The neurofibromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation

Neurofibromatosis-2 is an inherited disorder characterized by the development of benign schwannomas and other Schwann-cell-derived tumors associated with the central nervous system. The Neurofibromatosis-2 tumor suppressor gene encodes Merlin, a member of the Protein 4.1 superfamily most closely related to Ezrin, Radixin and Moesin. This discovery suggested a novel function for Protein 4.1 family members in the regulation of cell proliferation; proteins in this family were previously thought to function primarily to link transmembrane proteins to underlying cortical actin. To understand the basic cellular functions of Merlin, we are investigating a Drosophila Neurofibromatosis-2 homologue, Merlin. Loss of Merlin function in Drosophila results in hyperplasia of the affected tissue without significant disruptions in differentiation. Similar phenotypes have been observed for mutations in another Protein 4.1 superfamily member in Drosophila, expanded. Because of the phenotypic and structural similarities between Merlin and expanded, we asked whether Merlin and Expanded function together to regulate cell proliferation. In this study, we demonstrate that recessive loss of function of either Merlin or expanded can dominantly enhance the phenotypes associated with mutations in the other. Consistent with this genetic interaction, we determined that Merlin and Expanded colocalize in Drosophila tissues and cells, and physically interact through a conserved N-terminal region of Expanded, characteristic of the Protein 4.1 family, and the C-terminal domain of Merlin. Loss of function of both Merlin and expanded in clones revealed that these proteins function to regulate differentiation in addition to proliferation in Drosophila. Further genetic analyses suggest a role for Merlin and Expanded specifically in Decapentaplegic-mediated differentiation events. These results indicate that Merlin and Expanded function together to regulate proliferation and differentiation, and have implications for understanding the functions of other Protein 4.1 superfamily members.     

Shedding light on Merlin's wizardry

Inactivation of the tumor suppressor Merlin, encoded by the NF2 (Neurofibromatosis type 2) gene, contributes to malignant conversion in many cell types. Merlin is an Ezrin-Radixin-Moesin protein and localizes underneath the plasma membrane at cell-cell junctions and other actin-rich sites. Recent studies indicate that Merlin mediates contact inhibition of proliferation by blocking recruitment of Rac to the plasma membrane. In mitogen-stimulated cells, p21-activated kinase phosphorylates Ser518 in the C-terminus of Merlin, inactivating the growth suppressive function of the protein. Furthermore, the myosin phosphatase MYPT1-PP1delta, has been identified as a direct activator of Merlin and its inhibition has been linked to malignant transformation. Finally, studies in the fruit fly Drosophila melanogaster have revealed that Merlin functions together with the band 4.1 protein Expanded to promote [corrected] the endocytosis of many signaling receptors, limiting [corrected] their accumulation at the plasma membrane, and to activate [corrected] the Hippo signaling pathway. Here, we review these recent findings and their relevance to the tumor suppressor function of Merlin.     

The tumor-suppressor gene fat controls tissue growth upstream of expanded in the hippo signaling pathway

BACKGROUND: The tight control of cell proliferation and cell death is essential to normal tissue development, and the loss of this control is a hallmark of cancers. Cell growth and cell death are coordinately regulated during development by the Hippo signaling pathway. The Hippo pathway consists of the Ste20 family kinase Hippo, the WW adaptor protein Salvador, and the NDR kinase Warts. Loss of Hippo signaling in Drosophila leads to enhanced cell proliferation and decreased apoptosis, resulting in massive tissue overgrowth through increased expression of targets such as Cyclin E and Diap1. The cytoskeletal proteins Merlin and Expanded colocalize at apical junctions and function redundantly upstream of Hippo. It is not clear how they regulate growth or how they are localized to apical junctions. RESULTS: We find that another Drosophila tumor-suppressor gene, the atypical cadherin fat, regulates both cell proliferation and cell death in developing imaginal discs. Loss of fat leads to increased Cyclin E and Diap1 expression, phenocopying loss of Hippo signaling. Ft can regulate Hippo phosphorylation, a measure of its activation, in tissue culture. Importantly, fat is needed for normal localization of Expanded at apical junctions in vivo. Genetic-epistasis experiments place fat with expanded in the Hippo pathway. CONCLUSIONS: Together, these data suggest that Fat functions as a cell-surface receptor for the Expanded branch of the conserved Hippo growth control pathway.     

A systematic screen for dominant second-site modifiers of Merlin/NF2 phenotypes reveals an interaction with blistered/DSRF and scribbler

Merlin, the Drosophila homologue of the human tumor suppressor gene Neurofibromatosis 2 (NF2), is required for the regulation of cell proliferation and differentiation. To better understand the cellular functions of the NF2 gene product, Merlin, recent work has concentrated on identifying proteins with which it interacts either physically or functionally. In this article, we describe genetic screens designed to isolate second-site modifiers of Merlin phenotypes from which we have identified five multiallelic complementation groups that modify both loss-of-function and dominant-negative Merlin phenotypes. Three of these groups, Group IIa/scribbler (also known as brakeless), Group IIc/blistered, and Group IId/net, are known genes, while two appear to be novel. In addition, two genes, Group IIa/scribbler and Group IIc/blistered, alter Merlin subcellular localization in epithelial and neuronal tissues, suggesting that they regulate Merlin trafficking or function. Furthermore, we show that mutations in scribbler and blistered display second-site noncomplementation with one another. These results suggest that Merlin, blistered, and scribbler function together in a common pathway to regulate Drosophila wing epithelial development.     

