There have been some interesting reports relating to NF2 signaling in the past year. Dr. Vijaya Ramesh (Harvard Medical School/ Massachusetts General Hospital) and Dr. Marco Giovannini (House Ear Institute) chaired a session focused on this area. The session began with a talk by Dr. Anthony Bretscher (Weill Institute of Cell and Molecular Biology, Cornell University) that focused on the biology and structure of ezrin-radixin-moesin (ERM) proteins, actin binding proteins and structural relatives of the NF2 protein Merlin. Dr. Bretscher asked the question what can we learn about Merlin from ERM proteins? He focused on the role of the ERM binding protein, ERM-binding phosphoprotein 50 (EBP50, also known as NHERF1) and its binding protein EPI64, in morphogenesis and in the regulation of membrane trafficking. Based on the X-ray structure, the ~300 amino FERM domain of ERM has three subdomains: F1, F2, and F3. The FERM domain is masked in the dormant form, but when activated, it opens and binds to ligands. Data from biochemical and structural studies suggest that multiples signal transduction pathways contribute to the complete opening of ERMs to activate their membrane and cytoskeleton linking activity. Functionally important intermediate states of ERM activation also appear to exist. A distinct site on the ERM-FERM domain binds to EBP50; and knockdown of EBP50 results in disappearance of microvilli, suggesting that EBP50 is required for microvilli formation. Specifically, the PDZ1 domain, but not the PDZ2 domain, of EBP50 is required for microvilli formation.
EBP50 is a substrate for many kinases, and at least three of its sites are phosphorylated by PKC Dr. Bretscher showed that phosphorylation can regulate the accessibility of ligands to the PDZ1 domain. EPI64, a protein with TBC/RabGAP domains encoding a conserved DTYL at the C-terminal end, binds PDZ1. EPI64 is found at the base of the microvilli. An L-to-A mutant of EPI64 can no longer bind to EBP50, and when this EPI64 mutant is expressed in cells, it results in loss of microvilli. Exogenous expression of EPI64 leads to the formation of actin-covered vacuoles resembling cells expressing Arf6-GTP (Q67L). This observation led to the finding that the EPI64 TBC domain can bind Arf6-GTP, and that overexpression of EPI64 leads to increased Arf6 GTP/GDP ratio. Site-directed mutagenesis studies confirmed that TBC region of EPI64 is a functional Rab-GAP. Among the Rab proteins tested for the Gap activity of EPI64, Rab8 appears to be the key one as EPI64 (R160A) mutant increased Rab8-GTP and EPI64 (WT) decreased Rab8-GTP. Taken together, this data shows that EPI64 regulates a clathrin-independent endocytosis pathway. However, how this relates to the formation of microvilli remains unknown.
Dr. Bretscher also examined the similarities and differences between ERMs and Merlin. While ERMs exist as tightly regulated closed and open forms, the transition between closed and open forms of Merlin is not tightly regulated. ERMs are active at the cell cortex, and the active sites in Merlin are unknown. It may also have a nuclear function. While ERMs regulate membrane traffic through EPI64, and are suggested to be growth promoters, Merlin regulates endocytosis and functions as a tumor suppressor. It remains essential to understand the interaction between ERMs and Merlin [further reading: Fehon et al., 2010
Dr. Andrea McClatchey (Harvard Medical School/Massachusetts General Hospital) reviewed her findings that Merlin localizes to cadherin-containing cell junctions known as adherens junctions (AJs) will associate with the AJ complex, and is necessary for the formation of stable AJs in several cell types [Lallemand et al., 2003
]. Merlin can associate with the epidermal growth factor receptor (EGFR) and prevents its internalization and signaling in a cell contact-dependent manner [Curto et al., 2007
]. These data suggested that Merlin physically coordinates the establishment of cell junctions via inhibition of EGFR signaling, providing insight into how Merlin mediates contact-dependent inhibition of proliferation. Dr. McClatchey’s group has found that the ability of Merlin to block proliferation and EGFR endocytosis is dependent upon the first 17 amino acids of the protein that precede the FERM domain, and direct it to both AJs and to the insoluble, cortical cytoskeleton [Cole et al., 2008
]. Dr. McClatchey’s group has now used these studies as a guide to study and understand how Merlin assembles and how it more generally regulates membrane protein complexes.
