P.B. Oncogene. 1995; 11: 2477-2486PubMed Google Scholar). The cDNA synthesis was carried out in a total volume of 20 μl containing 1 μg of DNA-free total RNA, 50 mm Tris-Cl (pH 8.0), 100 mm KCl, 2 mm MgCl2, 20 μm dNTP, 1 μm oligo(dT)20 primer (GenHunter), and 200 units of Moloney murine leukemia virus reverse transcriptase (GenHunter) at 37 °C for 1 h. After 5 min at 75 °C to inactivate the Moloney murine leukemia virus reverse transcriptase, 1/10 of the reverse transcriptase reaction was used as template for PCR amplification of mda-7 using 0.2 μm of each LMda-7 primer (5′-AAGATCTGAGGCTGCTT-3′) and RMda-7 primer (5′-CCAGATCTAGACATTCAGAGCTTGTAG-3′). The PCR was carried out in 40 μl containing 4 μl of 10× PCR buffer, 1.6 μl of 1.25 mmdNTP, and 0.4 μl of Taq polymerase (Qiagen). After an initial denaturation period of 3 min at 94 °C, 30 cycles consisting of 15 s at 94 °C, 40 s at 56 °C, and 2 min at 72 °C followed. After the final 5-min extension at 72 °C, the reverse transcriptase-PCR products were analyzed on a 1.5% agarose gel. The amplified mda-7 cDNA was cloned into the PCR-TRAP vector (GenHunter) and completely sequenced. A 507-bpBamHI-HindIII fragment, containing the rat Mob-5 coding region without its N-terminal 23 signal peptides was generated by PCR using the cloned Mob-5 cDNA as a template. The primers used were Lhis (5′-CCAGAGCTTCAGGGTC-3′) and Rhis (5′-AAGCTTTCCGGATTGGCAAT-3′). The PCR product was first subcloned into the PCR-TRAP vector (GenHunter, Nashville, TN) and theBamHI-HindIII insert was then excised, purified, and ligated into corresponding sites of the 6X-His tag expression vector pQE32 (Qiagen, Chatsworth, CA). For the expression of recombinant Mda-7–6XHis-tagged protein, the coding region of Mda-7 excluding the putative signal peptide sequence (codon 1–44) was amplified by PCR using primers Lhis-hMob5 (5′-GGATCCABBTATCAGGGG-3′) and Rhis-hMob5 (5′-AAGCTTGTAGAATTTCTGCATC-3′). The PCR product was cloned into the PCR-TRAP cloning vector and then excised as aBamHI-HindIII fragment before ligating into the corresponding sites of pQE32. The recombinant plasmids were transformed into Escherichia coli TG1 cells to produce N-terminal 6X-His tagged Mob-5 and Mda-7. Upon induction with 2 mm IPTG (Life Technology, Inc.), the recombinant His-tagged Mob-5 and Mda-7 were overproduced as insoluble proteins in the bacteria and purified on a nickel-Sepharose column under denaturing conditions according to the manufacturer's protocol (Qiagen). The purified proteins were dialyzed in PBS to remove the denaturant and then injected into New Zealand White rabbits to produce polyclonal antibodies (HRP Inc., Denver, PA). A 680-bp BspHI-BglII restriction fragment containing the entire coding region of mob-5 was generated by PCR using the cloned mob-5 cDNA as a template. The targeting construct was subsequently inserted between the NcoI and BamI sites of retroviral vector pMFG-S (36Danos O. Mulligan R.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6460-6464Crossref PubMed Scopus (805) Google Scholar, 37Dranoff G. Jaffee E. Lazenby A. Golumbek P. Levitsky H. Brose K. Jackson V. Hamada H. Pardoll D. Mulligan R.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3539-3543Crossref PubMed Scopus (2658) Google Scholar) and sequenced to ensure the sequence accuracy. Both the entire Mob-5 and Mda-7 coding regions including their signal peptides were amplified by PCR and subsequently inserted into the BglII site of APtag2 cloning vector (26, GenHunter Corp., Nashville TN) to allow the C-terminal fusion of the AP. All constructs were sequenced to ensure sequence accuracy. The recombinant plasmids and APtag4, which express the secreted AP alone (26Flanagan J.G. Leder P. Cell. 1990; 63: 185-194Abstract Full Text PDF PubMed Scopus (633) Google Scholar), were stably transfected into 293T cells. The expression of the fusion proteins, AP