Loss of PTB or Negative Regulation of Notch mRNA Reveals Distinct Zones of Notch and Actin Protein Accumulation in Drosophila Embryo

Polypyrimidine Tract Binding (PTB) protein is a regulator of mRNA processing and translation. Genetic screens and studies of wing and bristle development during the post-embryonic stages of Drosophila suggest that it is a negative regulator of the Notch pathway. How PTB regulates the Notch pathway is unknown. Our studies of Drosophila embryogenesis indicate that (1) the Notch mRNA is a potential target of PTB, (2) PTB and Notch functions in the dorso-lateral regions of the Drosophila embryo are linked to actin regulation but not their functions in the ventral region, and (3) the actin-related Notch activity in the dorso-lateral regions might require a Notch activity at or near the cell surface that is different from the nuclear Notch activity involved in cell fate specification in the ventral region. These data raise the possibility that the Drosophila embryo is divided into zones of different PTB and Notch activities based on whether or not they are linked to actin regulation. They also provide clues to the almost forgotten role of Notch in cell adhesion and reveal a role for the Notch pathway in cell fusions

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17

<div><p>The respiratory epithelium is subject to continuous environmental stress and its responses to injury or infection are largely mediated by transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling cascades. Based on previous studies indicating involvement of ATP-dependent activation of the NADPH oxidase homolog DUOX1 in epithelial wound responses, the present studies were performed to elucidate the mechanisms by which DUOX1-derived H₂O₂ participates in ATP-dependent redox signaling and EGFR transactivation. ATP-mediated EGFR transactivation in airway epithelial cells was found to involve purinergic P2Y₂ receptor stimulation, and both ligand-dependent mechanisms as well as ligand-independent EGFR activation by the non-receptor tyrosine kinase Src. Activation of Src was also essential for ATP-dependent activation of the sheddase ADAM17, which is responsible for liberation and activation of EGFR ligands. Activation of P2Y₂R results in recruitment of Src and DUOX1 into a signaling complex, and transient siRNA silencing or stable shRNA transfection established a critical role for DUOX1 in ATP-dependent activation of Src, ADAM17, EGFR, and downstream wound responses. Using thiol-specific biotin labeling strategies, we determined that ATP-dependent EGFR transactivation was associated with DUOX1-dependent oxidation of cysteine residues within Src as well as ADAM17. In aggregate, our findings demonstrate that DUOX1 plays a central role in overall epithelial defense responses to infection or injury, by mediating oxidative activation of Src and ADAM17 in response to ATP-dependent P2Y₂R activation as a proximal step in EGFR transactivation and downstream signaling.</p> </div

ATP-mediated wound responses and EGFR activation depend on DUOX1.

<p>(A) H292 cells were pre-incubated with DUOX1- or DUOX2-targeted siRNA or with control NS siRNA for 72 hrs, and subsequently serum-starved overnight and subjected to scratch wounding for analysis of wound closure after 8 hrs (A), stimulated with exogenous ATP (100 µM) for 8 hrs for analysis of ATP-dependent increases in mRNA expression of MMP-9 or IL-8 (B), or stimulated with ATP (100 µM) or EGF (100 ng/ml) for 10 min for analysis for phosphorylated and unphosphorylated forms of EGFR, c-Src, or STAT3 (C). Representative blots from 2–3 separate experiments are shown. Efficiency of siRNA-dependent silencing of DUOX1 or DUOX2 was evaluated by RT-PCR (D).</p

ATP stimulation induces DUOX1-dependent cysteine oxidation in Src.

<p>(A) Serum-starved H292 cells were stimulated with ATP (100 µM) for indicated times and lysed in the presence of Iodoacetyl-LC-Biotin, followed by purification of biotin-labelled protein with NeutrAvidin agarose, and western blot analysis of biotinylated proteins and whole lysates for Src, reflecting Src-SH and total Src, respectively. (B) Densitometry analysis of Src-SH status from 3 separate experiments. *p<0.05 compared to untreated control. (C) Similar analysis of Src-SH status in H292-shDUOX1 or corresponding H292-CTL cells after 10-min stimulation with ATP (100 µM). (D) Densitometry analysis of Src-SH relative to total Src based on Western blot analysis in panel C. Mean values of 4 replicates from 2 independent experiments are shown. *p<0.05 compared to untreated control.</p

Schematic representation of proposed involvement of DUOX1 in ATP-mediated EGFR transactivation.

<p>ATP-mediated activation of P2Y₂R promotes Ca²⁺ and protein kinase C (PKC) and thereby activates DUOX1-dependent H₂O₂ production. DUOX1-derived H₂O₂ promotes activation of Src and ADAM-17 by cysteine oxidation (1), and Src activation also promotes ADAM-17 activity by phosphorylation and directly activates EGFR by phosphorylation at Y845 (2). In addition, ADAM-17 activation promotes EGFR ligand shedding resulting in further EGFR activation (3). Activated EGFR can in turn promote activation of Src and ADAM-17 in a feed-forward mechanism to enhance EGFR signaling and downstream pathways such as ERK1/2 and STAT3 (4).</p

ATP-mediated EGFR phosphorylation is both ligand- and Src-dependent.

<p>(A) Confluent H292 cells were serum-starved overnight and pre-treated with α-EGFR mAb (4 µg/ml) for 30 min prior to treatment with ATP (100 µM) or EGF (100 ng/ml) for 10 min, and lysates were analyzed by Western blot using p-EGFR and total EGFR antibodies. (B) H292 cells were pre-incubated with c-Src siRNA or control non-specific (NS) siRNA for 72 hrs, and subsequently serum-starved overnight and stimulated with ATP or EGF, and cell lysates were analyzed for phosphorylated EGFR and total EGFR, c-Src and β-actin. Representative blots from 2–3 separate experiments are shown.</p

ATP stimulation induced DUOX1-dependent oxidation of ADAM-17 at the cell surface.

<p>(A) Confluent H292-shDUOX1 or corresponding H292-CTL cells were stimulated with ATP (10 min; 100 µM), and surface proteins were labeled with sulfo-NHS-biotin or MPB for labeling of surface NH₂ groups or cysteine-SH residues, respectively. Avidin-purified biotinylated proteins were probed for ADAM-17 by western blot. Representative blot of 2 separate experiments are shown. Densitometry analysis of SH labeling relative to NH₂ labeling of upper 120 kD bands (B) and lower 100 kD bands (C) are shown from 4 replicates from 2 independent experiments. *p<0.05 compared to untreated control.</p