Regulation of NF-κB by PML and PML-RARα

Promyelocytic Leukemia (PML) is a nuclear protein that forms sub-nuclear structures termed nuclear bodies associated with transcriptionally active genomic regions. PML is a tumour suppressor and regulator of cell differentiation. We demonstrate that PML promotes TNFα-induced transcriptional responses by promoting NF-κB activity. TNFα-treated PML−/− cells show normal IκBα degradation and NF-κB nuclear translocation but significantly reduced NF-κB DNA binding and phosphorylation of NF-κB p65. We also demonstrate that the PML retinoic acid receptor-α (PML-RARα) oncofusion protein, which causes acute promyelocytic leukemia, inhibits TNFα induced gene expression and phosphorylation of NF-κB. This study establishes PML as an important regulator of NF-κB and demonstrates that PML-RARα dysregulates NF-κB

Rab-coupling protein coordinates recycling of α5β1 integrin and EGFR1 to promote cell migration in 3D microenvironments

Here we show that blocking the adhesive function of αvβ3 integrin with soluble RGD ligands, such as osteopontin or cilengitide, promoted association of Rab-coupling protein (RCP) with α5β1 integrin and drove RCP-dependent recycling of α5β1 to the plasma membrane and its mobilization to dynamic ruffling protrusions at the cell front. These RCP-driven changes in α5β1 trafficking led to acquisition of rapid/random movement on two-dimensional substrates and to a marked increase in fibronectin-dependent migration of tumor cells into three-dimensional matrices. Recycling of α5β1 integrin did not affect its regulation or ability to form adhesive bonds with substrate fibronectin. Instead, α5β1 controlled the association of EGFR1 with RCP to promote the coordinate recycling of these two receptors. This modified signaling downstream of EGFR1 to increase its autophosphorylation and activation of the proinvasive kinase PKB/Akt. We conclude that RCP provides a scaffold that promotes the physical association and coordinate trafficking of α5β1 and EGFR1 and that this drives migration of tumor cells into three-dimensional matrices

Rab32 interacts with SNX6 and affects retromer-dependent Golgi trafficking

The Rab family of small GTPases regulate various aspects of cellular dynamics in eukaryotic cells. Membrane trafficking has emerged as central to the functions of leucine-rich repeat kinase 2 (LRRK2), which is associated with inherited and sporadic forms of Parkinson’s disease (PD). Rabs act as both regulators of the catalytic activity and targets for serine/threonine phosphorylation by LRRK2. Rab32, Rab38 and Rab29 have been shown to regulate LRRK2 sub-cellular localization through direct interactions. Recently, Rab29 was shown to escort LRRK2 to the Golgi apparatus and activate the phosphorylation of Rab8 and Rab10. Rab32 is linked to multiple cellular functions including endosomal trafficking, mitochondrial dynamics, and melanosome biogenesis. A missense mutation in Rab32 has also recently been linked to PD. Here, we demonstrate that Rab32 directly interacts with sorting nexin 6 (SNX6). SNX6 is a transient subunit of the retromer, an endosome-Golgi retrieval complex whose Vps35 subunit is strongly associated with PD. We could further show that localization of cation-independent mannose-6-phosphate receptors, which are recycled to the trans-Golgi network (TGN) by the retromer, was affected by both Rab32 and SNX6. These data imply that Rab32 is linked to SNX6/retromer trafficking at the Golgi, and also suggests a possible connection between the retromer and Rab32 in the trafficking and biological functions of LRRK2

Insulin promotes Rip11 accumulation at the plasma membrane by inhibiting a dynamin- and PI3-kinase-dependent, but Akt-independent, internalisation event

Rip11 is a Rab11 effector protein that has been shown to be important in controlling the trafficking of several intracellular cargoes, including the fatty acid transporter FAT/CD36, V-ATPase and the glucose transporter GLUT4. We have previously demonstrated that Rip11 translocates to the plasma membrane in response to insulin and here we examine the basis of this regulated phenomenon in more detail. We show that Rip11 rapidly recycles between the cell interior and surface, and that the ability of insulin to increase the appearance of Rip11 at the cell surface involves an inhibition of Rip11 internalisation from the plasma membrane. By contrast the hormone has no effect on the rate of Rip11 translocation towards the plasma membrane. The ability of insulin to inhibit Rip11 internalisation requires dynamin and class I PI3-kinases, but is independent of the activation of the protein kinase Akt; characteristics which are very similar to the mechanism by which insulin inhibits GLUT4 endocytosis

Tumor Susceptibility Gene 101 (TSG101) Is a Novel Binding-Partner for the Class II Rab11-FIPs

