The NF2 tumor suppressor regulates microtubule-based vesicle trafficking via a novel Rac, MLK and p38SAPK pathway

© Macmillan Publishers, 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Oncogene 32 (2013): 1135–1143, doi:10.1038/onc.2012.135.Neurofibromatosis type 2 patients develop schwannomas, meningiomas and ependymomas resulting from mutations in the tumor suppressor gene, NF2, encoding a membrane-cytoskeleton adapter protein called merlin. Merlin regulates contact inhibition of growth and controls the availability of growth factor receptors at the cell surface. We tested if microtubule-based vesicular trafficking might be a mechanism by which merlin acts. We found that schwannoma cells, containing merlin mutations and constitutive activation of the Rho/Rac family of GTPases, had decreased intracellular vesicular trafficking relative to normal human Schwann cells. In Nf2−/− mouse Schwann (SC4) cells, re-expression of merlin as well as inhibition of Rac or its effector kinases, MLK and p38SAPK, each increased the velocity of Rab6 positive exocytic vesicles. Conversely, an activated Rac mutant decreased Rab6 vesicle velocity. Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38SAPK kinase. Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.This work was supported by NIH R01 CA118032 (to NR), and MBL research fellowships (to NR and GM), NIH R01 NS23868 (to STB)

Merlin tumor suppressor function is regulated by PIP2-mediated dimerization

Neurofibromatosis Type 2 is an inherited disease characterized by Schwann cell tumors of cranial and peripheral nerves. The NF2 gene encodes Merlin, a member of the ERM family consisting of an N-terminal FERM domain, a central α-helical region, and a C-terminal domain. Changes in the intermolecular FERM-CTD interaction allow Merlin to transition between an open, FERM accessible conformation and a closed, FERM-inaccessible conformation, modulating Merlin activity. Merlin has been shown to dimerize, but the regulation and function Merlin dimerization is not clear. We used a nanobody based binding assay to show that Merlin dimerizes via a FERM-FERM interaction, orientated with each C-terminus close to each other. Patient derived and structural mutants show that dimerization controls interactions with specific binding partners, including HIPPO pathway components, and correlates with tumor suppressor activity. Gel filtration experiments showed that dimerization occurs after a PIP2 mediated transition from closed to open conformation monomers. This process requires the first 18 amino acids of the FERM domain and is inhibited by phosphorylation at serine 518. The discovery that active, open conformation Merlin is a dimer represents a new paradigm for Merlin function with implications for the development of therapies designed to compensate for Merlin loss

Merlin tumor suppressor function is regulated by PIP2-mediated dimerization.

Neurofibromatosis Type 2 is an inherited disease characterized by Schwann cell tumors of cranial and peripheral nerves. The NF2 gene encodes Merlin, a member of the ERM family consisting of an N-terminal FERM domain, a central α-helical region, and a C-terminal domain. Changes in the intermolecular FERM-CTD interaction allow Merlin to transition between an open, FERM accessible conformation and a closed, FERM-inaccessible conformation, modulating Merlin activity. Merlin has been shown to dimerize, but the regulation and function Merlin dimerization is not clear. We used a nanobody based binding assay to show that Merlin dimerizes via a FERM-FERM interaction, orientated with each C-terminus close to each other. Patient derived and structural mutants show that dimerization controls interactions with specific binding partners, including HIPPO pathway components, and correlates with tumor suppressor activity. Gel filtration experiments showed that dimerization occurs after a PIP2 mediated transition from closed to open conformation monomers. This process requires the first 18 amino acids of the FERM domain and is inhibited by phosphorylation at serine 518. The discovery that active, open conformation Merlin is a dimer represents a new paradigm for Merlin function with implications for the development of therapies designed to compensate for Merlin loss

Retinoblastoma Tumor Suppressor: Analyses of Dynamic Behavior in Living Cells Reveal Multiple Modes of Regulation

The retinoblastoma tumor suppressor, RB, assembles multiprotein complexes to mediate cell cycle inhibition. Although many RB binding partners have been suggested to underlie these functions, the validity of these interactions on the behavior of RB complexes in living cells has not been investigated. Here, we studied the dynamic behavior of RB by using green fluorescent protein-RB fusion proteins. Although these proteins were universally nuclear, phosphorylation or oncoprotein binding mediated their active exclusion from the nucleolus. In vivo imaging approaches revealed that RB exists in dynamic equilibrium between a highly mobile and a slower diffusing species, and genetic lesions associated with tumorigenesis increased the fraction of RB in a highly mobile state. The RB complexes dictating cell cycle arrest were surprisingly dynamic and harbored a relatively short residence time on chromatin. In contrast, this rapid exchange was attenuated in cells that are hypersensitive to RB, suggesting that responsiveness may inversely correlate with mobility. The stability of RB dynamics within the cell was additionally modified by the presence and function of critical corepressors. Last, the RB-assembled complexes present in living cells were primarily associated with E2F binding sites in chromatin. In contrast to RB, E2F1 consistently maintained a stable association with E2F sites regardless of cell type. Together, these results elucidate the kinetic framework of RB tumor suppressor action in transcriptional repression and cell cycle regulation

RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult

Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute RB deletion. Using this system, we demonstrated that loss of RB disrupted the DNA damage checkpoint elicited by either cisplatin or camptothecin exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (γH2AX) foci, which indicate DNA double-strand breaks. The formation of γH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-deficient cells. Additionally, peak γH2AX accumulation occurred in S-phase cells undergoing DNA replication in the presence of damage, and these γH2AX foci co-localized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage-induced cell cycle arrest to induce γH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability

DEK over-expression promotes mitotic defects and micronucleus formation

<p>The <i>DEK</i> gene encodes a nuclear protein that binds chromatin and is involved in various fundamental nuclear processes including transcription, RNA splicing, DNA replication and DNA repair. Several cancer types characteristically over-express DEK at the earliest stages of transformation. In order to explore relevant mechanisms whereby DEK supports oncogenicity, we utilized cancer databases to identify gene transcripts whose expression patterns are tightly correlated with that of DEK. We identified an enrichment of genes involved in mitosis and thus investigated the regulation and possible function of DEK in cell division. Immunofluorescence analyses revealed that DEK dissociates from DNA in early prophase and re-associates with DNA during telophase in human keratinocytes. Mitotic cell populations displayed a sharp reduction in DEK protein levels compared to the corresponding interphase population, suggesting DEK may be degraded or otherwise removed from the cell prior to mitosis. Interestingly, DEK overexpression stimulated its own aberrant association with chromatin throughout mitosis. Furthermore, DEK co-localized with anaphase bridges, chromosome fragments, and micronuclei, suggesting a specific association with mitotically defective chromosomes. We found that DEK over-expression in both non-transformed and transformed cells is sufficient to stimulate micronucleus formation. These data support a model wherein normal chromosomal clearance of DEK is required for maintenance of high fidelity cell division and chromosomal integrity. Therefore, the overexpression of DEK and its incomplete removal from mitotic chromosomes promotes genomic instability through the generation of genetically abnormal daughter cells. Consequently, DEK over-expression may be involved in the initial steps of developing oncogenic mutations in cells leading to cancer initiation</p