Vertices with the Second Neighborhood Property in Eulerian Digraphs

The Second Neighborhood Conjecture states that every simple digraph has a vertex whose second out-neighborhood is at least as large as its first out-neighborhood, i.e. a vertex with the Second Neighborhood Property. A cycle intersection graph of an even graph is a new graph whose vertices are the cycles in a cycle decomposition of the original graph and whose edges represent vertex intersections of the cycles. By using a digraph variant of this concept, we prove that Eulerian digraphs which admit a simple dicycle intersection graph have not only adhere to the Second Neighborhood Conjecture, but have a vertex of minimum outdegree that has the Second Neighborhood Property.Comment: fixed an error in an earlier version and made structural change

Ubiquitination and Degradation of CFTR by the E3 Ubiquitin Ligase MARCH2 through Its Association with Adaptor Proteins CAL and STX6

<div><p>Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent binding, ubiquitination, and degradation of mature CFTR. We found that MARCH2 not only co-immunoprecipitated and co-localized with CAL and STX6, but its binding to CAL was also enhanced by STX6, suggesting a synergistic interaction. In vivo ubiquitination assays demonstrated the ubiquitination of CFTR by MARCH2, and overexpression of MARCH2, like that of CAL and STX6, led to a dose-dependent degradation of mature CFTR that was blocked by bafilomycin A1 treatment. A catalytically dead MARCH2 RING mutant was unable to promote CFTR degradation. In addition, MARCH2 had no effect on a CFTR mutant lacking the PDZ motif, suggesting that binding to the PDZ domain of CAL is required for MARCH2-mediated degradation of CFTR. Indeed, silencing of endogenous CAL ablated the effect of MARCH2 on CFTR. Consistent with its Golgi localization, MARCH2 had no effect on ER-localized ΔF508-CFTR. Finally, siRNA-mediated silencing of endogenous MARCH2 in the CF epithelial cell line CFBE-CFTR increased the abundance of mature CFTR. Taken together, these data suggest that the recruitment of the E3 ubiquitin ligase MARCH2 to the CAL complex and subsequent ubiquitination of CFTR are responsible for the CAL-mediated lysosomal degradation of mature CFTR.</p> </div

Model of ubiquitination of CFTR by the E3 ligase MARCH2.

<p>The CAL complex is formed by the PDZ domain-mediated interaction of CAL and CFTR and the coiled-coil domain-mediated interaction between STX6 and CAL. The CAL complex recruits the E3 ubiquitin ligase MARCH2 through an interaction between STX6 and MARCH2. The recruitment of MARCH2 leads to the association, ubiquitination and degradation of CFTR in the lysosome.</p

The PDZ binding motif of MARCH2 is not required for MARCH2 degradation of CFTR.

<p>(<b>A</b>) HEK293 cells were co-transfected with 3µg myc-MARCH2 or myc-ΔTPV-MARCH2 together with 3µg of HA-STX6 or several HA-CAL construct (see below), as indicated. After 48h, cell lysates were harvested and immunoprecipitated with an HA-affinity matrix. Cell lysates and immunoprecipitated materials were subjected to immunoblot analysis with the indicated antibodies. HA-CAL constructs used: HA-CAL (HA-tagged full length CAL), HA-CAL-PDZ (the HA-tagged PDZ domain of CAL) and HA-CAL-PCT (the HA-tagged PDZ and carboxyl domains of CAL) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068001#B18" target="_blank">18</a>] (<b>B</b>) HEK293 cells were co-transfected with 3µg CFTR (pCMV-CFTR) and increasing amounts of HA-ΔTPV-MARCH2 as indicated. After 48 h, cell lysates were harvested and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of at least three independent experiments.</p

Co-localization of MARCH2, STX6, and CAL.

<p>HEK293 cells grown on coverslips were co-transfected with 3µg GFP-STX6, 3µg myc-CAL, and 1µg HA-MARCH2. After 24 h, cells were fixed and subjected to indirect fluorescent immunocytochemical staining with an anti-myc rabbit polylonal antibody and an anti-HA mouse monoclonal antibody followed by goat anti-rabbit Alexa Fluor 647-conjugated secondary antibody and goat anti-mouse Alexa Fluor 594-conjugated secondary antibody. (<b>A</b>) GFP-STX6 appears in green. (<b>B</b>) HA-MARCH2 appears in red. (<b>C</b>) myc-CAL appears in magenta. (<b>D</b>) Overlay of GFP-STX6, HA-MARCH2 and myc-CAL. Data shown are representative of at least three independent experiments.</p

Degradation of wild-type CFTR, but not <i>Δ</i>TRL or Δ508 CFTR, by MARCH2.