activity assay, and receptor binding were essentially as described before (26Flanagan J.G. Leder P. Cell. 1990; 63: 185-194Abstract Full Text PDF PubMed Scopus (633) Google Scholar). Specifically, for receptor binding assays, the cells were seeded at 5 × 105/well in 6-well plates in duplicates. After cells reach confluence, receptor binding assay was carried out with 1 ml of either the AP fusion protein containing medium or AP alone containing medium (all were 0.7 unit/ml) produced by the 293T cells. Recombinant plasmids were transfected into target cells by the standard calcium phosphate precipitation method. For retroviral packaging, pMFG-S-Mob-5 was cotransfected with pCMV-Neo vector at a rate of 10:1 (10 μg of pcmv-mob-5, 1 μg of pCMV-Neo) into the CRIP packaging cells to produce G418 (1 mg/ml)-resistant stable cell lines. Retroviral infection was carried out essentially as described previously (35Zhang R. Averboukh L. Zhu W. Zhang H. Jo H. Dempsey P.J. Coffey R., A. Pardee A.B. Liang P. Mol. Cell. Biol. 1998; 18: 6131-6141Crossref PubMed Scopus (131) Google Scholar). For protein expression of Mob-5-AP, Mda-7-AP, and AP alone, the corresponding plasmids were co-transfected into the 293T cells, with a puromycin resistant vector, pBabe-puro, at a ratio of 10:1, and stable clones were selected by 10 μg/ml puromycin. Aliquots of 30 μl of each cell culture medium were separated by electrophoresis on a sodium dodecyl sulfate-12% polyacrylamide gel (National Diagnostics, Atlanta, GA) and blotted onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). For immunblotting, a 1:2000 dilution of either Mob-5-, Mda-7- or AP-specific antibodies (GenHunter Corp., Nashville, TN) was used. The secondary antibody was anti-rabbit IgG conjugated with horseradish peroxidase (Amersham Pharmacia Biotech). Western blot for Ras was carried out using anti-Ras antibody (Oncogene Science) and 50 μg/lane of total cellular protein extracted from Rat-1:iRas cells before and after IPTG treatment. Reactive proteins were visualized with an ECL kit (Amersham Pharmacia Biotech) following the manufacturer's protocol. Using automated differential display screening with rational primer designs (16Liang P. Zhu W. Zhang X. Guo Z. O'Connell R. Averboukh L. Wang F. Pardee A.B. Nucleic Acids Res. 1994; 22: 5763-5764Crossref PubMed Scopus (309) Google Scholar), two paradigms, one with Rat-1:iRas cells containing IPTG inducible oncogenic Ha-ras (9McCarthy S.A. Samuels M.L. Pritchard C.A. Abraham J.A. McMahon M. Genes Dev. 1995; 9: 1953-1964Crossref PubMed Scopus (170) Google Scholar, 11Zhang R. Zhang H. Zhu W. Coffey R. Pardee A.B. Liang P. Oncogene. 1997; 14: 1607-1610Crossref PubMed Scopus (27) Google Scholar) and the other with oncogenic Ha-ras transformed Rat-1 cells before and after treatment of a MAP kinase kinase inhibitor PD98059 (19Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar), were set up for the systematic screening of oncogenic ras target genes. After screening through 90 combinations of primers representing 70% coverage of the genes expressed in a cell (20Liang P. Bauer D. Averboukh L. Warthoe P. Rohrwild M. Muller H. Strauss M. Pardee A.B. Methods Enzymol. 1995; 254: 304-321Crossref PubMed Scopus (117) Google Scholar), a total of 14ras inducible genes and 7 ras repressible genes were identified (21Jo H. Zhang H. Zhang R. Liang P. Methods. 1998; 16: 365-372Crossref PubMed Scopus (10) Google Scholar). 2P. Liang, unpublished results.Four of these ras inducible genes, transin/stromelysin-1 (5Matrisian L.M. Glaichenhaus N. Gesnel M-C. Breathnach R. EMBO J. 1985; 4: 1435-1440Crossref PubMed Scopus (227) Google Scholar), osteopontin (22Craig A. Nemir M. Mukherjee B.B. Chambers A.F. Denhardt D.T. Biochem. Biophys. Res. Commun. 1988; 157: 166-173Crossref PubMed Scopus (98) Google Scholar), Cox-2 (23Sheng H. Williams C.S. Shao J. Liang P. Dubois R.N. Beauchamp R.D J. Biol. Chem. 1998; 273: 22120-22127Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), and Pai-2 (8Cohen L.R. Niclas J. Lee W.M.F. Wun T.-C. Crowley C.W. Levinson A.D. Sadler J.E. Shuman M.A. J. Biol. Chem. 1989; 264: 8375-8383Abstract Full Text PDF PubMed Google Scholar), were also identified by other methods in previous studies as ras targets. One of the 14 ras inducible genes, designated mob-5, appeared to be novel, and the gene was identified in both screenings using either an inducible ras oncogene or inhibitor, which blocksras signaling (Fig. 1,A and B). The 394-bp mob-5 cDNA was reamplified from the differential display gels, cloned, and used as a probe to successfully verify the Ha-ras induction of the gene (Fig. 2 A). Mob-1, a knownras target gene, previously (10Liang P. Averboukh L. Zhu W. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12515-12519Crossref PubMed Scopus (59) Google Scholar, 11Zhang R. Zhang H. Zhu W. Coffey R. Pardee A.B. Liang P. Oncogene. 1997; 14: 1607-1610Crossref PubMed Scopus (27) Google Scholar) was used as a control for ras induction. The IPTG treatment of Rat-1:iRas led to the appearance of the oncogenic Ha-Ras protein 4 h post IPTG induction, with concomitant rapid induction of mob-5 mRNA. This result suggests that mob-5 is an early target gene of oncogenic Ha-ras.Figure 2Northern blot confirmation of mob-5 as an oncogenic Ha-ras-specific target gene. A, total RNA from Rat-1:iRas cells before and after IPTG induction of Ha-ras were analyzed by Northern blot using mob-5 cDNA as a probe. Another ras target gene, mob-1, was also analyzed as a positive control. The induction of oncogenic Ha-Ras protein was confirmed by Western blot as indicated from the same cells. Note the induction of mutant Ha-Ras and mob-5 at 4 h after IPTG addition. B, mob-5 expression cannot be activated by serum growth factors. Normal Rat-1 cells were starved for serum (0.5% BCS) for 48 h and then stimulated with 10% bovine calf serum in the absence or presence of protein synthesis inhibitor, cyclohexamide, as indicated. Total cellular RNAs were analyzed by Northern blot using the same 32P-labeled cDNA probes as used in Fig. 2 A. Note the superinduction of mob-1, but not mob-5, by serum and protein synthesis inhibitor, cyclohexamide (CHX). C, inhibition of mob-5 expression in Ha-ras-transformed cells by FTI. Exponentially growing Rat-1(ras) cells were treated with 5 μm of FTI 739,749 for the period indicated, and the total cellular RNA was analyzed by Northern blot using mob-5 cDNA as a probe. Ribosomal RNA was shown as a control for loading.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previous studies showed that ras is required for the immediate early response to external growth factor stimulation and normal cell proliferation (12Dobrowolski S. Harter M. Stacey D.W. Mol. Cell. Biol. 1994; 14: 5441-5449Crossref PubMed Scopus (99) Google Scholar, 13Durkin J.P. Whitfield J.F. Mol. Cell. Biol. 1986; 6: 1386-1392Crossref PubMed Scopus (32) Google Scholar, 14Taylor S.J. Shalloway D. Curr. Biol. 1996; 6: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Although mob-5 expression was not detected in nontransformed Rat-1 cells in continuous culture, one would predict that it could be expressed in response to serum stimulation if the gene is a downstream target of the Ras signaling pathway. Consistent with this prediction were findings that almost all of the previously identified ras target genes including mob-1 (16Liang P. Zhu W. Zhang X. Guo Z. O'Connell R. Averboukh L. Wang F. Pardee A.B. Nucleic Acids Res. 1994; 22: 5763-5764Crossref PubMed Scopus (309) Google Scholar), heparin-binding epidermal growth factor (9McCarthy S.A. Samuels M.L. Pritchard C.A. Abraham J.A. McMahon M. Genes Dev. 1995; 9: 1953-1964Crossref PubMed Scopus (170) Google Scholar), Transin (5Matrisian L.M. Glaichenhaus N. Gesnel M-C. Breathnach R. EMBO J. 1985; 4: 1435-1440Crossref PubMed Scopus (227) Google Scholar), osteopontin (22Craig A. Nemir M. Mukherjee B.B. Chambers A.F. Denhardt D.T. Biochem. Biophys. Res. Commun. 