Dr. McClatchey’s most recent work has dissected the molecular and biochemical basis of how Merlin communicates with and stabilizes the AJ, and how Merlin controls EGFR endocytosis. Her group recently found that Merlin’s ability to physically link the AJ component α-catenin to the polarity protein Par3 is necessary for organizing cell junctions in the developing skin [Gladden et al., in press
]. This in turn is necessary for normal cell polarity and asymmetric division in basal epidermal cells. These studies suggest that Merlin, like other FERM-domain containing proteins, may play fundamental roles in establishing membrane asymmetry; in fact, Dr. McClatchey’s group has recently found that Merlin can organize the membrane of single cells.
Dr. Helen Morrison (Leibniz Institute for Aging) has previously shown that ERM proteins can act as counterplayers in Ras activation. While Merlin is inhibitory for Ras, ERM proteins appear to enhance Ras activity. In their Ras-controlling state, these proteins are specifically targeted to their relevant sites of activity via interaction with plasma membrane proteins. Dr. Morrison’s recent data demonstrates that from these plasma membrane docking sites, ERM proteins serve as essential components in the conformational regulation and activation of Son of sevenless (SOS), a major Ras guanine nucleotide exchange factor (GEF). Merlin cannot bind and regulate SOS but can antagonize this newly identified ezrin–SOS complex relevant for Ras activation. While Merlin antagonizes this ezrin function, this research has revealed an additional active role of Merlin in regulating Ras activity via GAPs. Merlin can complex and regulate p120RasGAP, an important GAPs for the downregulation of Ras activity. The functional relevance of these findings is currently being dissected in vitro and in vivo. The outcome of these experiments will address a novel role of Merlin in the regulation of p120RasGAP function during contact inhibition of growth.
Dr. Filippo Giancotti (Memorial Sloan-Kettering Cancer Center) provided new insights into Merlin’s tumor suppressor function through its nuclear localization. He presented data demonstrating that Merlin specifically interacts with the E3 ubiquitin ligase CRL4DCAF1
in the nucleus and inhibits its activity. The closed form of Merlin accumulates in the nucleus and interacts with CRL4DCAF1
, while the open form of Merlin is predominantly present in the cytoplasm and does not interact with CRL4DCAF1
. The FERM domain of Merlin binds to the C-terminal segment of CRL4DCAF1
. Expression of wild-type Merlin inhibits CRL4DCAF1
; however, expression of patient-derived mutations does not inhibit CRL4DCAF1
, strongly suggesting that Merlin is a negative regulator of DCAF1. Dr. Giancotti showed that there are three classes of NF2 mutants: (1) mutants that fail to localize to the nucleus; (2) mutants that do not interact with DCAF1; and (3) mutants that can go to the nucleus and can bind DCAF1, but fail to suppress the Ub ligase activity. DCAF1 is required for hyperproliferation of Merlin-deficient mesothelioma cells and shRNA-mediated silencing of DCAF1 in primary human schwannoma cells derived from NF2 patients suppressed the ability of these cells to progress through G1 and enter into S phase in response to mitogen. Silencing of DCAF1 decreased tumorigenicity in a xenograft model. Although the physiological substrates of DCAF1 have not been identified Dr. Giancotti proposed that Merlin functions as a tumor suppressor by controlling a wide gene expression program through inhibition of CRL4DCAF1
. However, how the inhibition of CRL4DCAF1
E3 ubiquitin ligase by Merlin relates to contact inhibition, RTK signaling and Hippo signaling where Merlin has been implicated is yet to be determined [further reading: Li et al., 2010
NF2 signaling updates also featured heavily in a number of the selected abstracts presented, including one from Wei Li (Memorial-Sloan Kettering Cancer Center) from Dr. Giancotti’s group. Dr. Li described new studies to unravel further how Merlin is transported into the nucleus which has included identification of a four amino acid peptide in Merlin which appears to be essential for the protein to accumulate in the nucleus. Dr. Li also described a novel approach that allows induction of Merlin accumulation in nucleus or cytoplasm which should be useful for further elucidation of Merlin transport and function in the cell.
