Regulation of Interleukin-10 Receptor Ubiquitination and Stability by Beta-TrCP-Containing Ubiquitin E3 Ligase

Interleukin-10 (IL-10) initiates potent anti-inflammatory effects via activating its cell surface receptor, composed of IL-10R1 and IL-10R2 subunits. The level of IL-10R1 is a major determinant of the cells' responsiveness to IL-10. Here, via a series of biochemical analyses using 293T cells reconstituted with IL-10R1, we identify the latter as a novel substrate of βTrCP-containing ubiquitin E3 ligase. Within the intracellular tail of IL-10R1, a canonical (318DpSGFGpS) and a slightly deviated (369DpSGICLQEP) βTrCP recognition motif can additively recruit βTrCP in a phosphorylation-dependent manner. βTrCP recruitment leads to ubiquitination, endocytosis and degradation of IL-10R1, subsequently reducing the cellular responsiveness to IL-10. Our study uncovers a novel negative regulatory mechanism that may potentially affect IL-10 function in target cells under physiological or pathological conditions

Distinct ubiquitin binding modes exhibited by SH3 domains: Molecular determinants and functional implications

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination. © 2013 Ortega Roldan et al

Dynein Light Chain Tctex-Type 1 Modulates Orexin Signaling through Its Interaction with Orexin 1 Receptor

Orexins (OX-A, OX-B) are neuropeptides involved in the regulation of the sleep-wake cycle, feeding and reward, via activation of orexin receptors 1 and 2 (OX1R, OX2R). The loss of orexin peptides or functional OX2R has been shown to cause the sleep disorder, narcolepsy. Since the regulation of orexin receptors remains largely undefined, we searched for novel protein partners of the intracellular tail of orexin receptors. Using a yeast two-hybrid screening strategy in combination with co-immunoprecipitation experiments, we found interactions between OX1R and the dynein light chains Tctex-type 1 and 3 (Dynlt1, Dynlt3). These interactions were mapped to the C-terminal region of the dynein light chains and to specific residues within the last 10 amino acids of OX1R. Hence, we hypothesized that dynein light chains could regulate orexin signaling. In HEK293 cells expressing OX1R, stimulation with OX-A produced a less sustained extracellular signal-regulated kinases 1/2 (ERK1/2) activation when Dynlt1 was co-expressed, while it was prolonged under reduced Dynlt1 expression. The amount of OX1R located at the plasma membrane as well as the kinetics and extent of OX-A-induced internalization of OX1R (disappearance from membrane) were not altered by Dynlt1. However, Dynlt1 reduced the localization of OX1R in early endosomes following initial internalization. Taken together, these data suggest that Dynlt1 modulates orexin signaling by regulating OX1R, namely its intracellular localization following ligand-induced internalization

The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

<p>Abstract</p> <p>Background</p> <p>Host cell invasion by the foodborne pathogen <it>Campylobacter jejuni </it>is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin^-/-, integrin-beta1^-/-, focal adhesion kinase (FAK)^-/-and Src/Yes/Fyn^-/-deficient mice, and wild-type control cells, to investigate <it>C. jejuni</it>-induced mechanisms leading to Cdc42 activation and bacterial uptake.</p> <p>Results</p> <p>Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and <it>C. jejuni </it>invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2^-/-knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using <it>C. jejuni </it>mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry.</p> <p>Conclusion</p> <p>Collectively, our findings led us propose that <it>C. jejuni </it>infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.</p

Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation

Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle

Interplay of Structural Disorder and Short Binding Elements in the Cellular Chaperone Function of Plant Dehydrin ERD14.

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 °C × 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo

The Tyrosine Kinase c-Src Directly Mediates Growth Factor-Induced Notch-1 and Furin Interaction and Notch-1 Activation in Pancreatic Cancer Cells

The proteolytic activity of Furin responsible for processing full length Notch-1 (p300) plays a critical role in Notch signaling. The amplitude and duration of Notch activity can be regulated at various points in the pathway, but there has been no report regarding regulation of the Notch-1-Furin interaction, despite its importance. In the present study, we found that the Notch-1-Furin interaction is regulated by the non-receptor tyrosine kinase, c-Src. c-Src and Notch-1 are physically associated, and this association is responsible for Notch-1 processing and activation. We also found that growth factor TGF-α, an EGFR ligand, and PDGF-BB, a PDGFR ligand, induce the Notch-1-Furin interaction mediated by c-Src. Our results support three new and provocative conclusions: (1) The association between Notch-1 and Furin is a well-regulated process; (2) Extracellular growth factor signals regulate this interaction, which is mediated by c-Src; (3) There is cross-talk between the plasma growth factor receptor-c-Src and Notch pathways. Co-localization of Notch-1 and c-Src was confirmed in xenograft tumor tissues and in the tissues of pancreatic cancer patients. Our findings have implications for the mechanism by which the Notch and growth factor receptor-c-Src signaling pathways regulate carcinogenesis and cancer cell growth

Diverse Roles of Eph/ephrin Signaling in the Mouse Lens

Recent genetic studies show that the Eph/ephrin bidirectional signaling pathway is associated with both congenital and age-related cataracts in mice and humans. We have investigated the molecular mechanisms of cataractogenesis and the roles of ephrin-A5 and EphA2 in the lens. Ephrin-A5 knockout (-/-) mice often display anterior polar cataracts while EphA2(-/-) lenses show very mild cortical or nuclear cataracts at weaning age. The anterior polar cataract of ephrin-A5(-/-) lenses is correlated with multilayers of aberrant cells that express alpha smooth muscle actin, a marker for mesenchymal cells. Only select fiber cells are altered in ephrin-A5(-/-) lenses. Moreover, the disruption of membrane-associated β-catenin and E-cadherin junctions is observed in ephrin-A5(-/-) lens central epithelial cells. In contrast, EphA2(-/-) lenses display normal monolayer epithelium while disorganization is apparent in all lens fiber cells. Immunostaining of ephrin-A5 proteins, highly expressed in lens epithelial cells, were not colocalized with EphA2 proteins, mainly expressed in lens fiber cells. Besides the previously reported function of ephrin-A5 in lens fiber cells, this work suggests that ephrin-A5 regulates β-catenin signaling and E-cadherin to prevent lens anterior epithelial cells from undergoing the epithelial-to-mesenchymal transition while EphA2 is essential for controlling the organization of lens fiber cells through an unknown mechanism. Ephrin-A5 and EphA2 likely interacting with other members of Eph/ephrin family to play diverse functions in lens epithelial cells and/or fiber cells