The mob as tumor suppressor gene is essential for early development and regulates tissue growth in Drosophila

Studies in Drosophila have defined a new growth inhibitory pathway mediated by Fat (Ft), Merlin (Mer), Expanded (Ex), Hippo (Hpo), Salvador (Sav)/Shar-pei, Warts (Wts)/Large tumor suppressor (Lats), and Mob as tumor suppressor (Mats), which are all evolutionarily conserved in vertebrate animals. We previously found that the Mob family protein Mats functions as a coactivator of Wts kinase. Here we show that mats is essential for early development and is required for proper chromosomal segregation in developing embryos. Mats is expressed at low levels ubiquitously, which is consistent with the role of Mats as a general growth regulator. Like mammalian Mats, Drosophila Mats colocalizes with Wts/Lats kinase and cyclin E proteins at the centrosome. This raises the possibility that Mats may function together with Wts/Lats to regulate cyclin E activity in the centrosome for mitotic control. While Hpo/Wts signaling has been implicated in the control of cyclin E and diap1 expression, we found that it also modulates the expression of cyclin A and cyclin B. Although mats depletion leads to aberrant mitoses, this does not seem to be due to compromised mitotic spindle checkpoint function.     

The Drosophila tumor suppressors Expanded and Merlin differentially regulate cell cycle exit, apoptosis, and Wingless signaling

Mutations that inactivate either merlin (mer) or expanded (ex) result in increased cell growth and proliferation in Drosophila. Both Mer and Ex are members of the Band 4.1 protein superfamily, and, based on analyses of mer ex double mutants, they are proposed to function together in at least a partially redundant manner upstream of the Hippo (Hpo) and Warts (Wts) proteins to regulate cell growth and division. By individually analyzing ex and mer mutant phenotypes, we have found important qualitative and quantitative differences in the ways Mer and Ex function to regulate cell proliferation and cell survival. Though both mer and ex restrict cell and tissue growth, ex clones exhibit delayed cell cycle exit in the developing eye, while mer clones do not. Conversely, loss of mer substantially compromises normal developmental apoptosis in the pupal retina, while loss of ex has only mild effects. Finally, ex has a role in regulating Wingless protein levels in the eye that is not obviously shared by either mer or hpo. Taken together, our data suggest that Mer and Ex differentially regulate multiple downstream pathways.     

The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis

Merlin, the protein product of the Neurofibromatosis type-2 gene, acts as a tumour suppressor in mice and humans. Merlin is an adaptor protein with a FERM domain and it is thought to transduce a growth-regulatory signal. However, the pathway through which Merlin acts as a tumour suppressor is poorly understood. Merlin, and its function as a negative regulator of growth, is conserved in Drosophila, where it functions with Expanded, a related FERM domain protein. Here, we show that Drosophila Merlin and Expanded are components of the Hippo signalling pathway, an emerging tumour-suppressor pathway. We find that Merlin and Expanded, similar to other components of the Hippo pathway, are required for proliferation arrest and apoptosis in developing imaginal discs. Our genetic and biochemical data place Merlin and Expanded upstream of Hippo and identify a pathway through which they act as tumour-suppressor genes.     

Molecular insights into NF2/Merlin tumor suppressor function

The FERM domain protein Merlin, encoded by the NF2 tumor suppressor gene, regulates cell proliferation in response to adhesive signaling. The growth inhibitory function of Merlin is induced by intercellular adhesion and inactivated by joint integrin/receptor tyrosine kinase signaling. Merlin contributes to the formation of cell junctions in polarized tissues, activates anti-mitogenic signaling at tight-junctions, and inhibits oncogenic gene expression. Thus, inactivation of Merlin causes uncontrolled mitogenic signaling and tumorigenesis. Merlin’s predominant tumor suppressive functions are attributable to its control of oncogenic gene expression through regulation of Hippo signaling. Notably, Merlin translocates to the nucleus where it directly inhibits the CRL4^DCAF1 E3 ubiquitin ligase, thereby suppressing inhibition of the Lats kinases. A dichotomy in NF2 function has emerged whereby Merlin acts at the cell cortex to organize cell junctions and propagate anti-mitogenic signaling, whereas it inhibits oncogenic gene expression through the inhibition of CRL4^DCAF1 and activation of Hippo signaling. The biochemical events underlying Merlin’s normal function and tumor suppressive activity will be discussed in this Review, with emphasis on recent discoveries that have greatly influenced our understanding of Merlin biology.     

