RASSF2 Is a Novel K-Ras-specific Effector and Potential Tumor Suppressor

Abstract

Ras proteins regulate a wide range of biological processes by interacting with a broad assortment of effector proteins. Although activated forms of Ras are frequently associated with oncogenesis, they may also provoke growth-antagonistic effects. These include senescence, cell cycle arrest, differentiation, and apoptosis. The mechanisms that underlie these growth-inhibitory activities are relatively poorly understood. Recently, two related novel Ras effectors, NORE1 and RASSF1, have been identified as mediators of apoptosis and cell cycle arrest. Both of these proteins exhibit many of the properties normally associated with tumor suppressors. We now identify a novel third member of this family, designated RASSF2. RASSF2 binds directly to K-Ras in a GTP-dependent manner via the Ras effector domain. However, RASSF2 only weakly interacts with H-Ras. Moreover, RASSF2 promotes apoptosis and cell cycle arrest and is frequently down-regulated in lung tumor cell lines. Thus, we identify RASSF2 as a new member of the RASSF1 family of Ras effectors/tumor suppressors that exhibits a specificity for interacting with K-Ras. Ras proteins regulate a wide range of biological processes by interacting with a broad assortment of effector proteins. Although activated forms of Ras are frequently associated with oncogenesis, they may also provoke growth-antagonistic effects. These include senescence, cell cycle arrest, differentiation, and apoptosis. The mechanisms that underlie these growth-inhibitory activities are relatively poorly understood. Recently, two related novel Ras effectors, NORE1 and RASSF1, have been identified as mediators of apoptosis and cell cycle arrest. Both of these proteins exhibit many of the properties normally associated with tumor suppressors. We now identify a novel third member of this family, designated RASSF2. RASSF2 binds directly to K-Ras in a GTP-dependent manner via the Ras effector domain. However, RASSF2 only weakly interacts with H-Ras. Moreover, RASSF2 promotes apoptosis and cell cycle arrest and is frequently down-regulated in lung tumor cell lines. Thus, we identify RASSF2 as a new member of the RASSF1 family of Ras effectors/tumor suppressors that exhibits a specificity for interacting with K-Ras. The Ras family of oncoproteins is intimately involved in the regulation of a wide variety of biological processes (1Malumbres M. Pellicer A. Front. Biosci. 1998; 3: d887-d912Crossref PubMed Scopus (11) Google Scholar, 2Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1122) Google Scholar, 3Rommel C. Hafen E. Curr. Opin. Genet. Dev. 1998; 8: 412-418Crossref PubMed Scopus (97) Google Scholar). This versatility is facilitated by the ability of Ras proteins to interact with a broad range of heterologous effector proteins (1Malumbres M. Pellicer A. Front. Biosci. 1998; 3: d887-d912Crossref PubMed Scopus (11) Google Scholar, 4Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (918) Google Scholar, 5Clark G.J. O'Bryan J.P. Der C.J. Gutkind J.S. Signaling Networks and Cell Cycle Control: The Molecular Basis of Cancer and Other Diseases. Humana Press Inc., Totowa, NJ2000: 213-227Google Scholar, 6Shields J.M. Pruitt K. McFall A. Shaub A. Der C.J. Trends Cell Biol. 2000; 10: 147-154Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Although best known for their role in mitogenesis and oncogenesis, Ras proteins can also promote growth arrest and cell death (7Bar-Sagi D. Feramisco J.R. Cell. 1985; 42: 841-848Abstract Full Text PDF PubMed Scopus (568) Google Scholar, 8Chen C.Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 9Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3912) Google Scholar, 10Shao J. Sheng H. DuBois R.N. Beauchamp R.D. J. Biol. Chem. 2000; 275: 22916-22924Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 11Joneson T. Bar-Sagi D. Mol. Cell. Biol. 1999; 19: 5892-5901Crossref PubMed Scopus (153) Google Scholar). In contrast to the Ras pathways mediating mitogenesis and transformation, those mediating growth-antagonistic effects remain relatively poorly characterized. RASSF1 has recently been identified as a potential tumor suppressor that can serve as a Ras effector (12Vos M.D. Ellis C.A. Bell A. Birrer M.J. Clark G.J. J. Biol. Chem. 2000; 275: 35669-35672Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 13Dammann R. Li C. Yoon J.H. Chin P.L. Bates S. Pfeifer G.P. Nat. Genet. 2000; 25: 315-319Crossref PubMed Scopus (998) Google Scholar). RASSF1 can induce apoptosis or cell cycle arrest (12Vos M.D. Ellis C.A. Bell A. Birrer M.J. Clark G.J. J. Biol. Chem. 2000; 275: 35669-35672Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 14Shivakumar L. Minna J. Sakamaki T. Pestell R. White M.A. Mol. Cell. Biol. 2002; 22: 4309-4318Crossref PubMed Scopus (349) Google Scholar) and is frequently down-regulated by promoter methylation during tumorigenesis (15Pfeifer G.P. Yoon J.H. Liu L. Tommasi S. Wilczynski S.P. Dammann R. Biol. Chem. 2002; 383: 907-914Crossref PubMed Scopus (111) Google Scholar). NORE1 is related to RASSF1 and can also induce a Ras-dependent apoptosis (16Khokhlatchev A. Rabizadeh S. Xavier R. Nedwidek M. Chen T. Zhang X.F. Seed B. Avruch J. Curr. Biol. 2002; 12: 253-265Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 17Vos M.D. Martinez A. Ellis C.A. Valecorsa T. Clark G.J. J. Biol. Chem. 2003; 278: 21938-21943Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Like RASSF1, NORE1 is frequently down-regulated in primary tumors and tumor cell lines (17Vos M.D. Martinez A. Ellis C.A. Valecorsa T. Clark G.J. J. Biol. Chem. 2003; 278: 21938-21943Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Data base analysis suggests that there may be more members of this family. We identified in the GenBank™ data base a human RASSF1-like hypothetical protein that was originally designated KIAA0168 but now is being referred to as RASSF2. We cloned the gene and sought to determine whether RASSF2 is also a Ras effector/tumor suppressor of the RASSF1 family. We now report that RASSF2 can bind directly to K-Ras in a GTP-dependent manner via the Ras effector domain. Therefore, RASSF2 demonstrates the basic characteristics of a Ras effector. The interaction of RASSF2 with Ras appears to be specific to K-Ras, as only a weak interaction could be detected with H-Ras. Rather than promoting transformation, over-expression of RASSF2 inhibits the growth of lung tumor cells. RASSF2-mediated growth inhibition is enhanced by activated K-Ras and appears to involve both apoptosis and cell cycle arrest. Analysis of RASSF2 protein expression in a series of human lung tumor cell lines shows that the protein is frequently down-regulated. Thus, we show that RASSF2 is a new member of the RASSF1 family and shares the properties of being a potential Ras effector/tumor suppressor. Identification of RASSF2—RASSF2 was identified by performing a tblastn search of the GenBank™ data base using the Ras association (RA) 1The abbreviations used are: RA, Ras association; HA, hemagglutinin; GST, glutathione S-transferase; EYFP, enhanced yellow fluorescent protein; GFP, green fluorescent protein. domain of RASSF1 as a query. The human hypothetical protein KIAA0168 was identified as a potential RASSF1-like protein. This protein is now being referred to as RASSF2/Rasfadin (18Comincini S. Castiglioni B.M. Foti G.M. Del Vecchio I. Ferretti L. Mamm. Genome. 