Tumor Suppressor Density-enhanced Phosphatase-1 (DEP-1) Inhibits the RAS Pathway by Direct Dephosphorylation of ERK1/2 Kinases

Density-enhanced phosphatase-1 (DEP-1) is a trans-membrane receptor protein-tyrosine phosphatase that plays a recognized prominent role as a tumor suppressor. However, the mechanistic details underlying its function are poorly understood because its primary physiological substrate(s) have not been firmly established. To shed light on the mechanisms underlying the anti-proliferative role of this phosphatase, we set out to identify new DEP-1 substrates by a novel approach based on screening of high density peptide arrays. The results of the array experiment were combined with a bioinformatics filter to identify eight potential DEP-1 targets among the proteins annotated in the MAPK pathway. In this study we show that one of these potential targets, the ERK1/2, is indeed a direct DEP-1 substrate in vivo. Pulldown and in vitro dephosphorylation assays confirmed our prediction and demonstrated an overall specificity of DEP-1 in targeting the phosphorylated tyrosine 204 of ERK1/2. After epidermal growth factor stimulation, the phosphorylation of the activation loop of ERK1/2 can be modulated by changing the concentration of DEP-1, without affecting the activity of the upstream kinase MEK. In addition, we show that DEP-1 contains a KIM-like motif to recruit ERK1/2 proteins by a docking mechanism mediated by the common docking domain in ERK1/2. ERK proteins that are mutated in the conserved docking domain become insensitive to DEP-1 de-phosphorylation. Overall this study provides novel insights into the anti-proliferative role of this phosphatase and proposes a new mechanism that may also be relevant for the regulation of density-dependent growth inhibition.DEP-1⁴ (also known as CD148, HPTPη, and PTPRJ) is a class III receptor protein-tyrosine phosphatase, characterized by eight fibronectin type III repeats within the extracellular domain, a trans-membrane region, and a single cytosolic catalytic domain (1, 2). DEP-1 is expressed in all human hematopoietic cell lineages and was shown to negatively regulate T cell activation. In addition, several epithelial cell types display DEP-1 on their cell membranes (3). Homozygous DEP-1 mutant mice die before embryonic day 11.5, displaying severe defects in vascular organization (4). Interestingly, DEP-1 expression levels were found to augment with increased cell density (2), suggesting a role for this tyrosine phosphatase in sensing cell-cell contacts and in density-dependent growth inhibition (5). Moreover, accumulating evidence supports a prominent role for DEP-1 as a tumor suppressor as it negatively regulates cell proliferation and is poorly expressed in many cancer cell lines (6–10). The observed anti-proliferative effect may be accounted for by the ability of DEP-1 to down-regulate growth factor signaling through the dephosphorylation of various receptor tyrosine kinases, such as PDGFR, VEGFR2, and MET (11–13), resulting in quenching of the downstream RAS-MAPK pathway. However, given the complex pleiotropic functions of DEP-1, it is also possible that additional regulatory circuits mediated by yet unknown DEP-1 substrates may play a functional role in contact inhibition and control of cell proliferation.A variety of in vivo and in vitro approaches has led us to propose a number of DEP-1 substrates as mediators of its function. These include PDGFR, p120 catenin (CTND1), hepatocyte growth factor receptor, SRC kinase, VEGFR2, phosphatidylinositol 3-kinase regulatory subunit α (P85A), and RET receptor kinase (5, 11–16).Here we report a novel, unbiased strategy based on the screening of high density phosphopeptide arrays for their ability to bind phosphatase trapping mutants. A large portion of the phosphoproteome could be explored by this approach, thus unveiling a long list of potential substrates. A selected list of potentially relevant substrates has been obtained by applying a bioinformatics context filter. In this study we report the detailed characterization of one of these substrates, and we propose that DEP-1 modulates the RAS pathway by directly dephosphorylating Tyr-204 of ERK1/2. In addition, we show that the efficient removal of the phosphate group from Tyr-204 requires the integrity of a docking site on the ERK1/2 proteins.     

EAACI position paper on occupational rhinitis

The present document is the result of a consensus reached by a panel of experts from European and non-European countries on Occupational Rhinitis (OR), a disease of emerging relevance which has received little attention in comparison to occupational asthma. The document covers the main items of OR including epidemiology, diagnosis, management, socio-economic impact, preventive strategies and medicolegal issues. An operational definition and classification of OR tailored on that of occupational asthma, as well as a diagnostic algorithm based on steps allowing for different levels of diagnostic evidence are proposed. The needs for future research are pointed out. Key messages are issued for each item.     

