Effect of merlin phosphorylation on neurofibromatosis 2 (NF2) gene function Ezequiel I Surace 1 , Carrie A Haipek 1 and David H Gutmann* ,1 1 Department of Neurology, Washington University School of Medicine, Box 8111, 660 S Euclid Avenue, St Louis, MO 63110, USA The neurofibromatosis 2 (NF2) tumor suppressor gene product, merlin, belongs to the ezrin–radixin–moesin (ERM) subgroup of the Protein 4.1 family, which links cell surface glycoproteins to the actin cytoskeleton. Previous studies have suggested that phosphorylation of merlin, similar to other ERM proteins, may regulate its function. To determine whether merlin phosphorylation has functional consequences for merlin suppression of cell growth and motility, we generated doxycycline-regulata- ble RT4 schwannoma cell lines that inducibly express full-length merlin with mutations at two potential phosphorylation sites (amino-acid residues S518 and T576). Whereas a mutation at S518 that mimics constitutive phosphorylation (S518D) abrogates the abil- ity of merlin to suppress cell growth and motility, the S518A merlin mutant, which mimics nonphosphorylated merlin, functions equivalently to wild-type merlin. Similar mutations involving T576, the analogous phosphorylation site in ERM proteins important for regulating their function, had no effect. In contrast to other functionally inactive missense merlin mutants, the regulated over- expression of S518D merlin resulted in dramatic changes in cell shape and the elaboration of filopodial extensions. These results provide the first direct demonstration that the S518D merlin mutation, which mimics merlin phosphorylation, impairs not only merlin growth and motility suppression but also leads to an acquisition of a novel phenotype previously ascribed to ERM proteins. Oncogene (2004) 23, 580–587. doi:10.1038/sj.onc.1207142 Keywords: merlin; schwannomin; ERM proteins; FERM; Protein 4.1; phosphorylation Introduction Neurofibromatosis 2 (NF2) is an autosomal dominant inherited cancer predisposition syndrome, in which affected individuals are prone to the development of schwannomas, meningiomas and ependymomas (Baser et al., 2003). The NF2 tumor suppressor gene was identified by positional cloning and found to encode a 595 amino-acid protein with significant sequence simi- larity to a family of proteins that links the actin cytoskeleton to cell surface glycoproteins (Rouleau et al., 1993; Trofatter et al., 1993). This family of proteins, which includes ezrin, radixin and moesin, are termed ERM molecules and share three conserved structural subregions important for their function (Tsukita et al., 1994). The amino terminal FERM domain mediates binding to cell surface glycoproteins, like CD44, while the carboxyl terminal domain binds actin. In addition, ERM proteins form both intramole- cular and intermolecular associations mediated by amino (FERM domain) to carboxyl terminal binding (Gary and Bretscher, 1993; Bretscher et al., 1995; Martin et al., 1995). In the closed conformation where FERM/C-term association occurs, ERM proteins are inactive and do not bind F-actin (Gary and Bretscher, 1995), whereas in the open, active conformation, ERM proteins are capable of inducing long membrane surface extensions (Henry et al., 1995; Martin et al., 1995). Like other ERM proteins, the NF2 gene product, merlin (or schwannomin), can associate with CD44 using FERM domain amino-acid residues (Morrison et al., 2001), but associates with actin by virtue of amino-acid residues in the amino terminus of the protein (Xu and Gutmann, 1998). In contrast to ERM proteins, merlin does not harbor the conventional ERM actin- binding domain in its carboxyl terminus. Merlin also forms both intramolecular and intermolecular FERM/ carboxyl terminal associations (Gonzalez-Agosti et al., 1999; Gutmann et al., 1999). Previous studies have shown that the ability of merlin to function as a negative growth regulator is dependent on its ability to form a productive head-to-tail intramolecular association (Sherman et al., 1997; Gutmann et al., 1998; Morrison et al., 2001). The regulation of ERM intra/intermolecular associa- tions and function is mediated by ERM phosphoryla- tion (Tsukita et al., 1997). ERM localization to the apical membrane and association with CD44 is dictated by phosphorylation mediated by Rho activation (Chen et al., 1995; Hirao et al., 1996; Matsui et al., 1998; Shaw et al., 1998a; Matsui et al., 1999). Rho activation induces the rapid phosphorylation of ERM proteins on a conserved threonine residue (T558, moesin; T564, radixin; T567, ezrin; T576, merlin) to promote the formation of microvillar structures (Oshiro et al., 1998; Shaw et al., 1998a). While phosphorylation of radixin on T564 has no effect on actin binding, it results in the Received 13 June 2003; revised 16 August 2003; accepted 16 August 2003 *Correspondence: DH Gutmann; E-mail: gutmannd@neuro.wustl.edu Oncogene (2004) 23, 580–587 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc