Tumor Suppressor Ras Association Domain Family 5 (RASSF5/NORE1) Mediates Death Receptor Ligand-induced Apoptosis
2010; Elsevier BV; Volume: 285; Issue: 45 Linguagem: Inglês
10.1074/jbc.m110.165506
ISSN1083-351X
AutoresJikyoung Park, Soo Im Kang, Sun‐Young Lee, Xian F. Zhang, Myoung Shin Kim, Lisa F. Beers, Dae‐Sik Lim, Joseph Avruch, Ho‐Shik Kim, Sean Bong Lee,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoEpigenetic silencing of RASSF (Ras association domain family) genes RASSF1 and RASSF5 (also called NORE1) by CpG hypermethylation is found frequently in many cancers. Although the physiological roles of RASSF1 have been studied in some detail, the exact functions of RASSF5 are not well understood. Here, we show that RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, TNF-α and TNF-related apoptosis-inducing ligand. Depletion of RASSF5 by siRNA significantly reduced TNF-α-mediated apoptosis, likely through its interaction with proapoptotic kinase MST1, a mammalian homolog of Hippo. Consistent with this, siRNA knockdown of MST1 also resulted in resistance to TNF-α-induced apoptosis. To further study the role of Rassf5 in vivo, we generated Rassf5-deficient mouse. Inactivation of Rassf5 in mouse embryonic fibroblasts (MEFs) resulted in resistance to TNF-α- and TNF-related apoptosis-inducing ligand-mediated apoptosis. Importantly, Rassf5-null mice were significantly more resistant to TNF-α-induced apoptosis and failed to activate Mst1. Loss of Rassf5 also resulted in spontaneous immortalization of MEFs at earlier passages than the control MEFs, and Rassf5-null immortalized MEFs, but not the immortalized wild type MEFs, were fully transformed by K-RasG12V. Together, our results demonstrate a direct role for RASSF5 in death receptor ligand-mediated apoptosis and provide further evidence for RASSF5 as a tumor suppressor. Epigenetic silencing of RASSF (Ras association domain family) genes RASSF1 and RASSF5 (also called NORE1) by CpG hypermethylation is found frequently in many cancers. Although the physiological roles of RASSF1 have been studied in some detail, the exact functions of RASSF5 are not well understood. Here, we show that RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, TNF-α and TNF-related apoptosis-inducing ligand. Depletion of RASSF5 by siRNA significantly reduced TNF-α-mediated apoptosis, likely through its interaction with proapoptotic kinase MST1, a mammalian homolog of Hippo. Consistent with this, siRNA knockdown of MST1 also resulted in resistance to TNF-α-induced apoptosis. To further study the role of Rassf5 in vivo, we generated Rassf5-deficient mouse. Inactivation of Rassf5 in mouse embryonic fibroblasts (MEFs) resulted in resistance to TNF-α- and TNF-related apoptosis-inducing ligand-mediated apoptosis. Importantly, Rassf5-null mice were significantly more resistant to TNF-α-induced apoptosis and failed to activate Mst1. Loss of Rassf5 also resulted in spontaneous immortalization of MEFs at earlier passages than the control MEFs, and Rassf5-null immortalized MEFs, but not the immortalized wild type MEFs, were fully transformed by K-RasG12V. Together, our results demonstrate a direct role for RASSF5 in death receptor ligand-mediated apoptosis and provide further evidence for RASSF5 as a tumor suppressor. IntroductionA small family of genes termed RASSF (Ras association domain family) has been recently described, the members of which are characterized by the presence of a Ras association (RA) 4The abbreviations used are: RARas associationMEFmouse embryonic fibroblastPARPpoly-ADP-ribose polymeraseCASPcaspaseTNF-R1TNF receptor 1TRAILTNF-related apoptosis-inducing ligandESembryonic stem. domain and a novel motif named the SARAH (Salvador, Rassf, Hippo) domain at the C terminus (reviewed in Refs. 1van der Weyden L. Adams D.J. Biochim. Biophys. Acta. 2007; 1776: 58-85Crossref PubMed Scopus (228) Google Scholar, 2Richter A.M. Pfeifer G.P. Dammann R.H. Biochim. Biophys. Acta. 2009; 1796: 114-128Crossref PubMed Scopus (247) Google Scholar, 3Avruch J. Xavier R. Bardeesy N. Zhang X.F. Praskova M. Zhou D. Xia F. J. Biol. Chem. 2009; 284: 11001-11005Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Among the members of the RASSF family, RASSF1 and RASSF5 (also known as NORE1, for novel Ras effector 1) share the closest homology, displaying 49% identity (66% similarity) at the protein level, and are frequently inactivated by CpG hypermethylation in human cancer cell lines and primary tumors (4Dammann 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, 5Morris M.R. Hesson L.B. Wagner K.J. Morgan N.V. Astuti D. Lees R.D. Cooper W.N. Lee J. Gentle D. Macdonald F. Kishida T. Grundy R. Yao M. Latif F. Maher E.R. Oncogene. 2003; 22: 6794-6801Crossref PubMed Scopus (94) Google Scholar, 6Hesson L. Dallol A. Minna J.D. Maher E.R. Latif F. Oncogene. 2003; 22: 947-954Crossref PubMed Scopus (112) Google Scholar). RASSF1 has been studied extensively (reviewed in Ref. 7Donninger H. Vos M.D. Clark G.J. J. Cell Sci. 2007; 120: 3163-3172Crossref PubMed Scopus (348) Google Scholar) and shown to play important roles in mitosis (8Song M.S. Song S.J. Ayad N.G. Chang J.S. Lee J.H. Hong H.K. Lee H. Choi N. Kim J. Kim H. Kim J.W. Choi E.J. Kirschner M.W. Lim D.S. Nat. Cell Biol. 2004; 6: 129-137Crossref PubMed Scopus (276) Google Scholar), microtubule and genomic stability (9Vos M.D. Martinez A. Elam C. Dallol A. Taylor B.J. Latif F. Clark G.J. Cancer Res. 2004; 64: 4244-4250Crossref PubMed Scopus (125) Google Scholar, 10van der Weyden L. Tachibana K.K. Gonzalez M.A. Adams D.J. Ng B.L. Petty R. Venkitaraman A.R. Arends M.J. Bradley A. Mol. Cell. Biol. 2005; 25: 8356-8367Crossref PubMed Scopus (86) Google Scholar, 11Liu L. Tommasi S. Lee D.H. Dammann R. Pfeifer G.P. Oncogene. 2003; 22: 8125-8136Crossref PubMed Scopus (164) Google Scholar), apoptosis (12Baksh S. Tommasi S. Fenton S. Yu V.C. Martins L.M. Pfeifer G.P. Latif F. Downward J. Neel B.G. Mol. Cell. 2005; 18: 637-650Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 13Matallanas D. Romano D. Yee K. Meissl K. Kucerova L. Piazzolla D. Baccarini M. Vass J.K. Kolch W. O'neill E. Mol. Cell. 2007; 27: 962-975Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar), and cell cycle (14Shivakumar L. Minna J. Sakamaki T. Pestell R. White M.A. Mol. Cell. Biol. 2002; 22: 4309-4318Crossref PubMed Scopus (348) Google Scholar), which are consistent with its function as a tumor suppressor. However, not much is known about the physiological roles of RASSF5.RASSF5 encodes multiple isoforms because of dual promoter usage and alternative splicing (15Tommasi S. Dammann R. Jin S.G. Zhang X.F. Avruch J. Pfeifer G.P. Oncogene. 2002; 21: 2713-2720Crossref PubMed Scopus (94) Google Scholar). The longest isoform, designated RASSF5A (NORE1A), is transcribed from the 5′-most promoter region containing CpG island and encodes a 418-amino acid protein containing the cysteine-rich diacylglycerol/phorbol ester-binding domain (also called protein kinase C conserved region 1, C1), the RA domain, and the C-terminal SARAH domain. Through alternative splicing, another isoform RASSF5B (NORE1Aβ), lacking the SARAH domain is produced. The shortest isoform, RASSF5C (NORE1B, also called RAPL), lacking the N-terminal C1 domain, is produced from a downstream CpG-containing promoter that is less frequently methylated in primary tumors and cancer cell lines than the 5′ upstream promoter (6Hesson L. Dallol A. Minna J.D. Maher E.R. Latif F. Oncogene. 2003; 22: 947-954Crossref PubMed Scopus (112) Google Scholar). Similar to RASSF1A, CpG methylation of RASSF5A has been found in various cancer cell lines and primary tumors of lung, breast, colon, liver, and kidney (6Hesson L. Dallol A. Minna J.D. Maher E.R. Latif F. Oncogene. 2003; 22: 947-954Crossref PubMed Scopus (112) Google Scholar, 16Calvisi D.F. Ladu S. Gorden A. Farina M. Conner E.A. Lee J.S. Factor V.M. Thorgeirsson S.S. Gastroenterology. 2006; 130: 1117-1128Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar), although less frequently than RASSF1A. These findings suggest that RASSF5A likely functions as a tumor suppressor.RASSF5A was first identified as the interacting partner of the active GTP-bound form of RAS or other RAS-like GTPases, such as Rap1, M-Ras, and R-Ras/R-Ras3, through the RA domain (17Vavvas D. Li X. Avruch J. Zhang X.F. J. Biol. Chem. 1998; 273: 5439-5442Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). RASSF5C (NORE1B/RAPL) was also shown to interact with Rap1 following T-cell antigen or chemokine receptor activation, and the RASSF5C-Rap1 complex promotes recruitment of integrin LFA-1 to the T-cell leading edge, enhancing the T-cell affinity for intercellular adhesion molecule (ICAM) (18Katagiri K. Ohnishi N. Kabashima K. Iyoda T. Takeda N. Shinkai Y. Inaba K. Kinashi T. Nat. Immunol. 2004; 5: 1045-1051Crossref PubMed Scopus (161) Google Scholar). Thus, RASSF5C has been identified as a critical molecule for lymphocyte and dendritic cell trafficking. In addition to the RA domain, RASSF5A, through its N-terminal domain, forms a heterodimer with RASSF1A (19Ortiz-Vega S. Khokhlatchev A. Nedwidek M. Zhang X.F. Dammann R. Pfeifer G.P. Avruch J. Oncogene. 2002; 21: 1381-1390Crossref PubMed Scopus (201) Google Scholar), although whether the diacylglycerol/phorbol ester domain of RASSF5A is necessary for this binding is not clear. The C-terminal SARAH domain of RASSF5 also mediates interaction with other SARAH-containing proteins (20Scheel H. Hofmann K. Curr. Biol. 2003; 13: R899-R900Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), such as the mammalian sterile 20-like kinases MST1/2 (21Praskova M. Khoklatchev A. Ortiz-Vega S. Avruch J. Biochem. J. 2004; 381: 453-462Crossref PubMed Scopus (276) Google Scholar, 22Khokhlatchev 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 (327) Google Scholar) and WW45/SAV1 (23Hwang E. Ryu K.S. Pääkkönen K. Güntert P. Cheong H.K. Lim D.S. Lee J.O. Jeon Y.H. Cheong C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9236-9241Crossref PubMed Scopus (105) Google Scholar), which are the mammalian homologs of Drosophila Hippo and Salvador, respectively. MST1 kinase has been shown to be proapoptotic and is cleaved by Caspase 3 during apoptosis (24Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar, 25Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (322) Google Scholar). Cleaved MST1 translocates into the nucleus and exhibits significantly higher kinase activity than the unprocessed form (26Ura S. Masuyama N. Graves J.D. Gotoh Y. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 10148-10153Crossref PubMed Scopus (137) Google Scholar). Activated MST1 phosphorylates histone H2B on Ser14 (27Cheung W.L. Ajiro K. Samejima K. Kloc M. Cheung P. Mizzen C.A. Beeser A. Etkin L.D. Chernoff J. Earnshaw W.C. Allis C.D. Cell. 2003; 113: 507-517Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar) and the pro-apoptotic Forkhead box-containing transcription factor, FOXO3a (Forkhead box O3a) (28Lehtinen M.K. Yuan Z. Boag P.R. Yang Y. Villén J. Becker E.B. DiBacco S. de la Iglesia N. Gygi S. Blackwell T.K. Bonni A. Cell. 2006; 125: 987-1001Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar), in mediating its apoptotic effects. The anti-apoptotic AKT kinase has been shown to phosphorylate MST1 and inhibit MST1-mediated FOXO3a phosphorylation and apoptosis (29Jang S.W. Yang S.J. Srinivasan S. Ye K. J. Biol. Chem. 2007; 282: 30836-30844Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Despite these studies, the exact role of RASSF5 in apoptosis has not been fully demonstrated. In this study, we demonstrate that RASSF5 is required for TNF-α-mediated apoptosis and for full activation of Mst1 in vivo.DISCUSSIONIn the present study, we demonstrate that RASSF5 is an important downstream effector of TNF-α- and TRAIL-induced apoptosis. Depletion of RASSF5, MST1, and YAP1, but not WW45 and LATS1, resulted in reduced TNF-α-induced apoptosis, indicating that not all components of the Hippo pathway are required for TNF-α-induced apoptosis mediated by RASSF5 and MST1. RASSF5 interacts strongly with MST1 but only weakly with other components of the mammalian Hippo signaling pathway. In Drosophila, dRASSF competes with Salvador (WW45 in human) for Hippo (MST1), and thus, the dRASSF-Hippo complex is thought to be mutually exclusive with the Salvador-Hippo complex (38Polesello C. Huelsmann S. Brown N.H. Tapon N. Curr. Biol. 2006; 16: 2459-2465Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). However, a recent study has shown that mammalian RASSF6 is capable of forming a tripartite complex with MST1 and WW45 (39Ikeda M. Kawata A. Nishikawa M. Tateishi Y. Yamaguchi M. Nakagawa K. Hirabayashi S. Bao Y. Hidaka S. Hirata Y. Hata Y. Sci. Signal. 2009; 2: ra59Crossref PubMed Scopus (82) Google Scholar). Therefore, further studies will be needed to determine whether RASSF5 is also capable of forming a multi-complex with the members of the Hippo pathway.The effector role of RASSF5 downstream of TNF-α was further supported by our in vivo studies. Inactivation of Rassf5 in the mouse led to a significant protection from TNF-α-mediated apoptosis of the liver. Our results further showed that Rassf5 is required for the activation of the proapoptotic kinase Mst1 after TNF-α stimulation in vivo, as measured by the absence of Mst1 cleavage and H2B (Ser14) phosphorylation in Rassf5-null cells. A shorter isoform, Rassf5C (Nore1B/Rapl), has also been shown to be necessary for the activation of Mst1 during immune cell trafficking (18Katagiri K. Ohnishi N. Kabashima K. Iyoda T. Takeda N. Shinkai Y. Inaba K. Kinashi T. Nat. Immunol. 2004; 5: 1045-1051Crossref PubMed Scopus (161) Google Scholar, 35Katagiri K. Imamura M. Kinashi T. Nat. Immunol. 2006; 7: 919-928Crossref PubMed Scopus (187) Google Scholar). Chemokines and T-cell receptor ligation activate T-cells and result in a rapid phosphorylation and activation of Mst1. It was shown that in the absence of Rassf5C, chemokine- or T-cell receptor-mediated activation of Mst1 in T-cells was undetectable or greatly reduced (18Katagiri K. Ohnishi N. Kabashima K. Iyoda T. Takeda N. Shinkai Y. Inaba K. Kinashi T. Nat. Immunol. 