Phosphorylation of Merlin by Aurora A kinase appears necessary for mitotic progression.

Although Merlin's function as a tumor suppressor and regulator of mitogenic signaling networks such as the Ras/rac, Akt, and Hippo pathways is well-documented, in mammals as well as in insects, its role during cell cycle progression remains unclear. In this study, using a combination of approaches, including FACS analysis, time-lapse imaging, immunofluorescence microscopy, and co-immunoprecipitation, we show that Ser-518 of Merlin is a substrate of the Aurora protein kinase A during mitosis and that its phosphorylation facilitates the phosphorylation of a newly discovered site, Thr-581. We found that the expression in HeLa cells of a Merlin variant that is phosphorylation-defective on both sites leads to a defect in centrosomes and mitotic spindles positioning during metaphase and delays the transition from metaphase to anaphase. We also show that the dual mitotic phosphorylation not only reduces Merlin binding to microtubules but also timely modulates ezrin interaction with the cytoskeleton. Finally, we identify several point mutants of Merlin associated with neurofibromatosis type 2 that display an aberrant phosphorylation profile along with defective α-tubulin-binding properties. Altogether, our findings of an Aurora A-mediated interaction of Merlin with α-tubulin and ezrin suggest a potential role for Merlin in cell cycle progression.

Determinants of lentiviral Vpx-CRL4 E3 ligase-mediated SAMHD1 degradation in the substrate adaptor protein DCAF1.

The lentiviral accessory protein Vpx enhances viral replication in macrophages, dendritic cells and resting CD4+ T cells by utilizing the host CRL4-DCAF1 E3 ligase to trigger the degradation of the intrinsic antiviral factor SAMHD1. Distinct from the species-specific recognition of either the N or C-terminus of SAMHD1 by Vpx proteins of different HIV-2 and SIV lineages, Vpx recruits SAMHD1 onto the same CRL4-DCAF1 complex. However, the determinants in DCAF1 that are required for Vpx-mediated SAMHD1 degradation have not been well characterized. Here, we demonstrate that the viral protein Vpx is resistant to suppression by a cellular inhibitor of the CRL4-DCAF1 E3 ligase, Merlin/NF2, through targeting a separate binding region in DCAF1. The Merlin binding-deficient DCAF1 truncation mutant (1-1417) is sufficient for Vpx-CRL4-DCAF1 E3 ligase assembly and SAMHD1 degradation. We found that the carboxyl-terminus ED-rich region (1312-1417) of DCAF1 is required for the nuclear localization of DCAF1 and for the Vpx-DCAF1 interaction. We identified the DCAF1 (1-1311) truncation mutant as a dominant negative mutant of wild-type DCAF1 that inhibits Vpx-mediated SAMHD1 degradation. These results suggest a unique strategy by which Vpx exploits DCAF1 to counteract this host restriction factor.

Functional role of glycosphingolipids in contact inhibition of growth in a human mammary epithelial cell line.

We have demonstrated previously the involvement of certain glycosphingolipids (GSLs) in 'contact inhibition' (dependent on cell-to-cell contact) of cell growth. Here, we examined the roles of specific GSLs in contact inhibition of the human epithelial cell line MCF10A. Contact-inhibited cells show increased expression of the ganglioside GD3 and the globo-series GSL Gb3, and of the mRNAs for the corresponding sialyltransferases ST8SIA1 (GD3 synthase) and galactosyltransferase A4GALT (Gb3 synthase). siRNA knockdown (KD) of ST8SIA1 and/or A4GALT significantly suppresses contact inhibition. Exogenous addition of GD3 or Gb3 inhibits proliferation of low-density cells. Our findings suggest that GSLs play functional roles in contact inhibition of these cells and that Merlin/NF2, a tumor suppressor protein, is involved in the GSL function.

Merlin/NF2-Lin28B-let-7 Is a Tumor-Suppressive Pathway that Is Cell-Density Dependent and Hippo Independent.

Contact inhibition of proliferation is critical for tissue organization, and its dysregulation contributes to tumorigenesis. Merlin/NF2 is a tumor suppressor that governs contact inhibition. Although Merlin/NF2 inhibits YAP1 and TAZ, which are paralogous Hippo pathway transcriptional co-activators and oncoproteins, it is not fully understood how Merlin/NF2-mediated signal transduction triggered by cell-cell contact exerts tumor suppression. Here, we identify Lin28B, an inhibitor of let-7 microRNAs (miRNAs), as an important downstream target of Merlin/NF2. Functional studies revealed that, at low cell density, Merlin/NF2 is phosphorylated and does not bind to Lin28B, allowing Lin28B to enter the nucleus, bind to pri-let-7 miRNAs, and inhibit their maturation in a YAP1/TAZ-independent manner. This inhibition of pri-let-7 maturation then promotes cell growth. However, cell-cell contact triggers Merlin/NF2 dephosphorylation, which sequesters Lin28B in the cytoplasm and permits pri-let-7 maturation. Our results reveal that Merlin/NF2-mediated signaling drives a tumor-suppressive pathway that is cell-density dependent and Hippo independent.

The role of angiomotin phosphorylation in the Hippo pathway during preimplantation mouse development.

The Hippo signaling pathway regulates a number of cellular events, including the control of cell fates in preimplantation mouse embryos. The inner and outer cells of the embryo show high and low levels of Hippo signaling, respectively. This position-dependent Hippo signaling promotes the specification of distinct cell fates. In a recent paper, we identified the molecular mechanism that controls Hippo signaling in preimplantation embryos. The junction-associated scaffold protein Angiomotin (Amot) plays a key role in this mechanism. At the adherens junctions of the inner cells, Amot activates the Hippo pathway by recruiting and activating the protein kinase large tumor suppressor (Lats). In contrast, Amot at the apical membrane of the outer cells suppresses Hippo signaling by interacting with F-actin. The phosphorylation of Amot inhibits its interaction with F-actin and activates Hippo signaling. We propose that Amot acts as a molecular switch for the Hippo pathway and links F-actin with Lats activity.