2001; 12: 150-156Crossref PubMed Scopus (9) Google Scholar), and we will conform to this convention. Sequences were aligned using ClustalW. DNA and Plasmids—RASSF2 was identified as IMAGE Consortium clone 22950 distributed by the American Type Culture Collection (ATCC; Manassas, VA). The RASSF2 coding region was PCR-cloned using oligomers 5′-ggatccatggactacagccaccaaac and 3′-caattgtcagattgttgctggggtc, which added a BamHI site to the 5′ end and an MfeI site to the 3′ end. After sequencing to confirm fidelity, the gene was cloned into pZIPHA, pCDNAF (17Vos M.D. Martinez A. Ellis C.A. Valecorsa T. Clark G.J. J. Biol. Chem. 2003; 278: 21938-21943Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), and pEGFP (Clontech, Palo Alto, CA) as a BamHI/MfeI fragment. The RA domain of RASSF2 (nucleotides 535–789) was cloned as a BamHI/MfeI fragment into pMal (New England BioLabs, Beverly, MA) and pGEX2T (Amersham Biosciences). Activated K-Ras was cloned into the BamHI site of pCGNHA (19Fiordalisi J.J. Johnson R.L. Ulku A.S. Der C.J. Cox A.D. Methods Enzymol. 2001; 332: 3-36Crossref PubMed Scopus (46) Google Scholar), and effector mutants were generated using a QuikChange kit (Stratagene, La Jolla, CA). Ras Binding Assays—In vivo assays were performed by transfecting 293-T cells with 5 μg of each plasmid using LipofectAMINE 2000 (Invitrogen). After 48 h, the cells were lysed in modified radioimmune precipitation assay buffer (20Ellis C.A. Vos M.D. Howell H. Vallecorsa T. Fults D.W. Clark G.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9876-9881Crossref PubMed Scopus (53) Google Scholar) and immunoprecipitated with HA beads (Sigma). The immunoprecipitate was then subjected to Western analysis with an anti-FLAG monoclonal (Sigma) to measure the co-precipitation of RASSF2. In vitro binding assays were performed using purified GST fusion protein of the RA domain of RASSF2 and purified K-Ras derived from baculovirus-infected Sf9 cells (a generous gift of D. Stokoe, University of California, San Francisco, CA). K-Ras protein was loaded with the GTP analog guanylyl-5-imidodiphosphate (Sigma) by incubating in 100 mm Tris, pH 8, 50 mm NaCl, and 10 mm EDTA with 6 mm GTP analog (Sigma) for 10 min at room temperature. The loaded protein was then stabilized with 10 mm MgCl2. 2 pg of loaded protein were then added to 100 ng of fusion protein on Sepharose beads in phosphate-buffered saline with 1× protease inhibitors (Pharmingen), 5 mm MgCl2, 1 μm ZnCl2, and 0.01% Tween 20. The proteins were rotated at 4 °C for 1 h, washed three times, and then subjected to Western analysis. Growth Inhibition Assays—Cell lines were obtained from the ATCC. A549 human lung carcinoma cells were transfected with 1 μg of pZIPHA RASSF2 or empty vector and selected in 500 μg/ml of G418 (Invitrogen) for 2 weeks. Cells were fixed and stained with crystal violet. Transient growth inhibition assays were performed in 293-T cells by transfecting with LipofectAMINE 2000 (Invitrogen) as described previously (12Vos M.D. Ellis C.A. Bell A. Birrer M.J. Clark G.J. J. Biol. Chem. 2000; 275: 35669-35672Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Apoptosis Assays—RASSF2-induced apoptosis was examined by using the pCaspase3-Sensor system (Clontech) and fluorescent microscopy to measure caspase-3 activation in individual live cells. This system uses an enhanced yellow fluorescent protein (EYFP) fused with a nuclear export signal and a nuclear localization signal. The nuclear export signal is dominant and separated from the EYFP by a caspase-3 cleavage site. In the absence of active caspase-3, the EYFP localizes to the cytosol. In the presence of active caspase-3, the nuclear export signal is cleaved off, and the nuclear localization signal promotes transport into the nucleus. Cells were transfected with 100 ng of pCaspase3-Sensor and/or red fluorescent protein-RASSF2. After 24 h, red cells were examined for the localization of EYFP. The detection of caspase-3 activation and induction of apoptosis using this method were quantified by identifying and counting cells that co-expressed the nuclear localized pCaspase3-Sensor protein and red fluorescent protein-RASSF2. In situ trypan blue uptake was performed on 293 HEK cells (ATCC). Cells were transfected with 5 μg of RASSF2 in the presence or absence of 50 ng of K-Ras12V. 72 h post-transfection trypan blue was added at a final concentration of 0.04%. Dye uptake was quantitated by counting the number of blue cells in three random 40× fields. Fluorescence-activated Cell Sorting Analysis—Cell cycle analysis was performed in 293-T cells transfected with 5 μg of pEGFP empty vector or pEGFP-RASSF2 using LipofectAMINE 2000 (Invitrogen). 