The N-Terminal Domain of the Drosophila Retinoblastoma Protein Rbf1 Interacts with ORC and Associates with Chromatin in an E2F Independent Manner

The retinoblastoma (Rb) tumor suppressor protein can function as a DNA replication inhibitor as well as a transcription factor. Regulation of DNA replication may occur through interaction of Rb with the origin recognition complex (ORC).We characterized the interaction of Drosophila Rb, Rbf1, with ORC. Using expression of proteins in Drosophila S2 cells, we found that an N-terminal Rbf1 fragment (amino acids 1-345) is sufficient for Rbf1 association with ORC but does not bind to dE2F1. We also found that the C-terminal half of Rbf1 (amino acids 345-845) interacts with ORC. We observed that the amino-terminal domain of Rbf1 localizes to chromatin in vivo and associates with chromosomal regions implicated in replication initiation, including colocalization with Orc2 and acetylated histone H4.Our results suggest that Rbf1 can associate with ORC and chromatin through domains independent of the E2F binding site. We infer that Rbf1 may play a role in regulating replication directly through its association with ORC and/or chromatin factors other than E2F. Our data suggest an important role for retinoblastoma family proteins in cell proliferation and tumor suppression through interaction with the replication initiation machinery

TRAF4 is a novel phosphoinositide-binding protein modulating tight junctions and favoring cell migration

Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration

Actomyosin-generated tension on cadherin is similar between dividing and non-dividing epithelial cells in early Xenopus laevis embryos

Epithelia represent a unique situation where polarized cells must maintain sufficiently strong cellcell contacts to guarantee the epithelial integrity indispensable for barrier functions. Nevertheless, epithelia must also keep sufficient plasticity which is crucial during development and morphogenesis. Adherens junctions and mechanical forces produced by the actomyosin cytoskeleton are major players for epithelial integrity maintenance and plasticity regulations. To understand how the epithelium is able to meet such a challenge, it is indispensable to determine how cellular junctions and mechanical forces acting at adherens junctions are regulated. Here, we investigate the tensile forces acting on adherens junctions via cadherin during cell division in the Xenopus embryos epithelium. Using the recently developed E-cadherin FRET tension sensor and a fastFLIM prototype microscope, we were able to measure mechanical forces applied on cadherin at cell-cell junctions. We have shown that the Xenopus epithelium is under tension, approximately 3 pN which remains stable, indicating that tensile forces acting on cadherin at the adherens junction are at equilibrium. Unexpectedly, mechanical tension across cadherin was similar between dividing and non-dividing epithelial cells.</p

β-arrestin 2 oligomerization controls the Mdm2-dependent inhibition of p53

β-arrestins (β-arrs), two ubiquitous proteins involved in serpentine heptahelical receptor regulation and signaling, form constitutive homo- and heterooligomers stabilized by inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Monomeric β-arrs are believed to interact with receptors after agonist activation, and therefore, β-arr oligomers have been proposed to represent a resting biologically inactive state. In contrast to this, we report here that the interaction with and subsequent titration out of the nucleus of the protooncogene Mdm2 specifically require β-arr2 oligomers together with the previously characterized nucleocytoplasmic shuttling of β-arr2. Mutation of the IP6-binding sites impair oligomerization, reduce interaction with Mdm2, and inhibit p53-dependent antiproliferative effects of β-arr2, whereas the competence for receptor regulation and signaling is maintained. These observations suggest that the intracellular concentration of β-arr2 oligomers might control cell survival and proliferation