Umbelliferone aminoalkyl derivatives as inhibitors of human oxidosqualene-lanosterol cyclase

Human and murine lanosterol synthases (EC 5.4.99.7) were studied as targets of a series of umbelliferone aminoalkyl derivatives previously tested as inhibitors of oxidosqualene cyclases from other eukaryotes. Tests were carried out on cell cultures of human keratinocytes and mouse 3T3 fibroblasts incubated with radiolabeled acetate, and on homogenates prepared from yeast cells expressing human lanosterol synthase, incubated with radiolabeled oxidosqualene. In cell cultures of both human keratinocytes and mouse 3T3 fibroblasts, the observed inhibition of cholesterol biosynthesis was selective for oxidosqualene cyclase. The most active compounds bear an allylmethylamino chain in position-7 of the coumarin ring. The inhibition was critically dependent on the position and length of the inhibitor side chain, as well as on the type of aminoalkyl group inserted at the end of the same chain. Molecular docking analyses, carried out to clarify details of inhibitors/enzyme interactions, proved useful to explain the observed differences in inhibitory activities.     

Bayesian Modeling of the Yeast SH3 Domain Interactome Predicts Spatiotemporal Dynamics of Endocytosis Proteins

SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast two-hybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes.     

Helminth communities of loggerhead turtles (Caretta caretta) from Central and Western Mediterranean Sea: The importance of host's ontogeny

We investigated the factors providing structure to the helminth communities of 182 loggerhead sea turtles, Caretta caretta, collected in 6 localities from Central and Western Mediterranean. Fifteen helminth taxa (10 digeneans, 4 nematodes and 1 acanthocephalan) were identified, of which 12 were specialist to marine turtles; very low numbers of immature individuals of 3 species typical from fish or cetaceans were also found. These observations confirm the hypothesis that phylogenetic factors restrict community composition to helminth species specific to marine turtles. There were significant community dissimilarities between turtles from different localities, the overall pattern being compatible with the hypothesis that parasite communities reflect the ontogenetic shift that juvenile loggerheads undergo from oceanic to neritic habitats. The smallest turtles at the putative oceanic, pelagic-feeding stage harboured only the 2 digenean species that were regionally the most frequent, i.e. Enodiotrema megachondrus and Calycodes anthos; the largest turtles at the putative neritic, bottom-feeding stage harboured 11 helminth taxa, including 3 nematode species that were rare or absent in turtles that fed partially on pelagic prey. Mean species richness per host was low (range: 1.60–1.89) and did not differ between localities. Variance ratio tests indicated independent colonization of each helminth species. Both features are expected in ectothermic and vagrant hosts living in the marine environment.     

Demonstration of long-range interactions in a PDZ domain by NMR, kinetics, and protein engineering

Understanding the basis of communication within protein domains is a major challenge in structural biology. We present structural and dynamical evidence for allosteric effects in a PDZ domain, PDZ2 from the tyrosine phosphatase PTP-BL, upon binding to a target peptide. The NMR structures of its free and peptide-bound states differ in the orientation of helix alpha2 with respect to the remainder of the molecule, concomitant with a readjustment of the hydrophobic core. Using an ultrafast mixing instrument, we detected a deviation from simple bimolecular kinetics for the association with peptide that is consistent with a rate-limiting conformational change in the protein (k(obs) approximately 7 x 10(3) s(-1)) and an induced-fit model. Furthermore, the binding kinetics of 15 mutants revealed that binding is regulated by long-range interactions, which can be correlated with the structural rearrangements resulting from peptide binding. The homologous protein PSD-95 PDZ3 did not display a similar ligand-induced conformational change.     

Ferric ions inhibit proteolytic processing of progastrin

The gastrointestinal hormone gastrin is generated from an 80 amino acid precursor (progastrin) by cleavage after dibasic residues by prohormone convertase 1. Phosphorylation of Ser₇₅ has previously been suggested, on the basis of indirect evidence, to inhibit cleavage of progastrin after Arg₇₃Arg₇₄. Gastrins bind two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated gastrins in vitro and in vivo. This study directly investigated the effect of iron binding and of serine phosphorylation on the cleavage of synthetic progastrin-derived peptides. The affinity of synthetic progastrin_55–80 for ferric ions, and the rate of cleavage by prohormone convertase 1, were not affected by phosphorylation of Ser₇₅. In contrast, in the presence of ferric ions the rate of cleavage of both progastrin_55–80 and phosphoSer₇₅progastrin_55–80 by prohormone convertase 1 was significantly reduced. Hence iron binding to progastrin may regulate processing and secretion in vivo, and regulation may be particularly important in diseases with altered iron homeostasis.