24 h after transfection cells were harvested and resuspended at a concentration of 2 × 106 cells/ml in medium with low serum (2%). Hoechst 33342 (Sigma) was added to a final concentration of 3 μg/ml and incubated for 1 h at 37 °C. After incubation cells were immediately centrifuged in the cold, resuspended to a final concentration of 1 × 106 cells/ml, and subjected to fluorescence-activated cell sorting analysis. DNA content of GFP-positive cells was determined using a fluorescence-activated cell sorting Vantage S.E. (BD Biosciences). The software programs CellQuest (BD Biosciences) and ModFit (Verity Software House) were used for data and cell cycle of was obtained from The coding region of RASSF2 was used as a were performed as described previously (12Vos M.D. Ellis C.A. Bell A. Birrer M.J. Clark G.J. J. Biol. Chem. 2000; 275: 35669-35672Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). of RASSF2 in Cell was to the by and The with NORE1 or RASSF1 was by The was then used at a of to Western analysis of a series of human lung tumor cell lines. of RASSF1, and were aligned using The RA domain is in 1 and shows to that of and to that of RASSF2 the domain of NORE1 and Binding of K-Ras to determine whether Ras and RASSF2 could we performed in 293-T cells with RASSF2 and activated and K-Ras. After 48 h, cells were lysed and immunoprecipitated with beads (Sigma) for 1 After the immunoprecipitate was subjected to Western analysis using an anti-FLAG shows that RASSF2 with activated K-Ras. The of Ras and RASSF2 in the were determined by the with HA and We determined the interaction the effector domain of Ras by using a with a series of effector mutants of K-Ras. shows that in the effector domain of K-Ras the ability of the protein to bind RASSF2. a Western of the is at the of we examined the that RASSF2 bind K-Ras H-Ras. In we show that activated K-Ras the of RASSF2 with activated were in the are at the of confirm that the interaction is we GST fusion protein of the RA domain of RASSF2. This was used as an to K-Ras gift of D. Stokoe, University of California, San Francisco, CA). shows that the domain fusion protein can K-Ras from of GST fusion protein was by Western of a of the with an CA). that the domain fusion protein also were obtained with a protein fusion of the RASSF2 RA domain Therefore, RASSF2 binds directly to K-Ras in a GTP-dependent manner via the effector domain. RASSF2 Cell was cloned into a expression pZIPHA, and transfected into A549 human lung carcinoma cells. Cells were selected for 2 and then stained with crystal Cells transfected with RASSF2 to the vector a we were to cell lines we to to determine the effects of Ras on RASSF2-mediated growth 293-T cells were transfected with RASSF2 activated Ras and examined after 48 h this growth inhibition was detected with the RASSF2 but this was enhanced by the presence of activated K-Ras. on this determine whether the growth inhibition by RASSF2 was in and whether the presence of activated Ras enhanced this we transfected 293 cells with RASSF2 K-Ras12V. 72 h post-transfection we added trypan blue directly to the cells and quantitated cells by 6 shows that RASSF2 on promotes cell but in the presence of activated K-Ras cell death is is in cell death is enhanced by 293 cells were transfected with RASSF2 activated K-Ras. blue was added after 72 h, and uptake was RASSF2 promotes a cell death that is enhanced in the presence of activated in situ trypan blue of and Dye uptake is enhanced RASSF2 is co-expressed with activated K-Ras. of the in RASSF2 Apoptosis and Cell Cycle determine