ABSTRACT
The family of tumor necrosis factor receptors (TNFRs) and their ligands form a regulatory signaling network that controls immune responses. Various members of this receptor family respond differently to the soluble and membrane-bound forms of their respective ligands. However, the determining factors and underlying molecular mechanisms of this diversity are not yet understood. Using an established system of chimeric TNFRs and novel ligand variants mimicking the bioactivity of membrane-bound TNF (mTNF), we demonstrate that the membrane-proximal extracellular stalk regions of TNFR1 and TNFR2 are crucial in controlling responsiveness to soluble TNF (sTNF). We show that the stalk region of TNFR2, in contrast to the corresponding part of TNFR1, efficiently inhibits both the receptor's enrichment/clustering in particular cell membrane regions and ligand-independent homotypic receptor preassembly, thereby preventing sTNF-induced, but not mTNF-induced, signaling. Thus, the stalk regions of the two TNFRs not only have implications for additional TNFR family members, but also provide potential targets for therapeutic intervention.
Subject(s)
Receptors, Tumor Necrosis Factor, Type II/chemistry , Receptors, Tumor Necrosis Factor, Type II/metabolism , Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/metabolism , Amino Acid Sequence , Animals , Binding Sites/genetics , Cell Membrane/metabolism , Cells, Cultured , Glycosylation , HEK293 Cells , HeLa Cells , Humans , Ligands , Mice , Mice, Knockout , Molecular Sequence Data , Receptors, Tumor Necrosis Factor, Type I/deficiency , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type II/deficiency , Receptors, Tumor Necrosis Factor, Type II/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Signal Transduction , Solubility , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Deleted in liver cancer 1 (DLC1) is a Rho-GTPase-activating protein (GAP) that is downregulated in various tumor types. In vitro, DLC1 specifically inactivates the small GTPases RhoA, RhoB and RhoC through its GAP domain and this appears to contribute to its tumor suppressor function in vivo. Molecular mechanisms that control DLC1 activity have not so far been investigated. Here, we show that phorbol-ester-induced activation of protein kinase C and protein kinase D stimulates association of DLC1 with the phosphoserine/phosphothreonine-binding 14-3-3 adaptor proteins via recognition motifs that involve Ser327 and Ser431. Association with 14-3-3 proteins inhibits DLC1 GAP activity and facilitates signaling by active Rho. We further show that treatment of cells with phorbol ester or coexpression of 14-3-3 proteins, blocks DLC1 nucleocytoplasmic shuttling, probably by masking a previously unrecognized nuclear localization sequence. The binding to 14-3-3 proteins is thus a newly discovered mechanism by which DLC1 activity is regulated and compartmentalized.
Subject(s)
14-3-3 Proteins/metabolism , Cell Nucleus/metabolism , GTPase-Activating Proteins/metabolism , Protein Isoforms/metabolism , Tumor Suppressor Proteins/metabolism , 14-3-3 Proteins/genetics , Active Transport, Cell Nucleus/physiology , Amino Acid Sequence , Animals , Cell Line , Enzyme Activation , GTPase-Activating Proteins/antagonists & inhibitors , Humans , Molecular Sequence Data , Phorbol Esters/metabolism , Protein Binding , Protein Isoforms/genetics , Protein Kinase C/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Serine/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
Deleted in liver cancer (DLC) 1 and 2 are Rho GTPase-activating proteins that are frequently down-regulated in various types of cancer. Ectopic expression in carcinoma cell lines lacking these proteins has been shown to inhibit cell migration and invasion. However, whether the loss of DLC1 or DLC2 is the cause of aberrant Rho signaling in transformed cells has not been investigated. Here, we have down-regulated DLC1 and DLC2 expression in breast cancer cells using a RNA interference approach. Silencing of DLC1 led to the stabilization of stress fibers and focal adhesions and enhanced cell motility in wound-healing as well as chemotactic Transwell assays. We provide evidence that enhanced migration of cells lacking DLC1 is dependent on the Rho effector protein Dia1 but does not require the activity of Rho kinase. By contrast, DLC2 knockdown failed to affect the migratory behavior of cells, suggesting that the two proteins have distinct functions. This is most likely due to their differential subcellular localizations, with DLC1 found in focal adhesions and DLC2 being mainly cytosolic. Collectively, our data show that DLC1 is critically involved in the control of Rho signaling and actin cytoskeleton remodeling and that its cellular loss is sufficient for the acquisition of a more migratory phenotype of breast cancer cells.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Cell Movement/physiology , Signal Transduction/physiology , Tumor Suppressor Proteins/physiology , Base Sequence , Cell Line , DNA Primers , Formins , GTPase-Activating Proteins , Guanosine Triphosphate/metabolism , Humans , Microscopy, FluorescenceABSTRACT
The movement protein (MP) of Abutilon mosaic virus (AbMV, Geminiviridae) exhibited a complex band pattern upon gel electrophoresis indicating its post-translational modification when expressed in AbMV-infected plants or, ectopically, in fission yeasts. High-resolution separation according to charge and molecular weight in acetic acid/urea polyacrylamide or sodium dodecyl sulphate polyacrylamide gels followed by western blot analysis revealed a pattern of AbMV MP from infected plants more related to that from fission yeast than from bacteria. For this reason, expression in fission yeast was established as an experimental system to study post-translational modifications of AbMV MP. Metabolic labelling with 32P-orthophosphate confirmed a phosphorylation of all MP variants suggesting multiple phosphorylation sites. Treatment with calf intestinal alkaline phosphatase removed this label completely, but was unable to eliminate all protein bands with lower electrophoretic mobility. Thus, multiple phosphorylations contribute to the complex migration behaviour of MP, but additional post-translational modifications may occur as well.
