The Nf2 tumor suppressor gene codes for merlin, a protein whose function has been elusive. We describe a novel interaction between merlin and p21-activated kinase 1 (Pak1), which is dynamic and facilitated upon increased cellular confluence. Merlin inhibits the activation of Pak1, as the loss of merlin expression results in the inappropriate activation of Pak1 under conditions associated with low basal activity. Conversely, the overexpression of merlin in cells that display a high basal activity of Pak1 resulted in the inhibition of Pak1 activation. This inhibitory function of merlin is mediated through its binding to the Pak1 PBD and by inhibiting Pak1 recruitment to focal adhesions. This link provides a possible mechanism for the effect of loss of merlin expression in tumorigenesis.

The Nf2 tumor suppressor gene product merlin is related to the membrane-cytoskeleton linker proteins of the band 4.1 superfamily, including ezrin, radixin, and moesin (ERMs). Merlin is regulated by phosphorylation in a Rac/cdc42-dependent fashion. We report that the phosphorylation of merlin at serine 518 is induced by the p21-activated kinase PAK2. This is demonstrated by biochemical fractionation, use of active and dominantnegative mutants of PAK2, and immunodepletion. By using wild-type and mutated forms of merlin and phospho-directed antibodies, we show that phosphorylation of merlin at serine 518 leads to dramatic protein relocalization. Neurofibromatosis type 2 (NF2)1 is an inherited disorder characterized by the development of Schwann cell tumors of the eighth cranial nerve. Mutations and loss of heterozygosity of the NF2 gene have been detected in NF2 patients and in various sporadic tumors, including schwannomas, meningiomas, and ependymomas (1). In further support of a role for NF2 in tumor suppression, mice heterozygous for an Nf2 mutation are predisposed to a wide variety of tumors with high metastatic potential (2). In a separate model in which Nf2 is inactivated specifically in Schwann cells, mice develop schwannomas and Schwann cell hyperplasia (3).The longest and predominant splice form of the Nf2 gene codes for a 595-amino acid protein highly similar to the band 4.1 family of proteins. It is most closely related to the ERM proteins, moesin, ezrin, and radixin. The ERM proteins are thought to function as cell membrane-cytoskeleton linkers and are localized to cortical actin structures near the plasma membrane such as microvilli, membrane ruffles, and lamellipodia (4, 5). Likewise, merlin is localized to cortical actin structures, in patterns that partially overlap with the ERMs (1). It has been proposed that intramolecular binding of the N-terminal and C-terminal domains conformationally regulates the ERM proteins by masking binding sites for interacting proteins. The ERMs can also form homodimers and heterodimers, among themselves and with merlin, adding an additional level of complexity to the regulation of these proteins (6). The recently solved crystal structure of the moesin N/C-terminal complex strengthens this model of conformational regulation (7). Given the sequence and, most likely, structural similarities of merlin to the ERM proteins, it is possible that merlin itself could be regulated in a similar fashion.Recent studies (8, 9) have implicated additional factors in the regulation of the ERMs, including phospholipids and phosphorylation. Previous work from our group and others (10, 11) has shown that merlin is differentially phosphorylated as well and that merlin protein levels are affected by growth conditions such as cell confluency, loss of adhesion, or serum deprivation. Merlin is found in an hypophosphorylated form when the combination of cellular and environmental conditions are growthinhibitory (10). ERMs can be phosphorylated by Rho kinase, and this phosphorylation can affect ...

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