RASSF6 Is a Novel Member of the RASSF Family of Tumor Suppressors
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Oncogene (2007) 26, 6203–6211 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE RASSF6 is a novel member of the RASSF family of tumor suppressors NPC Allen1, H Donninger2, MD Vos1, K Eckfeld1, L Hesson3, L Gordon2, MJ Birrer1, F Latif3 and GJ Clark1,2 1Department of Cell and Cancer Biology, National Cancer Institute, Rockville, MD, USA; 2Molecular Targets Group, Department of Medicine, JG Brown Cancer Center, University of Louisville, Louisville, KY, USA and 3Section of Medical and Molecular Genetics, University of Birmingham, Birmingham, UK RASSF family proteins are tumor suppressors that are activated forms of Ras can also exhibit growth frequently downregulated during the development of antagonistic properties. These include the induction of human cancer. The best-characterizedmember of the senescence, cell cycle arrest and apoptosis (Mayo et al., family is RASSF1A, which is downregulated by promoter 1997; Serrano et al., 1997; Nicke et al., 2005). We can methylation in 40–90% of primary human tumors. We reconcile these apparently contradictory properties if we now identify and characterize a novel member of the consider the fact that Ras proteins can interact with a RASSF family, RASSF6. Like the other family members, wide variety of downstream effector proteins allowing a RASSF6 possesses a Ras Association domain and binds broad range of effector outputs (Malumbres and activatedRas. Exogenous expression of RASSF6 pro- Pellicer, 1998). Many Ras effector proteins contain motedapoptosis, synergizedwith activatedK-Ras to conserved structural regions that are responsible for induce cell death and inhibited the survival of specific mediating the interaction with Ras. These regions have tumor cell lines. Suppression of RASSF6 enhancedthe been designated the Ras Association (RA) domain tumorigenic phenotype of a human lung tumor cell line. (Ponting and Benjamin, 1996). Using a bioinformatics- Furthermore, RASSF6 is often downregulated in primary based approach to screen for RA domain containing human tumors. RASSF6 shares some similar overall proteins, we have previously identified several members properties as other RASSF proteins. However, there are of the RASSF family as potential Ras effectors that significant differences in biological activity between mediate growth inhibitory effects. RASSF6 and other family members including a discrete Four RASSF proteins have been characterized biolo- tissue expression profile, cell killing specificity andimpact gically thus far: RASSF1, RASSF2, RASSF4 and Nore1 on signaling pathways. Moreover, RASSF6 may play a (RASSF5) (Dammann et al., 2000; Vos et al., 2000, role in dictating the degree of inflammatory response to 2003a, b; Burbee et al., 2001; Khokhlatchev et al., 2002; the respiratory syncytial virus. Thus, RASSF6 is a novel Eckfeld et al., 2004; Agathanggelou et al., 2005). All RASSF family member that demonstrates the properties demonstrate biological properties compatible with a of a Ras effector andtumor suppressor but exhibits tumor suppressor function. These include inhibiting biological properties that are unique anddistinctfrom growth, promoting cell cycle arrest and apoptosis. They those of other family members. are all frequently downregulated during tumorigenesis by Oncogene (2007) 26, 6203–6211; doi:10.1038/sj.onc.1210440; promoter methylation (Agathanggelou et al., 2005; Akino published online 2 April 2007 et al., 2005). Moreover, RASSF1A knockout mice demon- strate enhanced tumor susceptibility (Tommasi et al., Keywords: Ras; RASSF6; tumor suppressor 2005) and loss of Nore1 (RASSF5) function is linked to a familial form of kidney cancer (Chen et al., 2003). Here, we identify and characterize a novel member of this family, RASSF6. RASSF6 interacts directly with K-Ras in a guanosine triphosphate (GTP)-dependent Introduction manner via its effector domain with an affinity compar- able to that of other known Ras effectors. RASSF6 Mutant Ras proteins play a critical role in the induces apoptosis and the ability of RASSF6 to kill development of over 30% of human cancers (Shields cells is enhanced by activated Ras. Overexpression et al., 2000; Malumbres and Barbacid, 2003). However, of RASSF6 inhibits the survival of specific tumor cell although promoting many aspects of transformation, lines and knockdown of RASSF6 by siRNA enhances the ability of tumor cell lines to grow in soft agar. In matched pair primary tumor samples, the levels Correspondence: Dr GJ Clark, JG Brown Cancer Center, Department of RASSF6 mRNA are often downregulated in the Medicine, Molecular Targets Group, University of Louisville, 580 S primary tumors. However, although RASSF6 has some Preston Street, Louisville, KY 40202, USA. E-mail: [email protected] similar overall properties as RASSF1A, the proteins Received 4 January 2007; revised 22 February 2007; accepted 22 demonstrate discrete expression profiles, and cell killing February 2007; published online 2 April 2007 specificity. RASSF6 is a potential Ras effector/tumor suppressor NPC Allen et al 6204 Recently, the RASSF6 locus has been implicated in RASSF6 and K-Ras, we generated a recombinant determining susceptibility to infection by the respiratory glutathione S-transferase (GST)-fusion of the RA domain syncytial virus (RSV) (Hull et al., 2004). RSV infection of RASSF6, designated GST-F6RA. GST-F6RA was activates the eukaryotic nuclear factor kB (NFkB) used as an affinity reagent in quantitative binding assays pathway and this activation may play a vital role in with purified, farnesylated K-Ras (Figure 3). Ras was the inflammatory response to infection. We have found loaded with GTP and incubated with 100 ng of GST or that RASSF6 expression inhibits the basal levels of GST-F6RA at decreasing concentrations for 4 h at 41Cin NFkB activity in a lung epithelial cell line, suggesting phosphate-buffered saline, 0.025% Tween-20 and 1 mM that defects in RASSF6 may facilitate viral NFkB MgCl2. GST-F6 beads were then washed and subjected to activation. Thus, we identify a further member of the western analysis using an anti-K-Ras antibody (F234 RASSF family as a novel potential Ras effector/tumor Santa Cruz Biotechnology, CA, USA). The apparent Kd suppressor with distinct biological characteristics. was taken as half the maximal binding. Binding was saturated by 300 nM,givinganapparentKd of less than or equal to 150 nM. Results Family alignment and tissue distribution of RASSF6 RASSF6 and RASSF1A exhibit different cell-specific TblastN searches of the EST database using the RA growth inhibitory properties domain of RASSF1A led to the identification of Tumor cell lines were transfected with pBabe expression RASSF6. During the preparation of this manuscript, constructs and selected in puromycin. Colony formation the gene was described in: http://www.genecards.org/ was scored after 2 weeks (Figure 4). Whereas neither cgi-bin/carddisp.pl?gene ¼ RASSF6. Sequences were RASSF1A nor RASSF6 inhibited the growth of the aligned using ClustalW (Figure 1a). RASSF6 shows H1299 human lung tumor cell line, both inhibited the most identity to RASSF2 (57%) and RASSF4 (55%). It growth of MCF-7 human breast tumor cells. However, lacks the N-terminal extension of Nore1 (RASSF5) and RASSF6 was significantly less effective at inhibiting the the microtubule association domain of RASSF1A (Vos growth and survival of A549 human lung tumor cells. et al., 2004; Agathanggelou et al., 2005) but shares the SARAH motif of other family members. Northern analysis of RASSF6 expression (Figure 1b) showed a RASSF6 and K-Ras synergize to induce cell death distinct pattern of expression that differs from other To determine the effects of activated Ras on the family members (Vos et al., 2000, 2003a, b). biological activity of RASSF6, we performed cotrans- fections in 293-T cells. After 48 h, the cells were stained RASSF6 interacts with Ras GTP in cells via its with trypan blue to detect cell death. A dramatic effector domain synergistic activation of cell death was observed when 293-T cells were cotransfected with FLAG-tagged the cells were transfected with activated K-Ras and RASSF6 and HA-tagged activated K-Ras (G12V) or RASSF6 together (Figure 5aand b). wild-type K-Ras. The cells were lysed after 48 h and immunoprecipitated with anti-Ras antibody for western RASSF6 induces apoptotic cell death analysis using anti-FLAG antibodies. RASSF6 pre- To determine if RASSF6 was proapoptotic, we used the ferentially associated with the active form of K-Ras as pCaspase3-Sensor system (Clontech, Mountain View, no association was seen with the inactive (wild-type) CA, USA) and fluorescent microscopy to measure protein (Figure 2a). caspase-3 activation in individual cells as described Ras associates with its effectors via its effector previously (Vos et al., 2003b). Quantification showed domain (Marshall 1993) therefore, effector mutants that addition of RASSF6 induced a fivefold increase of (White et al., 1995; Vos et al., 2003a) of activated active caspases over empty vector (Figure 6). K-Ras (G12V/35S) and (G12V/40C) were subjected to similar coprecipitation experiments with RASSF6. Figure 2b shows that mutations in the effector region of RASSF6 binds the proapoptotic protein MOAP-1 K-Ras strongly impaired its ability to interact with One of the proapoptotic pathways that has been RASSF6. identified for RASSF1A involves the direct interaction Farnesylation is important for Ras to bind to its with the protein modulator of apoptosis 1 (MOAP-1) effectors with