1. NF2/Merlin: THE MAGIC OF MERLIN
Sarah Speed BIOL 445 Spring 2015
Merlin is a unique protein in terms of the sorts of proteins we’ve learned about so far

2. a structural cytoskeleton-membrane linker
NF2 encodes Merlin,
a structural cytoskeleton-membrane linker
NF2 Locus
NF2 = Neurofibromin 2, a classical tumor suppressor gene located on the long arm of chromosome 22 in humans
NF2 encodes Merlin.
Expressed primarily in the central nervous system, particularly in Schwann cells which wrap around and insulate nerves.
Exhibits homology with ezrin-radixin-moesin (ERM) family of membrane-cytoskeleton linking proteins
Acts as a structural linker
Has three domains, FERM (N-term conserved in ERM family), extended alpha helix, and C-term.
In the active conformation, Merlin’s FERM domain self-associates with its C-term domain and forms a closed loop.
Phosphorylation of Ser518 in the C-term blocks intramolecular binding resulting in an open, inactive protein
Merlin
JCS November 1, 2002 vol. 115 no. 21 

3. Merlin localizes to membrane ruffles and cellular junctions
Localizes to areas of membrane remodeling
Membrane ruffles associated with cell motility
Filopodia
At Adherens junctions
At cell-ECM junctions
Acts as linker between the plasma membrane and the actin cytoskeleton
Recently, it has also been shown to translocate into nucleus
Unique location for a tumor suppressor, negatively modulates motility through actin reorganization

4. Merlin inhibits the Ras pathway at cell-ECM junctions
JCS November 1, 2002 vol. 115 no. 21 
Paxillin binds and recruits Merlin to the cell membrane to interact with cell surface proteins
Growth permissive state –
growth factor receptor is bound by its ligand
This activates a kinase cascade signaled by the transmembrane glycoprotein CD44.
CD44 activates Rac1, which in turn activates Paks, a kinase that hyperphosphorylates Merlin, leading to its inactivation
At the same time, the Rho kinase phosphorylates another ERM protein, which is then active and bound to CD44, promoting cell growth and motility
Growth restrictive state –
Hyaluronic acid is bound to CD44, thus the GFR is inactive, and the kinase cascade is not activated
Merlin remains closed and active, binding CD44
In this position, Merlin can inhibit the Ras pathway, ultimately inhibiting cell growth and motility
(In the case of Ras, merlin acts downstream of (Grb2)-SOS complex. However, merlin does not bind either SOS or Ras, but counteracts the ERM-dependent activation of Ras)

5. Merlin controls the stabilization of adherens junctions
Merlin and β-catenin colocalize at the plasma membrane
Merlin also plays a role in adherens junction stabilization
Keep in mind adherens junctions are an integral part of contact inhibition
Merlin and B-catenin colocalize at the plama membrane; Merlin is present at these adherens junctions
Mouse embryonic fibroblasts homozygous mutant for NF2
Perturbed actin organization
Diffuse B-catenin, indicating a lack of proper localization of adherens junction complexes in the absence of functional Merlin
Intro of virus with unphosphorylateable (always active) Merlin restored contact inhibition and adherens junction formation
Thus Merlin controls the assembly or stabilization of adherens-junction complexes
Nf2 mutants show disturbed actin organization and diffuse β-catenin
 Genes & development, 2003 vol. 17 no

6. In flies, Merlin regulates cell proliferation
and apoptosis through the Hippo signaling pathway
Wild-type
Mer;Ex
double mutant
Antenna
Thorax
The Hippo pathway controls organ size (modulates cell growth, proliferation, apoptosis)
Proteins include the cadherin Fat, Expanded and Merlin, the kinases Hippo, Warts, the adaptor molecules Salvador, Mats and transcriptional co-activator Yorkie.
Important target genes of Yorkie include cyclin E (which drives cell proliferation)
Hippo pathway components are conserved throughout evolution from yeast to humans, and the pathway has been implicated in the genesis of human cancers.
Talk about fly picture - Deregulation of this pathway in Drosophila melanogaster leads to dramatic increases in organ size.
 Nature Reviews Cancer 2007 vol. 7 no
Nature cell biology 2006 vol. 8 no

7. In mammals, Merlin inhibits the E3 ubiquitin ligase CRL4 in the nucleus
Recent studies in humans and mice have also elucidated a role for Merlin in the nucleus, in a fashion similar to the Hippo pathway.
Without Merlin, the E3 ubiquitin ligase CRL4 targets Lats 1/2, homologs of Warts, for ubiquitylation and thus destruction
This allows YAP/TAZ, homolog of Yorkie, to target genes for expression, as I mentioned for the Hippo pathway
Closed, active Merlin accumulates in the nucleus, inhibiting CRL4, allowing Lats1 and 2 normally inhibit YAP, and thus prevent oncogenic gene expression.
Cancer cell 2014 vol. 26 no

8. Summary of Merlin Function Cell-ECM Communication
Contact Inhibition
Nuclear regulation
We know that when cells contact one another, they experience contact inhibition which restricts growth. We also know that cells will not attempt to grow unless they have stable ECM contacts. Merlin plays a role in both of these processes:
Contact inhibition, cadherins inhibit Pak, leading to an active Merlin and negative regulation of cell growth.
Stable cell-ECM contacts but no contact inhibition, RTKs will activate Pak, leading to phosphorylated, inactive Merlin and signals are transduced that ultimately lead to cell growth.
Cell-ECM Communication
Integrins
ECM
Clin Cancer Res 2012 vol. 18 no

