The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin's growth-inhibitory activity depends on specific interaction with the cytoplasmic tail of CD44, a transmembrane HA receptor. At low cell density, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data indicate that merlin and CD44 form a molecular switch that specifies cell growth arrest or proliferation.

In several types of cells, the activation of the receptor tyrosine kinase c-Met by its ligand hepatocyte growth factor (HGF) requires the coreceptor CD44v6. The CD44 extracellular domain is necessary for c-Met autophosphorylation, whereas the intracellular domain is required for signal transduction. We have already shown that the CD44 cytoplasmic tail recruits ezrin, radixin and moesin (ERM) proteins to the complex of CD44v6, c-Met, and HGF. We have now defined the function of the ERM proteins and the step they promote in the signaling cascade. The association of ERM proteins to the coreceptor is absolutely required to mediate the HGF-dependent activation of Ras by the guanine nucleotide exchange factor Sos. The ERM proteins need, in addition, to be linked to the actin cytoskeleton to catalyze the activation of Ras. Thus, we describe here a new function of the cytoskeleton. It is part of a "signalosome" complex that organizes the activation of Ras by Sos. So far the cytoskeleton has mainly been identified as a "responder" to signal transduction. Here, we show now that F-actin acts as an "inducer" that actively organizes the signaling cascade.

The small G-protein Ras is a tightly controlled regulator of cell fate. Prolonged or persistent arrest in the activated GTP-loaded state by mutation of Ras as in lung cancer or in a Ras-GTPase-activating protein as in neurofibromatosis type 1 promotes tumorigenesis. We now show that the tumorsuppressor protein merlin (mutated in neurofibromatosis type 2) also controls Ras activity. Systematic analysis of growth factor signaling located the step of merlin interference to the activation of Ras and Rac. Merlin independently uncouples both Ras and Rac from growth factor signals. In the case of Ras, merlin acts downstream of the receptor tyrosine kinase-growth factor receptor binding protein 2 (Grb2)-SOS complex. However, merlin does not bind either SOS or Ras, but it counteracts the ERM (ezrin, radixin, moesin)-dependent activation of Ras, which correlates with the formation of a complex comprising ERM proteins, Grb2, SOS, Ras, and filamentous actin. Because efficient signaling from Ras requires Rac-p21-activated kinase-dependent phosphorylations of Raf and mitogen-activated protein/extracellular signal-regulated kinase kinase, merlin can also inhibit signal transfer from dominantly active Ras mutants. We propose that the interference of merlin with Ras-and Racdependent signal transfer represents part of the tumorsuppressive action of merlin. [Cancer Res 2007;67(2):520-7]

The tumour suppressor protein merlin (encoded by the neurofibromatosis type 2 gene NF2) is an important regulator of proliferation in many cell and tissue types [1][2][3][4] . Merlin is activated by dephosphorylation at serine 518 (S518), which occurs on serum withdrawal or on cell-cell or cell-matrix contact 5,6 . However, the relevant phosphatase that activates merlin's tumour suppressor function is unknown. Here we identify this enzyme as the myosin phosphatase (MYPT-1-PP1d). The cellular MYPT-1-PP1d-specific inhibitor CPI-17 causes a loss of merlin function characterized by merlin phosphorylation, Ras activation and transformation. Constitutively active merlin (S518A) reverses CPI-17-induced transformation, showing that merlin is the decisive substrate of MYPT-1-PP1d in tumour suppression. In addition we show that CPI-17 levels are raised in several human tumour cell lines and that the downregulation of CPI-17 induces merlin dephosphorylation, inhibits Ras activation and abolishes the transformed phenotype. MYPT-1-PP1d and its substrate merlin are part of a previously undescribed tumour suppressor cascade that can be hindered in two ways, by mutation of the NF2 gene and by upregulation of the oncoprotein CPI-17.We tested whether carboxy-terminal merlin (amino-acid residues 299-595; C-merlin) carrying the decisive phosphorylation site S518 (ref. 6) could be dephosphorylated and could therefore be used as a substrate in vitro. C-merlin expressed in rat schwannoma RT4 cells under a doxycycline-inducible promoter (RT4tet-C-merlin) 5 migrated as a distinct doublet. The species with decreased mobility was phosphorylated and converted by treatment with calf intestinal phosphatase to a faster-migrating dephosphorylated band (Fig. 1a) with the same mobility as the non-phosphorylatable S518A mutant protein (in which S518 has been replaced with alanine, in contrast with the pseudo-phosphorylated S518D mutant protein containing an aspartic residue; Fig. 1d). The dephosphorylation of C-merlin was regulated by the same cues as that of full-length merlin 5 , for example, high cell density (Fig. 1b), the addition of high-molecular-mass hyaluronan (HA) 5 or serum withdrawal (as shown in Fig. 1c by the change in the ratio of the upper to the lower band). Because dephosphorylation took less than 5 min after hyaluronan addition or serum withdrawal, we assumed that the activation of a phosphatase was involved rather than a change in stability of the phosphorylated band.To search for the phosphatase that activates merlin's tumour suppressor function, we tested several phosphatase inhibitors for their effects on merlin dephosphorylation. Merlin dephosphorylation induced by hyaluronan addition (not shown) or serum withdrawal was inhibited by high but not low concentration of okadaic acid (C-merlin in Fig. 1e; full-length merlin in Fig. 1f; merlin phosphorylation status was detected by a phospho-S518 antibody because full-length merlin resolves less well into two bands). Okadaic acid inhibits the protein serine/threonine phosphatase...