Meningiomas and schwannomas are the two principal types of tumor that occur in NF2 and they are, for the large part non-cancerous. Dr. Marianne James (Harvard Medical School/Massachusetts General Hospital) is focused on understanding the mechanisms that do confer these tumor types with malignant potential. Dr. James showed that the signaling mechanisms involved in this include the aberrant activation of mammalian target of rapamycin complex 1 (mTORC1) plus impaired mTORC2 signaling. Furthermore, mTORC1 and mTORC2 appear to have distinct downstream ‘molecular signatures’ in arachnoid cells (those from which meningiomas will arise) and Schwann cells (those from which schwannomas will arise). These findings may help inform the future development of effective treatments for the two tumor types.
Dr. Li Guo (Cincinnati Children’s Hospital Medical Center) from Dr. Nancy Ratner’s group described new NF2 mouse models in which Rac1 is inactivated in Schwann cells either alone, or in combination with deletion of the NF2 gene. Rac1 activity seems to be necessary for Schwann cell myelin formation, partially through inhibition of Merlin function. Rac1 may represent a new candidate drug target for NF2 therapies.
Close to half of all Merlin protein in the cell resides in membrane rafts, and Timmy Mani (University of Cincinnati), a doctoral student in Dr. Wallace Ip’s group, has been investigating how Merlin attaches to these rafts. Mr. Mani showed that Merlin binds phosphoinositides including PIP2, via a conserved binding motif in its FERM domain. Mutating this domain in Merlin blocks FERM domain mediated PIP2 binding and association with membrane rafts showing that FERM domain mediated phosphoinositide binding is required for Merlin raft association. Mr. Mani also showed that this mutated Merlin becomes cytosolic; is much more mobile than membrane-bound Merlin and loses its growth suppressive functions; and fails to repress cyclin D1 expression as compared to wild-type Merlin suggesting a loss of ability to inhibit cell cycle progression. In summary, Mr. Mani’s research shows that FERM domain mediated phosphoinositide binding and raft localization are critical for the growth regulatory function of Merlin.
Dr. Helen McNeill (Mount Sinai Hospital, Canada) works on Drosophila analogs of the Merlin pathway—specifically, the Hippo pathway and the large cadherin Fat, a cell surface receptor that controls growth in parallel to Merlin. The Hippo (Hpo) signaling pathway regulates organ size in both Drosophila and mammals. While a core kinase cascade leading from the protein kinase Hpo (Mst1 and Mst2 in mammals) to the transcription coactivator Yorkie (Yki) (YAP in mammals) has been established, upstream regulators of the Hippo kinase cascade are less well defined, especially in mammals. Dr. McNeil described how Fat controls growth with the FERM protein Expanded, and the kinase Discs overgrown/Casein Kinase II. Full activation of the Hippo pathway requires the recruitment of Casein Kinase II to a Fat signaling complex. Loss of Fat or Casein Kinase II lead to unrestrained tissue growth, and this overgrowth was significantly increased when Merlin/NF2 was mutated, indicating synergy between these pathways.
While previous studies in Drosophila have implicated Merlin/ NF2 as an upstream regulator of Hippo signaling, it remains to be established whether Merlin/NF2 regulates Hippo signaling in the context of normal mammalian physiology. Using conditional knockout mice, Dr. Duoja Pan (Johns Hopkins University) showed that the Merlin/Nf2 tumor suppressor and the YAP oncoprotein function antagonistically to regulate liver development. While inactivation of Yap led to loss of hepatocytes and biliary epithelial cells, inactivation of Nf2 led to hepatocellular carcinoma and bile duct hamartoma. Strikingly, the Nf2-deficient phenotypes in multiple tissues were largely suppressed by heterozygous deletion of Yap, suggesting that YAP is a major effector of Merlin/NF2 in growth regulation. Dr. Pan’s studies link Merlin/NF2 to mammalian Hippo signaling and implicate YAP activation as a mediator of pathologies relevant to the manifestation of clinical NF2.