Abstract
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 dominant-negative 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 theNF2 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 Nf2gene 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 growth-inhibitory (10). ERMs can be phosphorylated by Rho kinase, and this phosphorylation can affect intramolecular association and cellular localization. Phosphorylation and/or phospholipids may promote the transition of the proteins to an active form by “opening” intra- and intermolecular associations. These active monomers can then bind to other interacting proteins and the actin cytoskeleton and induce actin-rich membrane projections (5,8, 12, 13). The induction of merlin phosphorylation by activated alleles of the Rho family GTPases has also been examined. Interestingly, although activated Rho did not induce noticeable phosphorylation of merlin, activated forms of Rac and cdc42 did. The site of Rac-induced phosphorylation was determined to be a serine at position 518; mutation of serine 518 results in reduced basal phosphorylation and eliminated Rac-induced phosphorylation (11).
Although Rac and cdc42 are implicated in the regulation of many pathways, they are most associated with regulation of cytoskeleton reorganization and gene expression (for recent reviews see Refs.14-16). In light of the data demonstrating that activated Rac/cdc42 leads to phosphorylation and possible inactivation of merlin, the elucidation of the responsible effector pathways and their effects on merlin function are of major importance. Understanding this regulation of merlin could lead to a more complete appreciation of the effects of merlin loss in tumors.
Publication Date
1-8-2002
Journal Title
Journal of Biological Chemistry
Publisher
American Society for Biochemistry and Molecular Biology
Digital Object Identifier (DOI)
Document Type
Article
Recommended Citation
Kissil JL, Johnson KC, Eckman MS, Jacks T. Merlin phosphorylation by p21-activated kinase 2 and effects of phosphorylation on merlin localization. J Biol Chem. 2002 Mar 22;277(12):10394-9. PubMed PMID: 11782491.
Rights
This research was originally published in Kissil JL, Johnson KC, Eckman MS, Jacks T. Merlin phosphorylation by p21-activated kinase 2 and effects of phosphorylation on merlin localization. J Biol Chem. 2002 Mar 22;277(12):10394-9. © the American Society for Biochemistry and Molecular Biology. https://dx.doi.org/10.1074/jbc.M200083200