The Usp8 deubiquitination enzyme is post-translationally modified by tyrosine and serine phosphorylation

The ERBB1–ERBB4 receptors belong to a family of receptor tyrosine kinases that trigger a network of signaling pathways after ligand binding, thereby regulating cellular growth, differentiation and development. Ligand-induced signaling through ERBB1, also known as EGFR, is attenuated by the clathrin-dependent receptor-mediated endocytosis and RING E3-ligase Cbl-mediated receptor ubiquitination, which is followed by incorporation into multi-vesicular bodies (MVBs) and subsequent degradation in lysosomes. Before incorporation into MVBs, the EGFR is deubiquitinated by Usp8. We previously demonstrated that Usp8 is tyrosine phosphorylated in an EGFR- and SRC-kinase dependent manner. In the present study we show that overexpression of constitutively active SRC enhances constitutive and ligand-induced Usp8 tyrosine phosphorylation. We also show that enhanced endosomal recycling of the EGFR induced by TGFα stimulation is associated with decreased Usp8 tyrosine phosphorylation. We therefore hypothesize that tyrosine phosphorylation of Usp8 could regulate the function of Usp8. To identify Usp8 tyrosine phosphorylation site(s), we used Usp8 deletion constructs, site-directed mutagenesis of nine individual Usp8 tyrosine residues and mass spectrometry (MS) analysis. Our results demonstrate that the MIT-domain is necessary for ligand-induced tyrosine phosphorylation of Usp8 1–504. However, mutation of three MIT domain tyrosine residues did not abolish Usp8 tyrosine phosphorylation. Similar results were obtained upon mutation of six exposed tyrosine residues in the Rhod domain and linker region. Repeated MS analysis of both Usp8 WT and C748A mutants readily detected serine phosphorylation, including the S680 14–3–3 binding site, but did not reveal any phospho-tyrosine residues. Notably, mutation of the tyrosine residue in the Usp8 14–3–3 binding motif (Y679) did not abolish phosphoserine-dependent binding of 14–3–3 to Usp8. Our findings are most consistent with the model that MIT domain-dependent recruitment of Usp8 to endosomal membranes is important for low stoichiometry SRC-mediated tyrosine phosphorylation of multiple Usp8 tyrosines. Our findings demonstrate that Usp8 is a target for the post-translational serine and tyrosine phosphorylation, most likely characterized by low abundant tyrosine phosphorylation on multiple residues, and high abundant serine phosphorylation on several residues.     

RESPONSE OF MICROALGAL EPIPHYTE COMMUNITIES TO NITRATE ENRICHMENT IN AN EELGRASS (ZOSTERA MARINA) MEADOW1

The community structure and productivity of epiphytic microalgae on field populations of eelgrass (Zostera marina L.) from a high flow regime were characterized under water-column nitrate enrichment over a 30–d period during the autumn growing season for the macrophyte. Epiphyte communities in replicate low-nitrogen sites (LOW-N, median water-column nitrate concentrations below detection) were compared to communities in replicate N-enriched sites wherein nitrate was leached from clay pots filled with enriched agar (N-ENRICH, median concentration ca. 6 μM NO₃^?_-N; pots replaced at 8– to 12–d intervals). In experimental chambers, total epiphyte community productivity as ¹⁴C-bicarbonate uptake was determined from short-term (3–h) laboratory assays. Track light microscope-autoradiography enabled estimates of species-specific productivity for abundant algal taxa. After 6 d in the LOW-N and N-ENRICH communities, the crustose adnate red alga Sahlingia subintegra (Rosenvinge) Kornmann was dominant in terms of cell number and codominant in biovolume. Photosynthetic dinoflagellates, not previously reported as abundant in eelgrass epiphyte communities, were dominant in biovolume contribution after both 6 and 30 d in LOW-N communities. Nitrate enrichment stimulated the adnate monoraphid diatom Cocconeis placentula Ehr. but apparently inhibited dinoflagellates and the diatom Melosira sp. Total productivity of the epiphyte communities remained comparable in both the LOW-N and N-ENRICH sites. Shifts in community structure and species-specific productivity, however, indicated a controlling influence of nitrate supply on microalgal epiphytes in the field eelgrass community.     

A probability-based indicator of ecological condition

We introduce a new method for quantifying the ecological condition (C) of sites based on documented species’ responses to environmental stress. Preliminary research is needed to establish species-specific logistic functions, representing probabilities of finding individual species across an explicit reference gradient, ranging from maximally stressed (C = 0) to minimally stressed (C = 10) localities. Each function takes into account the species’ tolerance to stress, the species’ overall ubiquity, and the probability of detecting the species when it is present. Given a set of standardized species-specific functions, the ecological condition of any site can be derived by iteration, converging on the value of C that best “predicts” the species that are actually present. Species from multiple taxonomic groups can be included in the calculations, and results are not directly affected by species richness or sampling area. We demonstrate a successful application of this method for bird species assemblages in the U.S. portion of the Great Lakes coastal zone. Approximately, 28% of the bird species observed in the Eastern Deciduous Forest Ecological Province and 35% of the species in the Laurentian Mixed Forest Ecological Province showed strong relationships with a reference gradient of land cover variables. Functional stress–response relationships of these species can be used effectively to estimate ecological condition at new sites. The estimated condition based on bird species generally mirrors the reference condition, but deviations from the expected 1:1 relationship provide meaningful insights about ecological condition of the target areas. Sensitivity analysis using different numbers of species shows that our method is robust and can be applied consistently with 25–30 species exhibiting strong stress–response functions.     

Stimulation of pinocytosis in the macrophage by lymphocyte mediators: I. Description of the phenomenon

Macrophages incubated in supernatants from antigen or concanavalin A-stimulated lymphocytes will demonstrate enhanced pinocytosis of horseradish peroxidase (HRPO) compared with macrophages incubated in control supernatants. This readily reproducible increment in HRPO uptake is seen within 12 hr in suspension culture. This enhancement may represent an early cellular change preparatory to the increased effector function of activated macrophages.     

Use of microrespirometry to determine viability of immobilized Burkholderia cepacia G4

Embedding of Burkholderia cepacia G4 cells in a polyurethane-based foam decreased their culturability by more than four orders of magnitude. However, respiration rates of immobilized cells were at least 33-41% of unimmobilized cells. Embedded cells also degraded trichloroethylene. Therefore, respirometry is a more reliable indicator of viability of polyurethane immobilized bacteria than culturing methods.