2004; 5: 1045-1051Crossref PubMed Scopus (161) Google Scholar). Therefore, it appears that Rassf5 is necessary for Mst1 activation in response to different stimuli in vivo. Given the previous observations of enhanced MST1 activation when recruited to plasma membrane or in association with activated RAS and RASSF5 (21Praskova M. Khoklatchev A. Ortiz-Vega S. Avruch J. Biochem. J. 2004; 381: 453-462Crossref PubMed Scopus (276) Google Scholar, 22Khokhlatchev 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 (327) Google Scholar), it is possible that RASSF5 is responsible for physically recruiting MST1 to the sites where it can be activated via its interaction with activated RAS or with TNF-R1 (in response to TNF-α stimulation). Although the exact mechanisms of how RASSF5 mediates TNF-α-induced apoptosis need further investigation, the downstream activation of the NF-κB pathway following TNF-α stimulation appears unaffected by depletion of RASSF5, either in the siRNA-treated cells or in Rassf5-null MEFs, as evidenced by similar phosphorylation kinetics of IKK and IκB (supplemental Fig. S9). These results suggest that RASSF5 is unlikely to play a role in TNF-α-induced activation of NF-κB pathway.It was recently shown that following TNF-α stimulation, RASSF1A activated BAX through its interaction with BH3-like protein modulator of apoptosis-1 and subsequently triggered apoptosis (12Baksh S. Tommasi S. Fenton S. Yu V.C. Martins L.M. Pfeifer G.P. Latif F. Downward J. Neel B.G. Mol. Cell. 2005; 18: 637-650Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). RASSF1A also forms a complex with the components of mammalian Hippo pathway (40Guo C. Tommasi S. Liu L. Yee J.K. Dammann R. Pfeifer G.P. Curr. Biol. 2007; 17: 700-705Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and induces apoptosis following stimulation with other death receptor ligands, such as Fas-L or TRAIL (12Baksh S. Tommasi S. Fenton S. Yu V.C. Martins L.M. Pfeifer G.P. Latif F. Downward J. Neel B.G. Mol. Cell. 2005; 18: 637-650Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 13Matallanas D. Romano D. Yee K. Meissl K. Kucerova L. Piazzolla D. Baccarini M. Vass J.K. Kolch W. O'neill E. Mol. Cell. 2007; 27: 962-975Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 41Vichalkovski A. Gresko E. Cornils H. Hergovich A. Schmitz D. Hemmings B.A. Curr. Biol. 2008; 18: 1889-1895Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 42Oh H.J. Lee K.K. Song S.J. Jin M.S. Song M.S. Lee J.H. Im C.R. Lee J.O. Yonehara S. Lim D.S. Cancer Res. 2006; 66: 2562-2569Crossref PubMed Scopus (157) Google Scholar). Similarly, we also observed reduced apoptosis in response to TRAIL in Rassf5-null MEFs compared with the control. However, deletion of Rassf5 did not have any effects on other apoptotic stimuli such as staurosporine, methyl methanesulfonate, tamoxifen, and nocodazole (supplemental Fig. S10B). Together, these results indicate that death receptor-induced apoptosis, such as TNF-α and TRAIL, is mediated by RASSF5, as well as RASSF1 (12Baksh S. Tommasi S. Fenton S. Yu V.C. Martins L.M. Pfeifer G.P. Latif F. Downward J. Neel B.G. Mol. Cell. 2005; 18: 637-650Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). In this regard, we note that RASSF1 and RASSF5 can form a heterodimer (19Ortiz-Vega S. Khokhlatchev A. Nedwidek M. Zhang X.F. Dammann R. Pfeifer G.P. Avruch J. Oncogene. 2002; 21: 1381-1390Crossref PubMed Scopus (201) Google Scholar), raising the possibility that the two RASSF proteins might share overlapping functions.The results from this study suggest that inactivation of RASSF5 by epigenetic silencing will likely lead to tumorigenesis in conjunction with other oncogenic events. This is suggested by the precocious spontaneous immortalization of the Rassf5−/− MEFs compared with the wild type MEFs and by enhanced susceptibility of the mutant MEFs to oncogenic transformation by K-RasG12V. These results provide evidence for the role of Rassf5 as a tumor suppressor. However, loss of Rassf5 alone was not sufficient to fully transform MEFs, demonstrating that further genetic and/or epigenetic changes are required for tumorigenesis. This idea is consistent with the low spontaneous tumor incidence observed in Rassf5-null mice (supplemental Table S2). The low tumor incidence in Rassf5-null mice is similar to the Rassf1-null mice (10van der Weyden L. Tachibana K.K. Gonzalez M.A. Adams D.J. Ng B.L. Petty R. Venkitaraman A.R. Arends M.J. Bradley A. Mol. Cell. Biol. 2005; 25: 8356-8367Crossref PubMed Scopus (86) Google Scholar, 43Tommasi S. Dammann R. Zhang Z. Wang Y. Liu L. Tsark W.M. Wilczynski S.P. Li J. You M. Pfeifer G.P. Cancer Res. 2005; 65: 92-98PubMed Google Scholar). Given the high frequency of CpG hypermethylation of RASSF1 and RASSF5 in human cancers, it will be interesting to determine the tumor susceptibility of double Rassf1 and Rassf5 mutant mice. The generation of Rassf5-deficient cells and mice will further help to elucidate the roles of Rassf5 in other cellular processes. IntroductionA small family of genes termed RASSF (Ras association domain family) has been recently described, the members of which are characterized by the presence of a Ras association (RA) 4The abbreviations used are: RARas associationMEFmouse embryonic fibroblastPARPpoly-ADP-ribose polymeraseCASPcaspaseTNF-R1TNF receptor 1TRAILTNF-related apoptosis-inducing ligandESembryonic stem. domain and a novel motif named the SARAH (Salvador, Rassf, Hippo) domain at the C terminus (reviewed in Refs. 1van der Weyden L. Adams D.J. Biochim. Biophys. Acta. 2007; 1776: 58-85Crossref PubMed Scopus (228) Google Scholar, 2Richter A.M. Pfeifer G.P. Dammann R.H. Biochim. Biophys. Acta. 2009; 1796: 114-128Crossref PubMed Scopus (247) Google Scholar, 3Avruch J. Xavier R. Bardeesy N. Zhang X.F. Praskova M. Zhou D. Xia F. J. Biol. Chem. 2009; 284: 11001-11005Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Among the members of the RASSF family, RASSF1 and RASSF5 (also known as NORE1, for novel Ras effector 1) share the closest homology, displaying 49% identity (66% similarity) at the protein level, and are frequently inactivated by CpG hypermethylation in human cancer cell lines and primary tumors (4Dammann 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, 5Morris M.R. Hesson L.B. Wagner K.J. Morgan N.V. Astuti D. Lees R.D. Cooper W.N. Lee J. Gentle D. Macdonald F. Kishida T. Grundy R. Yao M. Latif F. Maher E.R. Oncogene. 2003; 22: 6794-6801Crossref PubMed Scopus (94) Google Scholar, 6Hesson L. Dallol A. Minna J.D. Maher E.R. Latif F. Oncogene. 2003; 22: 947-954Crossref PubMed Scopus (112) Google Scholar). RASSF1 has been studied extensively (reviewed in Ref. 7Donninger H. Vos M.D. Clark G.J. J. Cell Sci. 2007; 120: 3163-3172Crossref PubMed Scopus (348) Google Scholar) and shown to play important roles in mitosis (8Song M.S. Song S.J. Ayad N.G. Chang J.S. Lee J.H. Hong H.K. Lee H. Choi N. Kim J. Kim H. Kim J.W. Choi E.J. Kirschner M.W. Lim D.S. Nat. Cell Biol. 2004; 6: 129-137Crossref PubMed Scopus (276) Google Scholar), microtubule and genomic stability (9Vos M.D. Martinez A. Elam C. Dallol A. Taylor B.J. Latif F. Clark G.J. Cancer Res. 2004; 64: 4244-4250Crossref PubMed Scopus (125) Google Scholar, 10van der Weyden L. Tachibana K.K. Gonzalez M.A. Adams D.J. Ng B.L. Petty R. Venkitaraman A.R. Arends M.J. Bradley A. Mol. Cell. Biol. 2005; 25: 8356-8367Crossref PubMed Scopus (86) Google Scholar, 11Liu L. Tommasi S. Lee D.H. Dammann R. Pfeifer G.P. Oncogene. 