1988; 157: 166-173Crossref PubMed Scopus (98) Google Scholar), and glucose transporter (6Flier J. Mueckler M.M. Usher P. Lodish H.F. Science. 1987; 235: 1492-1495Crossref PubMed Scopus (688) Google Scholar) could be induced by serum growth factors. To test the serum inducibility of the mob-5 expression, Rat-1 cells were starved for serum and then restimulated with 10% fetal calf serum. Unlike mob-1, mob-5 expression was not detected following serum stimulation, either in the presence or absence of protein synthesis inhibitor (Fig. 2 B). In addition, Northern blot analysis failed to detect any mob-5 expression in normal tissues from adult rats, which included brain, heart, lung, kidney, stomach, pancreas, spleen, intestine, skin, skeletal muscle, and blood (data not shown). The tetrapeptide FTI has been shown to be able to inhibit Ha-ras-mediated cell transformation of rat embryo fibroblasts (18Kohl N.E. Mosser S.D. deSolms S.J. Giuliani E.A. Pompiano D.L. Graham S.L. Smith R.L. Scolnick E.M. Oliff A. Gibbs J.B. Science. 1993; 260: 1934-1937Crossref PubMed Scopus (619) Google Scholar). FTIs inhibit the farnesylation of Ras protein, thereby preventing its membrane localization and biological function. If mob-5 is indeed a target gene of ras, one would predict that FTIs would down-regulate mob-5 expression in oncogenic Ha-ras-transformed cells. To test this, exponentially growing Rat-1(ras) cells were treated with 5 μm of FTI, and the mob-5 mRNA expression and cellular morphology were monitored following the drug addition. As predicted, mob-5 mRNA expression was essentially abolished in 24 h (Fig. 2 C), when the cells began to lose the transformed cellular morphology (data not shown). Using the 394-bp cDNA probe obtained by differential display, the full-length mob-5 cDNA was isolated from a cDNA library of Ha-ras-transformed rat embryo fibroblasts (10Liang P. Averboukh L. Zhu W. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12515-12519Crossref PubMed Scopus (59) Google Scholar). The 1.2-kilobase mob-5 cDNA encodes an open reading frame of 183 amino acids with an in-frame stop codon 27 bases upstream of the putative translation initiation codon (Fig. 3 A). As predicted, the 394-bp mob-5 cDNA sequence identified by differential display lies at the 3′-end of the mob-5 full-length cDNA. The predicted Mob-5 protein exhibits 68% sequence identity to Mda-7, a gene isolated as a differentiation associated gene from human melanoma cells treated with interferon-β and Mezerein (24 and Fig.3 B). The only other gene that showed a significant degree of homology (23% identity) to Mob-5 over most of the coding regions is interleukin 10 (IL-10) from both viral or cellular origins, which is a potent anti-inflammatory cytokine (Fig.3 C and 31). The Mob-5 protein sequence contains a typical signal peptide sequence at its N terminus, suggesting that Mob-5 may be a secreted protein, although previous study failed to demonstrate so for Mda-7 (24Jiang H. Lin J.J. Su Z. Goldstein N.I. Fisher P.B. Oncogene. 1995; 11: 2477-2486PubMed Google Scholar). Overexpression of the full-length mob-5 cDNA with retroviral vector in a variety of rodent fibroblasts, including Rat-1 cells, led to a high level secretion of Mob-5 protein with a predicted molecular mass of 23 kDa. The Mob-5 protein secreted into the culture medium could be readily detected by Western blot using polyclonal antibody directed against the recombinant Mob-5 (Fig.4 A). However, overexpression of Mob-5 alone in Rat-1 cells did not lead to any detectable phenotypic changes, such as morphological transformation. To determine whether the secreted Mob-5 is a signaling molecule or a cytokine (like IL-10), it is important to know if its specific receptor(s) exist. To this end, APtag technology (26Flanagan J.G. Leder P. Cell. 1990; 63: 185-194Abstract Full Text PDF PubMed Scopus (633) Google Scholar) was employed to create a Mob-5-AP fusion protein, which can then be used as an affinity agent for the receptor analysis. This strategy has been successfully used to identify and clone a number of important cell surface receptors, including those of leptin (27Tartaglia L.A. Dembski M. Weng X. Deng N. Culpepper J. Dvos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3231) Google Scholar) and semaphorin III (28He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (970) Google Scholar). Like Mob-5 itself, Mob-5-AP was secreted at a high level from 293T cells stably transfected with the recombinant plasmid. The secreted 90-kDa Mob-5-AP can be readily detected from the culture medium by either the alkaline phosphatase assay or direct Western blot analysis using anti-Mob-5 antibody (Fig. 4 A). The construct of the Mob-5-AP fusion protein was also verified by Western blot using antibody to the 67-kDa human placental secreted AP (Fig. 4 A, right panel). For the Mob-5 receptor study, Rat-1 cells before and after transformation by oncogenic Ha-ras were incubated with the cell culture medium containing an equal amount of either the Mob-5-AP fusion protein or secreted AP itself. Unlike the AP alone control, Mob-5-AP was found to be preferentially bound to the cell surface of the Ha-ras-transformed cells but not of the parental Rat-1 control (Fig. 4 B). Because Ki-ras mutations have been tightly linked with cancers of gastric intestinal origins (3Kiaris H. Spandidos D.A. Int. J. Oncol. 1995; 7: 413-421PubMed Google Scholar), RIE cells were analyzed for the expression of mob-5. Northern blot analysis was conducted with RIE cells before and after transformation by oncogenic Ha-ras and Ki-ras (29Ko T.C. Sheng H.M. Reisman D. Thompson E.A. Beauchamp R.D. Oncogene. 1995; 10: 177-184PubMed Google Scholar). Consistent with the results obtained with fibroblasts, both oncogenic Ha-rasand Ki-ras were shown to be able to activate mob-5 expression in RIE cells (Fig.5 A). With a high degree of homology to Mda-7, Mob-5 may be the rat homolog of Mda-7. Supporting this hypothesis, mda-7 was found to be overexpressed in nearly all human colon cancer specimens analyzed.2 However, without any functional data on either protein, one cannot rule out that Mob-5 and Mda-7 are instead closely related members of a same gene family. Based on the published cDNA sequence of mda-7, the entire coding region of the gene was amplified from human colon cancer tissues by reverse transcriptase-PCR. DNA sequence analysis of the resulting mda-7 clone revealed a single amino acid (Ser) insertion between the codon 14 and 15 of the published Mda-7 sequence (24Jiang H. Lin J.J. Su Z. Goldstein N.I. Fisher P.B. Oncogene. 1995; 11: 2477-2486PubMed Google Scholar). The Mda-7-AP fusion protein was constructed and expressed in 293T cells. Unlike previous studies on Mda-7, here the Mda-7-AP directed by the signal peptide of Mda-7 was found to be secreted at a high level into the culture medium. The secreted Mda-7-AP could be readily detected by both AP activity assay (range from 0.7 to 1.7 unit/ml) and direct Western blot analysis using polyclonal antibody against Mda-7 (Fig. 5 B, left panel). Further confirmation of the 90-kDa Mda-7-AP fusion protein was obtained by Western blot using antibody to AP itself (Fig. 5 B,right panel). As in Rat-1 cells, the putative Mob-5 receptor was also found to be differentially expressed between the normal and Ha-ras-transformed RIE cells using Mob-5-AP fusion protein as a ligand (Fig. 5 C, left panel). To determine whether the Mda-7-AP could also preferentially bind to t
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