the of the RASSF2-mediated growth we used the pCaspase3-Sensor system (Clontech) and fluorescent microscopy to measure caspase-3 activation in individual cells. Cells were with pCaspase3-Sensor and red fluorescent protein-RASSF2. After 24 h, red cells were examined for the localization of EYFP. shows that in cells the EYFP However, in the of cells nuclear localization of EYFP, caspase-3 activation and apoptosis. The are quantified in We also performed cell cycle assays on 293-T cells. demonstrates that cells show an in the of the cell that the cells to arrest in the of RASSF2 in and determine the specificity of RASSF2 we performed analysis on a was detected at in The signal was in the and lung determine whether RASSF2 be down-regulated during we generated a We from a series of human lung tumor cell lines and examined for RASSF2 protein cell was used as a Western analysis that the RASSF2 protein was frequently down-regulated in the human lung tumor cell lines cell nuclear was used as an for protein protein expression is frequently down-regulated in human lung tumor cell lines. to RASSF2 was and used to a series of lung tumor cell lines by Western analysis. human cell was used as a lung tumor cell lines were for The was with cell nuclear as a Although Ras oncoproteins are in many D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1122) Google Scholar, 5Clark G.J. O'Bryan J.P. Der C.J. Gutkind J.S. Signaling Networks and Cell Cycle Control: The Molecular Basis of Cancer and Other Diseases. Humana Press Inc., Totowa, NJ2000: 213-227Google Scholar, G.J. Cox A.D. Der C.J. Methods Enzymol. PubMed Scopus Google Scholar), they may also provoke a variety of growth-antagonistic effects M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3912) Google Scholar), apoptosis J. Sheng H. DuBois R.N. Beauchamp R.D. J. Biol. Chem. 2000; 275: 22916-22924Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 11Joneson T. Bar-Sagi D. Mol. Cell. Biol. 1999; 19: 5892-5901Crossref PubMed Scopus (153) Google Scholar), and cell cycle arrest A.W. Lowe S.W. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). This to induce or death is to Ras but has also been described for oncoproteins as Trends Genet. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S.W. Lin A.W. 2000; PubMed Scopus Google Scholar). Thus, oncoproteins a the processes of and in a cell after activation will determine whether that cell or to a the pathways by may for Ras pathways mediating growth inhibition and death are as as those used to promote mitogenesis and However, the of the of Ras has to this RASSF1 and NORE1 have been to Ras-dependent apoptosis as as cell cycle arrest (12Vos M.D. Ellis C.A. Bell A. Birrer M.J. Clark G.J. J. Biol. Chem. 2000; 275: 35669-35672Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 14Shivakumar L. Minna J. Sakamaki T. Pestell R. White M.A. Mol. Cell. Biol. 2002; 22: 4309-4318Crossref PubMed Scopus (349) Google Scholar, A. Rabizadeh S. Xavier R. Nedwidek M. Chen T. Zhang X.F. Seed B. Avruch J. Curr. Biol. 2002; 12: 253-265Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 17Vos M.D. Martinez A. Ellis C.A. Valecorsa T. Clark G.J. J. Biol. Chem. 2003; 278: 21938-21943Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). only these proteins growth but they are also frequently down-regulated during tumor Moreover, of the into tumor cell lines in a of R. Li C. Yoon J.H. Chin P.L. Bates S. Pfeifer G.P. Nat. Genet. 