The Rab11-FIPs (Rab11-family interacting proteins; henceforth, FIPs) are a family of Rab11a/Rab11b/Rab25 GTPase effector proteins implicated in an assortment of intracellular trafficking processes. Through proteomic screening, we have identified TSG101 (tumor susceptibility gene 101), a component of the ESCRT-I (endosomal sorting complex required for transport) complex, as a novel FIP4-binding protein, which we find can also bind FIP3. We show that α-helical coiled-coil regions of both TSG101 and FIP4 mediate the interaction with the cognate protein, and that point mutations in the coiled-coil regions of both TSG101 and FIP4 abrogate the interaction. We find that expression of TSG101 and FIP4 mutants cause cytokinesis defects, but that the TSG101-FIP4 interaction is not required for localisation of TSG101 to the midbody/Flemming body during abscission. Together, these data suggest functional overlap between Rab11-controlled processes and components of the ESCRT pathway

Rab coupling protein mediated endosomal recycling of N-cadherin influences cell motility

Rab coupling protein (RCP) is a Rab GTPase effector that functions in endosomal recycling. The RCP gene is frequently amplified in breast cancer, leading to increased cancer aggressiveness. Furthermore, RCP enhances the motility of ovarian cancer cells by coordinating the recycling of alpha 5 beta 1 integrin and EGF receptor to the leading edge of migrating cells. Here we report that RCP also influences the motility of lung adenocarcinoma cells. Knockdown of RCP inhibits the motility of A549 cells in 2D and 3D migration assays, while its overexpression enhances migration in these assays. Depletion of RCP leads to a reduction in N-cadherin protein levels, which could be restored with lysosomal inhibitors. Trafficking assays revealed that RCP knockdown inhibits the return of endocytosed N-cadherin to the cell surface. We propose that RCP regulates the endosomal recycling of N-cadherin, and in its absence N-cadherin is diverted to the degradative pathway. The increased aggressiveness of tumour cells that overexpress RCP may be due to biased recycling of N-cadherin in metastatic cancer cells

Rab11 proteins in health and disease

Abstract Comprising over 60 members, Rab proteins constitute the largest branch of the Ras superfamily of lowmolecular-mass G-proteins. This protein family have been primarily implicated in various aspects of intracellular membrane trafficking processes. On the basis of distinct subfamily-specific sequence motifs, many Rabs have been grouped into subfamilies. The Rab11 GTPase subfamily comprises three members: Rab11a, Rab11b and Rab25/Rab11c, which, between them, have been demonstrated to bind more than 30 proteins. In the present paper, we review the function of the Rab11 subfamily. We describe their localization and primary functional roles within the cell and their implication, to date, in disease processes. We also summarize the protein machinery currently known to regulate or mediate their functions and the cargo molecules which they have been shown to transport

Bioregulatory Systems Medicine: An Innovative Approach to Integrating the Science of Molecular Networks, Inflammation and Systems Biology with the Patient’s Autoregulatory Capacity?

Bioregulatory systems medicine is a paradigm that aims to advance current medical practices. The basic scientific and clinical tenets of this approach embrace an interconnected picture of human health, supported largely by recent advances in systems biology and genomics, and focuses on the implications of multi-scale interconnectivity for improving therapeutic approaches to disease. This article introduces the formal incorporation of these scientific and clinical elements into a cohesive theoretical model of the bioregulatory systems medicine approach. The authors review this integrated body of knowledge and discuss how the emergent conceptual model offers the medical field a new avenue for extending the armamentarium of current treatment and healthcare, with the ultimate goal of improving population health

Rab11‐FIP3 Is Critical for the Structural Integrity of the Endosomal Recycling Compartment

Rab11‐FIP3 is an endosomal recycling compartment (ERC) protein that is implicated in the process of membrane delivery from the ERC to sites of membrane insertion during cell division. Here we report that Rab11‐FIP3 is critical for the structural integrity of the ERC during interphase. We demonstrate that knockdown of Rab11‐FIP3 and expression of a mutant of Rab11‐FIP3 that is Rab11‐binding deficient cause loss of all ERC‐marker protein staining from the pericentrosomal region of A431 cells. Furthermore, we find that fluorophore‐labelled transferrin cannot access the pericentrosomal region of cells in which Rab11‐FIP3 function has been perturbed. We find that this Rab11‐FIP3 function appears to be specific because expression of the equivalent Rab11‐binding deficient mutant of Rab‐coupling protein does not perturb ERC morphology. In addition, we find that other organelles such as sorting and late endosomes are unaffected by loss of Rab11‐FIP3 function. Finally, we demonstrate the presence of an extensive coiled‐coil region between residues 463 and 692 of Rab11‐FIP3, which exists as a dimer in solution and is critical to support its function on the ERC. Together, these data indicate that Rab11‐FIP3 is necessary for the structural integrity of the pericentrosomal ERC

Purification, crystallization and preliminary X-ray diffraction studies of Rab11 in complex with Rab11-FIP2

Crystals of Rab11 in complex with the Rab-binding domain (RBD) of its effector Rab11-FIP2 were grown and synchrotron diffraction data were collected at 2.8 Å resolution