Conserved cysteine and tryptophan residues of the endothelin-converting enzyme-1 CXAW motif are critical for protein maturation and enzyme activity

The neprilysin (NEP)/endothelin-converting enzyme (ECE) family of metalloproteases contains a highly conserved carboxyl-terminal tetrapeptide sequence, CXAW, where "C" is cysteine, "X" is a polar amino acid, "A" is an aliphatic residue, and "W" is tryptophan. Although this sequence strongly resembles a prenylation motif, human ECE-1 did not appear to be prenylated when labeled in vivo using various isoprenoid precursors in cell lines expressing ECE-1. We used site-directed mutagenesis to investigate the role of the CXAW motif and determined that the conserved cysteine residue of the CXAW motif in ECE-1, Cys(755), is critical for proper folding of the enzyme, its export from the endoplasmic reticulum, and its maturation in the secretory pathway. In addition, site-directed mutagenesis revealed that the conserved tryptophan residue of the sequence CEVW appears to be important for endoplasmic reticulum export and is essential for enzyme activity. Deletion of Trp(758) or substitution with alanine greatly slowed maturation of the enzyme, and resulted in more than a 90% loss of enzyme activity relative to the wild type. Conservative substitution of the tryptophan with phenylalanine did not reduce activity, whereas replacement with tyrosine, methionine, or leucine reduced enzyme activity by 50%, 75%, and 85%, respectively. Together, these data indicate that the conserved CEVW sequence does not serve as a prenylation signal and that both the conserved cysteine and tryptophan residues are necessary for proper folding and maturation of the enzyme. Furthermore, the conserved tryptophan appears to be critical for enzyme activity.     

Nudf, a fungal homolog of the human LIS1 protein, functions as a dimer in vivo

The NUDF protein is required for nuclear migration through the mycelium of the filamentous fungus Aspergillus nidulans. It is of particular interest, because it closely resembles a human protein, LIS1, that is required for development of the cerebral cortex. Both are approximately 50-kDa proteins with a short N-terminal predicted coiled coil and seven WD-40 domains in the C-terminal half of the molecule. They also interact with homologous proteins, suggesting that they may have similar biochemical functions. Here we describe the purification to homogeneity of NUDF protein in a single step from a cell-free extract of A. nidulans. We demonstrate that NUDF is a homodimer, that its dimerization occurs via the N-terminal coiled coil region of the molecule, and that it must be a dimer to support the growth of A. nidulans.     

Epidermal growth factor (EGF) receptor-dependent ERK activation by G protein-coupled receptors: a co-culture system for identifying intermediates upstream and downstream of heparin-binding EGF shedding

"Transactivation" of epidermal growth factor receptors (EGFRs) in response to activation of many G protein-coupled receptors (GPCRs) involves autocrine/paracrine shedding of heparin-binding EGF (HB-EGF). HB-EGF shedding involves proteolytic cleavage of a membrane-anchored precursor by incompletely characterized matrix metalloproteases. In COS-7 cells, alpha(2A)-adrenergic receptors (ARs) stimulate ERK phosphorylation via two distinct pathways, a transactivation pathway that involves the release of HB-EGF and the EGFR and an alternate pathway that is independent of both HB-EGF and the EGFR. We have developed a mixed culture system to study the mechanism of GPCR-mediated HB-EGF shedding in COS-7 cells. In this system, alpha(2A)AR expressing "donor" cells are co-cultured with "acceptor" cells lacking the alpha(2A)AR. Each population expresses a uniquely epitope-tagged ERK2 protein, allowing the selective measurement of ERK activation in the donor and acceptor cells. Stimulation with the alpha(2)AR selective agonist UK14304 rapidly increases ERK2 phosphorylation in both the donor and the acceptor cells. The acceptor cell response is sensitive to inhibitors of both the EGFR and HB-EGF, indicating that it results from the release of HB-EGF from the alpha(2A)AR-expressing donor cells. Experiments with various chemical inhibitors and dominant inhibitory mutants demonstrate that EGFR-dependent activation of the ERK cascade after alpha(2A)AR stimulation requires Gbetagamma subunits upstream and dynamin-dependent endocytosis downstream of HB-EGF shedding and EGFR activation, whereas Src kinase activity is required both for the release of HB-EGF and for HB-EGF-mediated ERK2 phosphorylation.     