9. NF2-knockout mice are embryonic lethal
Genes & development 1997 vol. 11 no
Wild-type
Nf2 -/-
Most homozygous mutant mice embryos are misshaped and lack a distinct extraembryonic-embryonic boundary
Talk about pics
Embryonic lethal at day 7
Merlin is important for organization of embryonic structures
A study created mice heterozygous for the NF2 locus that mimicked the germ-line mutations present in human NF2 patients.
In contrast to the limited spectrum of benign tumors associated with NF2 in humans, mice that are the genetic analogs of human NF2 patients develop a variety of malignant tumor types such as osteosarcomas, fibrosarcomas, or hepatocellular carcinomas, and therefore do not model human NF2.
Important to note that these mouse tumors were highly motile and exhibited high rates of metastasis, thus Merlin plays a role in negatively regulating these characteristics of cancer cells.
Mutants exhibit underdeveloped ectoplacental cones and unorganized extraembryonic ectoderm
Wild-type
Nf2 -/-
Nf2 -/-

10. NF2/Merlin is a bona fide tumor-suppressor whose loss-of-function leads to Neurofibromatosis type 2
Inactivation of NF2 occurs through Knudson’s “two-hit hypothesis”:
Sporadic Cancer:
2 acquired mutations
Specifically, loss-of function in NF2 leads to the autosomal dominant inherited disease Neurofibromatosis Type 2
As a classical tumor suppressor, NF2 requires loss of heterozygosity and thus inactivation of both alleles in order to lead to aberrant phenotypes.
Like the mechanisms behind the predisposition to Retinoblastoma, inactivation of NF2 occurs through Knudson’s “two-hit” mechanism – inheritance of a mutant allele then subsequent sporadic mutation of the second allele leading to LOH and eventually tumorigenesis.
Hereditary Cancer:
1 inherited and
1 acquired mutation

11. Neurofibromatosis Type 2 manifests as benign tumors of the nervous system
Tumors are slow-growing and benign
Incidence: 1 in 33,000
Tumor-types:
Schwannomas
Meningiomas
Ependymomas
Symptoms:
Hearing loss
Tinnitus
Balance problems
Cataracts
Face paralysis
Treatment:
Surgical removal
Gamma knife radiotherapy
Erlotinib
Bevacizumab
In the case of unresectable schwannomas, Erlotinib has shown promise in shrinking tumor size
Bevacizumab, an endothelial growth factor monoclonal antibody also showed some efficacy in the shrinkage of schwannomas
Meningioma
Schwannoma

12. References
Ammoun, Sylwia, et al. "Dissecting and targeting the growth factor–dependent and growth factor–independent extracellular signal-regulated kinase pathway in human schwannoma." Cancer research 68.13 (2008):
Benhamouche, Samira, et al. "Nf2/Merlin controls progenitor homeostasis and tumorigenesis in the liver." Genes & development 24.16 (2010):
Bretscher, Anthony, Kevin Edwards, and Richard G. Fehon. "ERM proteins and merlin: integrators at the cell cortex." Nature reviews Molecular cell biology 3.8 (2002):
Gutmann, David H., et al. "The diagnostic evaluation and multidisciplinary management of neurofibromatosis 1 and neurofibromatosis 2." Jama 278.1 (1997):
Hamaratoglu, Fisun, et al. "The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis." Nature cell biology 8.1 (2006):
Harvey, Kieran, and Nicolas Tapon. "The Salvador–Warts–Hippo pathway—an emerging tumour-suppressor network." Nature Reviews Cancer 7.3 (2007):
Kissil, Joseph L., et al. "Merlin phosphorylation by p21-activated kinase 2 and effects of phosphorylation on merlin localization." Journal of Biological Chemistry  (2002):
Lallemand, Dominique, et al. "NF2 deficiency promotes tumorigenesis and metastasis by destabilizing adherens junctions." Genes & development 17.9 (2003):
Li, Wei, et al. "Merlin/NF2 suppresses tumorigenesis by inhibiting the E3 ubiquitin ligase CRL4 DCAF1 in the nucleus." Cell 140.4 (2010):
Li, Wei, et al. "Merlin/NF2 Loss-Driven Tumorigenesis Linked to CRL4 DCAF1-Mediated Inhibition of the Hippo Pathway Kinases Lats1 and 2 in the Nucleus."Cancer cell  (2014):
McClatchey, Andrea I., et al. "The Nf2 tumor suppressor gene product is essential for extraembryonic development immediately prior to gastrulation."Genes & development 11.10 (1997):
McClatchey, Andrea I., et al. "Mice heterozygous for a mutation at the Nf2 tumor suppressor locus develop a range of highly metastatic tumors." Genes & development  (1998):
McClatchey, Andrea I. "Merlin and ERM proteins: unappreciated roles in cancer development?." Nature Reviews Cancer 3.11 (2003):
Rong, et al. "Neurofibromatosis 2 (NF2) tumor suppressor merlin inhibits phosphatidylinositol 3-kinase through binding to PIKE-L." Proceedings of the National Academy of Sciences  (2004):
Schmucker, B., W. G. Ballhausen, and M. Kressel. "Subcellular localization and expression pattern of the neurofibromatosis type 2 protein merlin/schwannomin." European journal of cell biology 72.1 (1997):
Sun, Chun-Xiao, Victoria A. Robb, and David H. Gutmann. "Protein 4.1 tumor suppressors: getting a FERM grip on growth regulation." Journal of cell science (2002):
Xiao, Guang‐Hui, Jonathan Chernoff, and Joseph R. Testa. "NF2: the wizardry of merlin." Genes, Chromosomes and Cancer 38.4 (2003): .