DAL-1/4.1Btumorsuppressorinteractswithproteinarginine N-methyltransferase3(PRMT3)andinhibitsitsabilitytomethylate substrates in vitro and in vivo Vinita Singh 1,5 , Tina Branscombe Miranda 2,5 , Wei Jiang 1 , Adam Frankel 2 , Martha E Roemer 1 , Victoria A Robb 3 , David H Gutmann 3 , Harvey R Herschman 4 , Steven Clarke 2 and Irene F Newsham* ,1 1 Department of Neurosurgery, David and Doreen Hermelin Laboratory of Molecular Oncogenetics, Hermelin Brain Tumor Center, Henry Ford Hospital, Detroit, MI 48202, USA; 2 Molecular Biology Institute and Department of Chemistry and Biochemistry, UCLA, CA 90095, USA; 3 Department of Neurology, Washington University School of Medicine, St Louis, MO 63110, USA; 4 Departments of Biological Chemistry and Molecular and Medical Pharmacology, UCLA, CA 90095, USA DAL-1( differentiallyexpressedin adenocarcinomaofthe lung)/4.1B is a tumor suppressor gene on human chromosome 18p11.3 whose expression is lost in 450% of primary non-small-cell lung carcinomas. Based on sequencesimilarity,DAL-1/4.1Bhasbeenassignedtothe Protein 4.1 superfamily whose members interact with plasma membrane proteins through their N-terminal FERM (4.1/Ezrin/Radixin/Moesin) domain, and cytos- keletal components via their C-terminal SAB (spectrin– actin binding) region. Using the DAL-1/4.1B FERM domain as bait for yeast two-hybrid interaction cloning, we identified protein arginine N-methyltransferase 3 (PRMT3) as a specific DAL-1/4.1B-interacting protein. PRMT3 catalyses the post-translational transfer of methyl groups from S-adenosyl-L-methionine to arginine residues of proteins. Coimmunoprecipitation experiments using lung and breast cancer cell lines confirmed this interaction in mammalian cells in vivo. In vitro binding assays demonstrated that this was an interaction occur- ringviatheC-terminalcatalyticcoredomainofPRMT3. DAL-1/4.1B was determined not to be a substrate for PRMT3-mediated methylation but its presence inhibits the in vitro methylationofaglycine-richandarginine-rich methyl-accepting protein, GST (glutathione-S-transfer- ase-GAR (glycine- and arginine-rich), which contains 14 ‘RGG’ consensus methylation sites. In addition, induced expressionofDAL-1/4.1BinMCF-7breastcancercells showedthattheDAL-1/4.1Bproteinsignificantlyinhibits PRMT3methylationofcellularsubstrates.Thesefindings suggestthatmodulationofpost-translationalmethylation may be an important mechanism through which DAL-1/ 4.1Baffectstumorcellgrowth. Oncogene (2004) 23, 7761–7771. doi:10.1038/sj.onc.1208057 Published online 30 August 2004 Keywords: PRMT3; DAL-1/4.1B; tumor suppressor; protein methylation Introduction The human tumor suppressor gene DAL-1/4.1B ( differ- entially expressed in adenocarcinoma of the lung) was identified using Differential Display PCR (DDPCR) as a gene whose expression was lacking in primary non- small-cell lung cancer (NSCLC) when compared with matched normal tissue (Tran et al., 1999). This gene product was determined to be a new member of the Protein 4.1 superfamily by virtue of the presence of a 336 amino-acid N-terminal region sharing significant identity to the FERM (4.1/Ezrin/Radixin/Moesin) domain present in all 4.1 family proteins (Chishti et al., 1998). Members of this family, which include the neurofibromatosis 2 tumor suppressor protein merlin or schwannomin (Rouleau et al., 1993; Trofatter et al., 1993), are proteins that localize to the cytoplasmic side of the plasma membrane and link membrane proteins with the spectrin/actin cytoskeleton (Tsukita et al., 1994). Like merlin and now 4.1R, which have been shown to suppress growth in schwannoma and menin- gioma cell lines (Gutmann et al., 2000; Robb et al., 2004), DAL-1/4.1B negatively regulates cell growth in several different cell types. For example, when intro- duced into DAL-1/4.1B-null lung and breast cancer cells, this new Protein 4.1 family member significantly suppresses growth (Tran et al., 1999; Charboneau et al., 2002). Similar results are obtained when DAL-1/4.1B is overexpressed in meningioma cell lines (Gutmann et al., 2000, 2001) and the glioma cell line U87 (Newsham, data not shown). DAL-1/4.1B maps to a region on human chromosome 18p11.3 that undergoes frequent loss of heterozygosity (LOH) in a significant proportion of lung, breast and brain tumors (Tran et al., 1998). Extensive analysis on Received 29 December 2003; revised 7 July 2004; accepted 19 July 2004; published online 30 August 2004 *Correspondence: IF Newsham, Department of Neurosurgery and Hermelin Brain Tumor Center, Henry Ford Hospital, E&R Bldg., Room 3096, 2799 W Grand Boulevard, Detroit, MI 48202, USA; E-mail: irene@bogler.net 5 These two authors contributed equally to this work Oncogene (2004) 23, 7761–7771 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc