Cyclic nucleotide-dependent protein kinases inhibit binding of 14-3-3 to the GTPase-activating protein Rap1GAP2 in platelets

GTPase-activating proteins are required to terminate signaling by Rap1, a small guanine nucleotide-binding protein that controls integrin activity and cell adhesion. Recently, we identified Rap1GAP2, a GTPase-activating protein of Rap1 in platelets. Here we show that 14-3-3 proteins interact with phosphorylated serine 9 at the N terminus of Rap1GAP2. Platelet activation by ADP and thrombin enhances serine 9 phosphorylation and increases 14-3-3 binding to endogenous Rap1GAP2. Conversely, inhibition of platelets by endothelium-derived factors nitric oxide and prostacyclin disrupts 14-3-3 binding. These effects are mediated by cGMP- and cAMP-dependent protein kinases that phosphorylate Rap1GAP2 at serine 7, adjacent to the 14-3-3 binding site. 14-3-3 binding does not change the GTPase-activating function of Rap1GAP2 in vitro. However, 14-3-3 binding attenuates Rap1GAP2 mediated inhibition of cell adhesion. Our findings define a novel crossover point of activatory and inhibitory signaling pathways in platelets.     

Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IV

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3–43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (K_D = 220 pM) and HER3-expressing tumor cells with EC₅₀ values in the low picomolar range (27 - 83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3–43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3–43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.     

ATP-dependent 6-phosphofructokinase from the hyperthermophilic bacterium Thermotoga maritima: characterization of an extremely thermophilic, allosterically regulated enzyme

The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic bacterium Thermotoga maritimawas purified 730-fold to homogeneity. The enzyme is a 140-kDa homotetramer composed of 34 kDa subunits. Kinetic constants were determined for all substrates in both reaction directions at pH 7 and at 75 degrees C. Rate dependence (forward reaction) on fructose 6-phosphate (F-6-P) showed sigmoidal kinetics with a half-maximal saturation constant ( S(0.5)) of 0.7 mM and a Hill coefficient of 2.2. The apparent K(m) for ATP was 0.2 mM and the apparent V(max) value was about 360 U/mg. The enzyme also catalyzed in vitro the reverse reaction with an apparent K(m) for fructose 1,6-bisphosphate and ADP of 7.6 mM and 1.4 mM, respectively, and an apparent V(max) of about 13 U/mg. Divalent cations were required for maximal activity; Mg(2+), which was most effective, could partially be replaced by Mn(2+) and Fe(2+). Enzyme activity was allosterically regulated by classical effectors of ATP-PFKs of Eukarya and Bacteria; it was activated by ADP and inhibited by PEP. The enzyme had a temperature optimum of 93 degrees C and showed a significant thermostability up to 100 degrees C. Using the N-terminal amino acid sequence of the subunit, the pfk gene coding for ATP-PFK was identified and functionally overexpressed in Escherichia coli. The purified recombinant ATP-PFK had identical kinetic and allosteric properties as the native enzyme purified from T. maritima. The deduced amino acid sequence showed high sequence similarity to members of the PFK-A family. In accordance with its allosteric properties, ATP-PFK of T. maritima contained the conserved allosteric effector-binding sites for ADP and PEP.     

A new species of <Emphasis Type="Italic">Laboulbenia</Emphasis> and new records from Panama

Species of Laboulbenia on ground beetles (Coleoptera, Carabidae) collected in a mountain rainforest in Western Panama are described and illustrated. A new species of Laboulbenia on carabids of the genus Platynus (Platynini) in Panama is proposed. It differs from the other species of Laboulbenia mainly by curved thalli and longitudinally twisted wall cells of the perithecia with lips oriented towards strongly branched appendages. L. decipiens, L. pseudomasei, L. subpunctata, and L. tenera are newly recorded for Panama. Only one species collected during the survey is already known for Panama, L. flagellata.     

TNF-α influences the lateral dynamics of TNF receptor I in living cells

In mammalian cells, inflammation is mainly mediated by the binding of tumor necrosis factor alpha to tumor necrosis factor receptor 1. In this study, we investigated lateral dynamics of TNF-R1 before and after ligand binding using high-density single-particle tracking in combination with photoactivated localization microscopy. Our single-molecule data indicates the presence of tumor necrosis factor receptor 1 with different mobilities in the plasma membrane, suggesting different molecular organizations. Cholesterol depletion led to a decrease of slow receptor species and a strong increase in the average diffusion coefficient. Moreover, as a consequence of tumor necrosis factor-alpha treatment, the mean diffusion coefficient moderately increased while its distribution narrowed. Based on our observation, we propose a refined mechanism on the structural arrangement and activation of tumor necrosis factor receptor 1 in the plasma membrane.     

Vertical distribution and dietary preferences of deep-sea copepods (Euchaetidae and Aetideidae; Calanoida) in the vicinity of the Antarctic Polar Front

Four Paraeuchaeta species and three aetideids were frequently encountered along 51°30′S in the Atlantic sector of the Southern Ocean. Paraeuchaeta antarctica was most abundant close to the Antarctic Polar Front. Within the genera Paraeuchaeta and Gaetanus, congeners usually partitioned the water column. Euchaetidae had high lipid (≤37% dry mass, DM in adult females) and wax ester contents (≤22% DM). Fatty acid composition of Paraeuchaeta spp. was dominated by monounsaturated moieties, especially 16:1(n-7) and 18:1(n-9), while fatty alcohols were mainly saturated. Surprisingly, only the bathypelagic P. barbata contained moderate amounts of 20:1(n-9) and 22:1(n-11) fatty acids (≤14%) and high levels of the respective fatty alcohols (≤50%), generally considered trophic biomarkers for calanid copepods as prey. Thus, herbivorous calanid copepods seem to be a readily available prey source at bathypelagic depths, indicating that their seasonal vertical migration provides a “trophic shortcut” from primary production at the surface to the interior of the ocean. Aetideidae also contained substantial levels of total lipid (14–36% DM), but wax esters contributed only up to 12% DM in copepodite stages C5 of Gaetanus spp., whereas other stages of Gaetanus and Aetideopsis minor only contained ≤6% DM of wax esters. The fatty acid compositions of Aetideidae were more balanced with 16:0, 18:1(n-9), 20:5(n-3), and 22:6(n-3) as important components, indicating a generally omnivorous feeding behaviour.