Tob-mediated cross-talk between MARCKS phosphorylation and ErbB-2 activation

The biochemical path for the activation of ErbB-2 by PKC activator was investigated in MDA-MB-231 human breast cancer cells. We found that PMA-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) increased its binding with Tob that exerts an anti-proliferative effect through the binding with ErbB-2. The phosphorylation site domain (PSD) of MARCKS was relevant to its interaction with Tob. Decreased binding of Tob with ErbB-2 and subsequent activation of ErbB-2 were observed in MDA-MB-231 cells in response to PMA treatment. The present study proposes that MARCKS phosphorylation by PKC removes Tob from ErbB-2 by increasing its binding affinity with Tob, and thereby activates the ErbB-2 mediated signal transduction.     

Polymerized liposome assemblies: bifunctional macromolecular selectin inhibitors mimicking physiological selectin ligands

Monomeric sialyl Lewis(X) (sLe(x)) and sLe(x)-like oligosaccharides are minimal structures capable of supporting selectin binding in vitro. However, their weak binding interactions do not correlate with the high-affinity binding interactions witnessed in vivo. The polyvalent display of carbohydrate groups found on cell surface glycoprotein structures may contribute to the enhanced binding strength of selectin-mediated adhesion. Detailed biochemical analyses of physiological selectin ligands have revealed a complicated composition of molecules that bind to the selectins in vivo and suggest that there are other requirements for tight binding beyond simple carbohydrate multimerization. In an effort to mimic the high-affinity binding, polyvalent scaffolds that contain multicomponent displays of selectin-binding ligands have been synthesized. Here, we demonstrate that the presentation of additional anionic functional groups in the form of sulfate esters, on a polymerized liposome surface containing a multimeric array of sLe(x)-like oligosaccharides, generates a highly potent, bifunctional macromolecular assembly. This assembly inhibits L-, E-, and P-selectin binding to GlyCAM-1, a physiological ligand better than sLe(x)-like liposomes without additional anionic charge. These multivalent arrays are 4 orders of magnitude better than the monovalent carbohydrate. Liposomes displaying 3'-sulfo Lewis(X)-like oligosaccharides, on the other hand, show slight loss of binding with introduction of additional anionic functional groups for E- and P-selectin and negligible change for L-selectin. The ability to rapidly and systematically vary the composition of these assemblies is a distinguishing feature of this methodology and may be applied to the study of other systems where composite binding determinants are important for high-affinity binding.     

Toxicity of the herbicide glufosinate-ammonium to predatory insects and mites of Tetranychus urticae (Acari: Tetranychidae) under laboratory conditions

The toxicities of the herbicide glufosinate-ammonium to three predatory insect and two predatory mite species of Tetranychus urticae Koch were determined in the laboratory by the direct contact application. At a concentration of 540 ppm (a field application rate for weed control in apple orchards), glufosinate-ammonium was almost nontoxic to eggs of Amblyseius womersleyi Schicha, Phytoseiulus persimilis Athias-Henriot, and T. urticae but highly toxic to nymphs and adults of these three mite species, indicating that a common mode of action between predatory and phytophagous mites might be involved. In tests with predatory insects using 540 ppm, glufosinate-ammonium revealed little or no harm to larvae and pupae of Chrysopa pallens Rambur but was slightly harmful to eggs (71.2% mortality), nymphs (65.0% mortality), and adults (57.7% mortality) of Orius strigicollis Poppius. The herbicide showed no direct effect on eggs and adults of Harmonia axyridis (Pallas) but was harmful, slightly harmful, and harmless to first instars (100% mortality), fourth instars (51.1% mortality), and pupae (24.5% mortality), respectively. The larvae and nymphs of predators died within 12 h after treatment, suggesting that the larvicidal and nymphicidal action may be attributable to a direct effect rather than an inhibitory action of chitin synthesis. On the basis of our data, glufosinate-ammonium caused smaller effects on test predators than on T. urticae with the exception of P. persimilis, although the mechanism or cause of selectivity remains unknown. Glufosinate-ammonium merits further study as a key component of integrated pest management.