<p>(<b>A</b>) HEK293 cells were co-transfected with 3 µg GFP-tagged wild-type (WT), ΔTRL, or Δ508 CFTR and 1µg HA-MARCH2 as indicated. After 48 h, cell lysates were harvested and subjected to immunoblot analysis. (<b>B</b>) Densitometric analysis of CFTR in (A). CFTR is normalized to GAPDH. Values are presented as mean +/- S.E. (<b>C</b>) HEK293 cells were first transfected with 20 nM CAL siRNA. After 24 h, the cells were transfected with 3µg pCMV-CFTR, with or without 1µg HA-MARCH2, as indicated. At 48 h after the second transfection, cell lysates were collected and subjected to SDS-PAGE and immunoblot analysis with the indicated antibodies. (<b>D</b>) HEK293 cells were first transfected with 20 nM MARCH2 siRNA. After 24 h, the cells were transfected with 3µg pCMV-CFTR, with or without 3µg HA-CAL, as indicated. At 48 h after the second transfection, cell lysates were collected and subjected to SDS-PAGE and immunoblot analysis with the indicated antibodies. Data shown are representative of at least three independent experiments.</p

In vivo ubiquitination of CFTR by MARCH2.

<p>(<b>A</b>) HEK293 cells were first co-transfected with 3µg CFTR (pCMV-CFTR) and 3µg His₆-ubiquitin. After 24 h, the cells were transfected again with 1µg HA-MARCH2 or the vector plasmid pRK5KS. Twenty-four hours after the second transfection, cell lysates were harvested under denaturing conditions (in a buffer containing 6 M guanidinium hydrochloride). Ubiquitinated proteins were affinity-purified with Ni^2+-NTA-agarose beads, eluted with Laemmli buffer, separated on SDS-PAGE, transferred to a PVDF membrane, and subjected to the immunoblot analysis with anti-CFTR mouse monoclonal antibody. (<b>B</b>) Same as in (A) except cells were treated with 400nM bafilomycin to inhibit lysosome activity 24 hours before harvest under denaturing conditions. Ubiquitinated proteins were affinity-purified with Ni^2+-NTA-agarose beads and subjected to the immunoblot analysis with anti-CFTR mouse monoclonal antibody. (<b>C</b>) Same as in (B) total cell lysates were subjected to immunoblot analysis with anti-CFTR antibody (<b>D</b>) The PVDF membrane in (C) was stripped and blotted with an anti-ubiquitinated proteins antibody FK2. Data shown are representative of at least three independent experiments. (<b>E</b>) HEK293 cells were co-transfected with 3µg CFTR (pCMV-CFTR) and 1µg HA-MARCH2 or HA-MARCH2 RING. Forty-eight hours after transfection, cell lysates were harvested and subjected to Western blot analysis with anti-CFTR mouse monoclonal antibody 217. Data shown are representative of at least three independent experiments.</p

Localization and interaction of MARCH2 with wild-type and <i>Δ</i>TRL CFTR.

<p>(<b>A</b>) CBFE cells grown on coverslips were co-transfected with 3µg GFP-CFTR or 3µg GFP-ΔTRL-CFTR and 0.5 µg HA-MARCH2. After 24h, the cells were fixed and subjected to indirect fluorescent immunocytochemical staining with an anti-HA mouse monoclonal antibody and anti-GFP rabbit polyclonal antibody. HA-MARCH2 appears red and GFP-CFTR green. (<b>B</b>) HEK293 cells were co-transfected with 3 µg GFP-CFTR or 3µg GFP-ΔTRL-CFTR and 1 µg HA-MARCH2, as indicated. After 48 h, cell lysates were harvested and immunoprecipitated with an anti-HA affinity matrix. Cell lysates and immunoprecipitated materials were subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of at least three independent experiments.</p

Co-localization and interaction of MARCH2 with STX6 and CAL.

<p>(<b>A</b>) HEK293 cells grown on coverslips were co-transfected with 3µg GFP-STX6 or 3µg GFP-CAL and 1µg HA-MARCH2. Twenty-four hours after transfection, cells were fixed and subjected to indirect fluorescent immunocytochemical staining with an anti-HA monoclonal antibody followed by Cy3-conjugated secondary antibody. HA-MARCH2 appears in red and GFP-STX6 and GFP-CAL are green. Arrow heads point to colocalization. (<b>B</b>) HEK293 cells were co-transfected with 3µg myc-CAL, 3µg myc-STX6, and 1µg HA-MARCH2 as indicated. After 48 h, cell lysates were harvested and immunoprecipitated with an anti-HA affinity matrix. Cell lysates and immunoprecipitated materials were subjected to immunoblot analysis with Myc or HA antibodies. Data shown are representative of at least three independent experiments.</p

Managerial perception on competition and strategic R&D decisions

The literature contends that managerial perception shapes firms' strategic decisions. Using a sample of high-tech firms in China, we examine the impact of managerial perception on competition (MPC), a learned response stemming from past market environment and measured by the textual analysis technique, on firms' strategic R&D decisions (R&D investments and R&D activities disclosure). We find that managers who perceived fierce competition from past market environment have strong incentive to enhance/sustain firms' competitive strength. Such incentive drives managers to calm on the surface (i.e., withholding R&D activities information) but strive underneath (i.e., making more R&D investments). Current product market competition, firms' market share leadership and CEO educational level moderate the effects of MPC on strategic R&D decisions due to their influence on managers' incentive or ability to compete. Finally, we find that firms with a high MPC obtain higher market valuations through engaging in strategic R&D decisions.</p