Interaction between Epsin/Yap180 adaptors and the scaffolds Ede1/Pan1 is required for endocytosis

The spatial and temporal regulation of the interactions among the approximately 60 proteins required for endocytosis is under active investigation in many laboratories. We have identified the interaction between monomeric clathrin adaptors and endocytic scaffold proteins as a critical prerequisite for the recruitment and/or spatiotemporal dynamics of endocytic proteins at early and late stages of internalization. Quadruple deletion yeast cells (DeltaDeltaDeltaDelta) lacking four putative adaptors, Ent1/2 and Yap1801/2 (homologues of epsin and AP180/CALM proteins), with a plasmid encoding Ent1 or Yap1802 mutants, have defects in endocytosis and growth at 37 degrees C. Live-cell imaging revealed that the dynamics of the early- and late-acting scaffold proteins Ede1 and Pan1, respectively, depend upon adaptor interactions mediated by adaptor asparagine-proline-phenylalanine motifs binding to scaffold Eps15 homology domains. These results suggest that adaptor/scaffold interactions regulate transitions from early to late events and that clathrin adaptor/scaffold protein interaction is essential for clathrin-mediated endocytosis.     

Positive and negative regulation of T-cell activation by adaptor proteins

Adaptor proteins, molecules that mediate intermolecular interactions, are now known to be as crucial for lymphocyte activation as are receptors and effectors. Extensive work from numerous laboratories has identified and characterized many of these adaptors, demonstrating their roles as both positive and negative regulators. Studies into the molecular basis for the actions of these molecules shows that they function in various ways, including: recruitment of positive or negative regulators into signalling networks, modulation of effector function by allosteric regulation of enzymatic activity, and by targeting other proteins for degradation. This review will focus on a number of adaptors that are important for lymphocyte function and emphasize the various ways in which these proteins carry out their essential roles.     

Adaptors, junction dynamics, and spermatogenesis

Adaptors are component proteins of junctional complexes in all epithelia, including the seminiferous epithelium of the mammalian testis. They recruit other regulatory and structural proteins to the site of both anchoring junctions (such as cell-cell actin-based adherens junctions [AJs], e.g., ectoplasmic specialization [ES] and tubulobulbar complex, which are both testis-specific cell-cell actin-based AJ types, and cell-cell intermediate filament-based desmosome-like junctions) and tight junctions (TJ). Furthermore, adaptors per se can be substrates and/or activators of kinases or phosphatases. As such, the integrity of cell junctions and the regulation of junction dynamics during spermatogenesis rely on adaptors for their ability to recruit and link different junctional components to the same site and to tether transmembrane proteins at both anchoring and TJ sites to the underlying cytoskeletons, such as the actin filaments, intermediate filaments, and microtubules. These protein-protein interactions are possible because adaptors are composed of conserved protein binding domains, which allow them to link to more than one structural or signaling protein, recruiting multi-protein complexes to the same site. Herein, we provide a timely review of adaptors recently found at the sites of AJ (e.g., ES) and TJ. In addition, several in vivo models that can be used to delineate the function of adaptors in the testis are described, and the role of adaptors in regulating junction dynamics pertinent to spermatogenesis is discussed.     

Phosphorylation of the LIR Domain of SCOC Modulates ATG8 Binding Affinity and Specificity

Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood.We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic.Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.     

Dynamic control of signaling by modular adaptor proteins

Adaptor proteins are composed exclusively of domains and motifs that mediate molecular interactions, and can thereby link signaling proteins such as activated cell-surface receptors to downstream effectors. Recent data supports the notion that adaptors are not simply coupling devices that hard-wire successive components of signaling pathways. Rather, they display highly dynamic properties that direct the flow of information through signaling networks. The binding activity of adaptors can be regulated by conformational reorganization, and by the cooperative association of domains within the same adaptor. Furthermore, an individual adaptor can deliver different outputs by utilizing distinct combinations of binding partners. Adaptors can also control the oligomerization of receptor signaling complexes, and the subcellular location and duration of signaling events, and act as coincidence detectors to enhance specificity in cellular responses.     

A mechanism for death receptor discrimination by death adaptors

The death domain and death effector domain are two common motifs that mediate protein-protein interactions between components of cell death signaling complexes. The mechanism by which these domains engage their binding partners has been explored by extensive mutagenesis of two death adaptors, FADD and TRADD, suggesting that a death adaptor can discriminate its intended binding partners from other proteins harboring similar motifs. Death adaptors are found to utilize one of two topologically conserved surfaces for protein-protein interaction, whether that partner is another adaptor or its cognate receptor. These surfaces are topologically related to the interaction between death domains observed in the x-ray crystal structure of the Drosophila adaptor Tube bound to Pelle kinase. Comparing the topology of protein-protein interactions for FADD complexes to TRADD complexes reveals that FADD uses a Tube-like surface in each of its death motifs to engage either CD95 or TRADD. TRADD reverses these roles, employing a Pelle-like surface to interact with either receptor TNFR1 or adaptor FADD. Since death adaptors display a Tube-like or Pelle-like preference for engaging their binding partners, Tube/Pelle-like pairing provides a mechanism for death adaptor discrimination of death receptors.     

APP intracellular domain formation and unaltered signaling in the presence of familial Alzheimer's disease mutations

One of the cardinal neuropathological findings in brains from Alzheimer's disease (AD) patients is the occurrence of amyloid beta-peptide (Abeta) deposits. The gamma-secretase-mediated intramembrane proteolysis event generating Abeta also results in the release of the APP intracellular domain (AICD), which may mediate nuclear signaling. It was recently shown that AICD starts at a position distal to the site predicted from gamma-secretase cleavage within the membrane. This novel site, the epsilon site, is located close to the inner leaflet of the membrane bilayer. The relationship between proteolysis at the gamma and epsilon sites has not been fully characterized. Here we studied AICD signaling in intact cells using a chimeric C99 molecule and a luciferase reporter system. We show that the release of AICD from the membrane takes place in a compartment downstream of the endoplasmic reticulum, is dependent on presenilin proteins, and can be inhibited by treatment with established gamma-secretase inhibitors. Moreover, we find that AICD signaling remains unaltered from C99 derivatives containing mutations associated with increased Abeta42 production and familial AD. These findings indicate that there are very similar routes for Abeta and AICD formation but that FAD-linked mutations in APP primarily affect gamma-secretase-mediated Abeta42 formation, and not AICD signaling.     

A novel motif in the yeast mitochondrial dynamin Dnm1 is essential for adaptor binding and membrane recruitment

To initiate mitochondrial fission, dynamin-related proteins (DRPs) must bind specific adaptors on the outer mitochondrial membrane. The structural features underlying this interaction are poorly understood. Using yeast as a model, we show that the Insert B domain of the Dnm1 guanosine triphosphatase (a DRP) contains a novel motif required for association with the mitochondrial adaptor Mdv1. Mutation of this conserved motif specifically disrupted Dnm1–Mdv1 interactions, blocking Dnm1 recruitment and mitochondrial fission. Suppressor mutations in Mdv1 that restored Dnm1–Mdv1 interactions and fission identified potential protein-binding interfaces on the Mdv1 β-propeller domain. These results define the first known function for Insert B in DRP–adaptor interactions. Based on the variability of Insert B sequences and adaptor proteins, we propose that Insert B domains and mitochondrial adaptors have coevolved to meet the unique requirements for mitochondrial fission of different organisms.     

Pik1-ing clathrin adaptors

Clathrin adaptor proteins are essential for clathrin-coated vesicle biogenesis, yet the mechanisms governing their recruitment and interactions remain incompletely defined. The clathrin adaptors Gga and AP-1 are now shown to be recruited sequentially to the trans-Golgi network in two waves of clathrin coat assembly, coupled by Pik1-mediated phosphatidylinositol-4-phosphate synthesis. These findings reveal mechanistic insights into the functional and regulatory relationships between these clathrin adaptors.     

Cooperative interactions between epigenetic modifications and their function in the regulation of chromosome architecture

Epigenetic information is encoded by DNA methylation and by covalent modifications of histone tails. While defined epigenetic modification patterns have been frequently correlated with particular states of gene activity, very little is known about the integration level of epigenetic signals. Recent experiments have resulted in the characterization of several epigenetic adaptors that mediate interactions between distinct modifications. These adaptors include methyl-DNA binding proteins, chromatin remodelling enzymes and siRNAs. Complex interactions between epigenetic modifiers and adaptors provide the foundation for the stability of epigenetic inheritance. In addition, they also provide an explanation for the long-range effects of epigenetic mechanisms. We propose that a major aspect of epigenetic regulation lies in the modification of chromosome architecture and that local changes in gene expression would be secondary consequences. This view is consistent with many results from recent genomic analyses.     

Endocytic sorting of transmembrane protein cargo

The accurate distribution and recycling of transmembrane proteins amongst the membrane-bound organelles of the cell is vital to ensure its correct functioning. Transmembrane protein cargo destined for clathrin-mediated endocytosis and transport along the endocytic pathway is sorted into transport vesicles by interactions with adaptors, which simultaneously link clathrin to the membrane. Clathrin adaptors recognize a variety of signals present in the cytoplasmic portions of cargo proteins; recent structural, biophysical and cell biological studies have elucidated new types of cargo-adaptor interactions and probed the molecular mechanisms regulating cargo selection and vesicle maturation. Here, we review this recent progress in the context of our existing knowledge of endocytic sorting mechanisms.     

Ligand-Dependent Activation of EphA4 Signaling Regulates the Proteolysis of Amyloid Precursor Protein Through a Lyn-Mediated Pathway

Alzheimer's disease is the most common dementia afflicting the elderly in modern society. This disease arises from the neurotoxicity elicited by abnormal aggregates of amyloid-β (Aβ) protein. Such aggregates form through the cleavage of amyloid precursor protein (APP) by β-secretase and the subsequent proteolysis of the APP C-terminal fragment (APP-βCTF or C99) by γ-secretase to yield Aβ and APP intracellular domain (AICD). Recent evidence suggests that C99 and AICD may exert harmful effects on cells, suggesting that the proteolytic products of APP, including Aβ, C99, and AICD, could play a pivotal role in neuronal viability. Here, we demonstrate that ligand-activated EphA4 signaling governs the proteostasis of C99, AICD, and Aβ, without significantly affecting γ-secretase activity. EphA4 induced accumulation of C99 and AICD through a Lyn-dependent pathway; activation of this pathway triggered phosphorylation of EphA4, resulting in positive feedback of C99 and AICD proteostasis. Inhibition of EphA4 by dasatinib, a receptor tyrosine kinase inhibitor, effectively suppressed C99 and AICD accumulation. Furthermore, EphA4 signaling controlled C99 and AICD proteolysis through the ubiquitin–proteasome system. In conclusion, we have identified an EphA4–Lyn pathway that is essential for the metabolism of APP and its proteolytic derivatives, thereby providing novel pharmacological targets for the development of anti-Aβ therapeutics for AD.     

Development of a ligand blot assay using biotinylated live cells

Adhesive interactions between cells are critical to a variety of processes, including host-pathogen relationships. The authors have developed a new technique for the observation of binding interactions in which molecules obtained from excised tissues are resolved by gel electrophoresis and transferred to a membrane. Biotinylated live cells are then kept in contact with that membrane, and their interactions with proteins of interest are detected by peroxidase-labeled streptavidin, followed by a biotin-streptavidin detection system. The adhesion proteins can eventually be identified by cutting the relevant band(s) and performing mass spectrometry or other amino acid-sequencing methods. The technique described here allows for the identification of both known and novel adhesion molecules capable of binding to live cells, among a complex mixture and without previous isolation or purification. This is especially important for the analysis of host-parasite interactions and may be extended to other types of cell-cell interactions.     

