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

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 3991-4000

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

Merlin inhibits the Ras pathway at cell-ECM junctions JCS November 1, 2002 vol. 115 no. 21 3991-4000 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)

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. 9 1090-1100.

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. 3 182-191. Nature cell biology 2006 vol. 8 no. 1 27-36.

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. 1 48-60.

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. 17 4485-4490

NF2-knockout mice are embryonic lethal Genes & development 1997 vol. 11 no. 10 1253-1265. 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 -/-

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

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

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