2003; 22: 8125-8136Crossref PubMed Scopus (164) Google Scholar), apoptosis (12Baksh S. Tommasi S. Fenton S. Yu V.C. Martins L.M. Pfeifer G.P. Latif F. Downward J. Neel B.G. Mol. Cell. 2005; 18: 637-650Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 13Matallanas D. Romano D. Yee K. Meissl K. Kucerova L. Piazzolla D. Baccarini M. Vass J.K. Kolch W. O'neill E. Mol. Cell. 2007; 27: 962-975Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar), and cell cycle (14Shivakumar L. Minna J. Sakamaki T. Pestell R. White M.A. Mol. Cell. Biol. 2002; 22: 4309-4318Crossref PubMed Scopus (348) Google Scholar), which are consistent with its function as a tumor suppressor. However, not much is known about the physiological roles of RASSF5.RASSF5 encodes multiple isoforms because of dual promoter usage and alternative splicing (15Tommasi S. Dammann R. Jin S.G. Zhang X.F. Avruch J. Pfeifer G.P. Oncogene. 2002; 21: 2713-2720Crossref PubMed Scopus (94) Google Scholar). The longest isoform, designated RASSF5A (NORE1A), is transcribed from the 5′-most promoter region containing CpG island and encodes a 418-amino acid protein containing the cysteine-rich diacylglycerol/phorbol ester-binding domain (also called protein kinase C conserved region 1, C1), the RA domain, and the C-terminal SARAH domain. Through alternative splicing, another isoform RASSF5B (NORE1Aβ), lacking the SARAH domain is produced. The shortest isoform, RASSF5C (NORE1B, also called RAPL), lacking the N-terminal C1 domain, is produced from a downstream CpG-containing promoter that is less frequently methylated in primary tumors and cancer cell lines than the 5′ upstream promoter (6Hesson L. Dallol A. Minna J.D. Maher E.R. Latif F. Oncogene. 2003; 22: 947-954Crossref PubMed Scopus (112) Google Scholar). Similar to RASSF1A, CpG methylation of RASSF5A has been found in various cancer cell lines and primary tumors of lung, breast, colon, liver, and kidney (6Hesson L. Dallol A. Minna J.D. Maher E.R. Latif F. Oncogene. 2003; 22: 947-954Crossref PubMed Scopus (112) Google Scholar, 16Calvisi D.F. Ladu S. Gorden A. Farina M. Conner E.A. Lee J.S. Factor V.M. Thorgeirsson S.S. Gastroenterology. 2006; 130: 1117-1128Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar), although less frequently than RASSF1A. These findings suggest that RASSF5A likely functions as a tumor suppressor.RASSF5A was first identified as the interacting partner of the active GTP-bound form of RAS or other RAS-like GTPases, such as Rap1, M-Ras, and R-Ras/R-Ras3, through the RA domain (17Vavvas D. Li X. Avruch J. Zhang X.F. J. Biol. Chem. 1998; 273: 5439-5442Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). RASSF5C (NORE1B/RAPL) was also shown to interact with Rap1 following T-cell antigen or chemokine receptor activation, and the RASSF5C-Rap1 complex promotes recruitment of integrin LFA-1 to the T-cell leading edge, enhancing the T-cell affinity for intercellular adhesion molecule (ICAM) (18Katagiri K. Ohnishi N. Kabashima K. Iyoda T. Takeda N. Shinkai Y. Inaba K. Kinashi T. Nat. Immunol. 2004; 5: 1045-1051Crossref PubMed Scopus (161) Google Scholar). Thus, RASSF5C has been identified as a critical molecule for lymphocyte and dendritic cell trafficking. In addition to the RA domain, RASSF5A, through its N-terminal domain, forms a heterodimer with RASSF1A (19Ortiz-Vega S. Khokhlatchev A. Nedwidek M. Zhang X.F. Dammann R. Pfeifer G.P. Avruch J. Oncogene. 2002; 21: 1381-1390Crossref PubMed Scopus (201) Google Scholar), although whether the diacylglycerol/phorbol ester domain of RASSF5A is necessary for this binding is not clear. 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