Regulation of activation-induced cell death of mature T-lymphocyte populations

Resting mature T lymphocytes are activated when triggered via their antigen-specific T-cell receptor (TCR) to elicit an appropriate immune response. In contrast, preactivated T cells may undergo activation-induced cell death (AICD) in response to the same signals. along with cell death induced by growth factor deprivation, AICD followed by the elimination of useless or potentially harmful cells preserves homeostasis, leads to the termination of cellular immune responses and ensures peripheral tolerance. T-cell apoptosis and AICD are controlled by survival cytokines such as interleukin-2 (IL-2) and by death factors such as tumor necrosis factor (TNF) and CD95 ligand (CD95L). In AICD-sensitive T cells, stimulation upregulates expression of one or several death factors, which in turn engage specific death receptors on the same or a neighboring cell. Death receptors are activated by oligomerization to rapidly assemble a number of adapter proteins and enzymes to result in an irreversible activation of proteases and nucleases that culminates in cell death by apoptosis. Increased knowledge of the molecular mechanisms that regulate AICD of lymphocytes opens new immunotherapeutic perspectives for the treatment of certain autoimmune diseases, and has implications in other areas such as transplantation medicine and AIDS research.     

Molecular basis of oligoubiquitin-dependent internalization of membrane proteins in Mammalian cells

Ubiquitination induced down-regulation of cell surface proteins by internalization and lysosomal targeting plays a fundamental role in cell physiology and pathogenesis of diseases. The molecular basis of a single ubiquitin (Ub) as an autonomous endocytic signal, the widely accepted mechanism, however, remains elusive in higher eukaryotes. Using Ub containing reporter proteins without signalling abilities, we present evidence that only multiple Ub moieties, linked either covalently or assembled as oligomers with an intact interface for recognition by Ub-interacting motifs (UIMs), are recognized by the endocytic machinery in vivo and associate with a subset of Ub-binding clathrin adaptors in vitro. Genetic and pharmacological approaches show that internalization of plasma membrane proteins harbouring multiple Ub moieties is clathrin-dependent, but caveolin-independent. Functional assays demonstrate the cargo-dependent involvement of eps15/15R and epsin, UIM containing clathrin adaptors, in the endocytosis of model proteins, CD4 and the activated beta(2)-adrenergic receptor complex, containing polymeric or oligomeric Ub. These results provide a paradigm for the clathrin-mediated uptake of ubiquitinated membrane proteins in mammalian cells, requiring the assembly of multiple UIM-Ub interactions to overcome the low affinity binding of mono-Ub to UIM.     

Dynamic regulation of cystic fibrosis transmembrane conductance regulator by competitive interactions of molecular adaptors

Disorganized ion transport caused by hypo- or hyperfunctioning of the cystic fibrosis transmembrane conductance regulator (CFTR) can be detrimental and may result in life-threatening diseases such as cystic fibrosis or secretory diarrhea. Thus, CFTR is controlled by elaborate positive and negative regulations for an efficient homeostasis. It has been shown that expression and activity of CFTR can be regulated either positively or negatively by PDZ (PSD-95/discs large/ZO-1) domain-based adaptors. Although a positive regulation by PDZ domain-based adaptors such as EBP50/NHERF1 is established, the mechanisms for negative regulation of the CFTR by Shank2, as well as the effects of multiple adaptor interactions, are not known. Here we demonstrate a physical and physiological competition between EBP50-CFTR and Shank2-CFTR associations and the dynamic regulation of CFTR activity by these positive and negative interactions using the surface plasmon resonance assays and consecutive patch clamp experiments. Furthermore whereas EBP50 recruits a cAMP-dependent protein kinase (PKA) complex to CFTR, Shank2 was found to be physically and functionally associated with the cyclic nucleotide phosphodiesterase PDE4D that precludes cAMP/PKA signals in epithelial cells and mouse brains. These findings strongly suggest that balanced interactions between the membrane transporter and multiple PDZ-based adaptors play a critical role in the homeostatic regulation of epithelial transport and possibly the membrane transport in other tissues.