The protein 4.1, ezrin, radixin, moesin (FERM) domain of Drosophila Coracle, a cytoplasmic component of the septate junction, provides functions essential for embryonic development and imaginal cell proliferation

Coracle is a member of the Protein 4.1 superfamily of proteins, whose members include Protein 4.1, the Neurofibromatosis 2 tumor suppressor Merlin, Expanded, the ERM proteins, protein tyrosine phosphatases, and unconventional myosins. Recent evidence suggests that members of this family participate in cell signaling events, including those that regulate cell proliferation and the cytoskeleton. Previously, we demonstrated that Coracle protein is localized to the septate junction in epithelial cells and is required for septate junction integrity. Loss of coracle function leads to defects in embryonic development, including failure in dorsal closure, and to proliferation defects. In addition, we determined that the N-terminal 383 amino acids define an essential functional domain possessing membrane-organizing properties. Here we investigate the full range of functions provided by this highly conserved domain and find that it is sufficient to rescue all embryonic defects associated with loss of coracle function. In addition, this domain is sufficient to rescue the reduced cell proliferation defect in imaginal discs, although it is incapable of rescuing null mutants to the adult stage. This result suggests the presence of a second functional domain within Coracle, a notion supported by molecular characterization of a series of coracle alleles.     

Phosphorylation and activity of the tumor suppressor Merlin and the ERM protein Moesin are coordinately regulated by the Slik kinase

Merlin and Moesin are closely related members of the 4.1 Ezrin/Radixin/Moesin domain superfamily implicated in regulating proliferation and epithelial integrity, respectively. The activity of both proteins is regulated by head to tail folding that is controlled, in part, by phosphorylation. Few upstream regulators of these phosphorylation events are known. In this study, we demonstrate that in Drosophila melanogaster, Slik, a Ste20 kinase, controls subcellular localization and phosphorylation of Merlin, resulting in the coordinate but opposite regulation of Merlin and Moesin. These results suggest the existence of a novel mechanism for coordinate regulation of cell proliferation and epithelial integrity in developing tissues.     

Contact-dependent inhibition of EGFR signaling by Nf2/Merlin

The neurofibromatosis type 2 (NF2) tumor suppressor, Merlin, is a membrane/cytoskeleton-associated protein that mediates contact-dependent inhibition of proliferation. Here we show that upon cell-cell contact Merlin coordinates the processes of adherens junction stabilization and negative regulation of epidermal growth factor receptor (EGFR) signaling by restraining the EGFR into a membrane compartment from which it can neither signal nor be internalized. In confluent Nf2(-/-) cells, EGFR activation persists, driving continued proliferation that is halted by specific EGFR inhibitors. These studies define a new mechanism of tumor suppression, provide mechanistic insight into the poorly understood phenomenon of contact-dependent inhibition of proliferation, and suggest a therapeutic strategy for NF2-mutant tumors.     

Tao-1 phosphorylates Hippo/MST kinases to regulate the Hippo-Salvador-Warts tumor suppressor pathway

Recent studies have shown that the Hippo-Salvador-Warts (HSW) pathway restrains tissue growth by phosphorylating and inactivating the oncoprotein Yorkie. How growth-suppressive signals are transduced upstream of Hippo remains unclear. We show that the Sterile 20 family kinase, Tao-1, directly phosphorylates T195 in the Hippo activation loop and that, like other HSW pathway genes, Tao-1 functions to restrict cell proliferation in developing imaginal epithelia. This relationship appears to be evolutionarily conserved, because mammalian Tao-1 similarly affects MST kinases. In S2 cells, Tao-1 mediates the effects of the upstream HSW components Merlin and Expanded, consistent with the idea that Tao-1 functions in tissues to regulate Hippo phosphorylation. These results demonstrate that one family of Ste20 kinases can activate another and identify Tao-1 as a component of the regulatory network controlling HSW pathway signaling, and therefore tissue growth, during development.     

Mob as tumor suppressor is activated at the cell membrane to control tissue growth and organ size in Drosophila

Growth inhibition mediated by Hippo (Hpo) signaling is essential for tissue growth and organ size control in Drosophila. However, the cellular mechanism by which the core components like Mob as tumor suppressor (Mats) and Warts (Wts) protein kinase are activated is poorly understood. In this work, we found that the endogenous Mats is located at the plasma membrane in developing tissues. Membrane targeting constitutively activates Mats to promote apoptosis and reduce cell proliferation, which leads to reduced tissue growth and organ size. Moreover, the ability of membrane-targeted Mats to inhibit tissue growth required the wts gene activity and Wts kinase activity was increased by the activated Mats in developing tissues. Consistent with the idea that Mats is a key component of the Hpo pathway, Mats is required and sufficient to regulate Yki nuclear localization. These results support a model in which the plasma membrane is an important site of action for Mats tumor suppressor to control tissue growth and organ size.