2000; 25: 315-319Crossref PubMed Scopus (998) Google Scholar, A. Rabizadeh S. Xavier R. Nedwidek M. Chen T. Zhang X.F. Seed B. Avruch J. Curr. Biol. 2002; 12: 253-265Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 17Vos M.D. Martinez A. Ellis C.A. Valecorsa T. Clark G.J. J. Biol. Chem. 2003; 278: 21938-21943Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Therefore, these proteins have the potential to serve as tumor suppressors that are directly activated by of of these growth-inhibitory pathways may as as of of pathways during tumor data base analysis suggests that there may be members of the family that remain We have now cloned and RASSF2 to determine whether as a Ras effector/tumor suppressor. of the Ras binding properties of RASSF2 that with K-Ras in a Moreover, the interaction was as the purified RA domain is the activated gene in human tumors G.J. Der C.J. L. in Inc., Scholar) and is the only gene that is for L. D. K. K. E. E. H. R. T. Dev. 1997; PubMed Scopus Google Scholar). Thus, appears that K-Ras protein is from the Ras and has a role in and has been that the for the properties of K-Ras is that there may be Ras effector proteins that are to tumorigenesis and that with K-Ras C.A. Clark G.J. Cell. 2000; 12: PubMed Scopus Google Scholar). However, in effector binding the Ras have been J. S. A. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), these involved only a in binding we an for K-Ras H-Ras. Thus, RASSF2 could a role in mediating of the biological properties of K-Ras. we in association of RASSF2 with we the three Ras effector and M.A. C. A. A. L. M. Cell. Full Text PDF PubMed Scopus Google Scholar) in the ability to interact with and this was Thus, the interaction of RASSF2 and is via the Ras effector domain. RASSF2 the basic for a potential Ras effector. Ras effector mutants have been used as to identify the Ras pathways that are for tumor E. I. L. K. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, J.H. Der C.J. Dev. 2002; PubMed Scopus Google Scholar). the of this of now into the potential of interaction with tumor suppressor effectors, as as as the of interaction with effectors, as The of of the RASSF1 family of proteins RASSF1 and NORE1 have been to promote apoptosis in cell (12Vos M.D. Ellis C.A. Bell A. Birrer M.J. Clark G.J. J. Biol. Chem. 2000; 275: 35669-35672Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, A. Rabizadeh S. Xavier R. Nedwidek M. Chen T. Zhang X.F. Seed B. Avruch J. Curr. Biol. 2002; 12: 253-265Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 17Vos M.D. Martinez A. Ellis C.A. Valecorsa T. Clark G.J. J. Biol. Chem. 2003; 278: 21938-21943Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), L. Minna J. Sakamaki T. Pestell R. White M.A. Mol. Cell. Biol. 2002; 22: 4309-4318Crossref PubMed Scopus (349) Google Scholar) they cell cycle arrest, in we to both for RASSF2. We that RASSF2 the activation of caspase-3 in cells. was in 293-T cells but the were in the cell to their Thus, RASSF2 can promote apoptosis. cell cycle we used fluorescence-activated cell sorting analysis of 293-T cells. These cells a in the in three This suggests that the cells are to arrest in Thus, RASSF2 can promote apoptosis and cell cycle arrest. potential for apoptosis was recently identified by (16Khokhlatchev A. Rabizadeh S. Xavier R. Nedwidek M. Chen T. Zhang X.F. Seed B. Avruch J. Curr. Biol. 2002; 12: 253-265Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar) was that NORE1 binds to the However, they were to that NORE1 activated the appears to and this is an the role of in cell death Analysis of RASSF2 in shows of the which show the of RASSF2 expression and the of RASSF1 expression in an (12Vos M.D. Ellis C.A. Bell A. Birrer M.J. Clark G.J. J. Biol. Chem. 2000; 275: 35669-35672Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Both show of in lung Analysis of RASSF2 expression in a of lung tumor cell lines that the protein is at in a human cell but to in of the tumor cell lines cell of RASSF2 This suggests that there is a in the RASSF2 that or that there is a in a of the RASSF2 that this cell to RASSF2-mediated growth These are being In we now identify a third member of the family that binds K-Ras with the characteristics of an effector. RASSF2 inhibits the growth of tumor and growth-inhibitory properties are enhanced by activated K-Ras. RASSF2 promotes both cell cycle arrest and may a role in the of