Phosphotyrosine interactome of the ErbB-receptor kinase family

Interactions between short modified peptide motifs and modular protein domains are central events in cell signal-transduction. We determined interaction partners to all cytosolic tyrosine residues of the four members of the ErbB-receptor family in an unbiased fashion by quantitative proteomics using pull-down experiments with pairs of phosphorylated and nonphosphorylated synthetic peptides. Each receptor had characteristic preferences for interacting proteins and most interaction partners had multiple binding sites on each receptor. EGFR and ErbB4 had several docking sites for Grb2, while ErbB3 was characterized by six binding sites for PI3K. We identified STAT5 as a direct binding partner to EGFR and ErbB4 and discovered new recognition motifs for Shc and STAT5. The overall pattern of interaction partners of EGFR and ErbB4 suggests similar roles during signaling through their respective ligands. Phosphorylation kinetics of several tyrosine resides was measured by mass spectrometry and correlated with interaction partner preference. Our results demonstrate that system-wide mapping of peptide-protein interactions sites is possible, and suggest shared and unique roles of ErbB-receptor family members in downstream signaling

Phosphotyrosine interactome of the ErbB-receptor kinase family

Interactions between short modified peptide motifs and modular protein domains are central events in cell signal-transduction. We determined interaction partners to all cytosolic tyrosine residues of the four members of the ErbB-receptor family in an unbiased fashion by quantitative proteomics using pull-down experiments with pairs of phosphorylated and nonphosphorylated synthetic peptides. Each receptor had characteristic preferences for interacting proteins and most interaction partners had multiple binding sites on each receptor. EGFR and ErbB4 had several docking sites for Grb2, while ErbB3 was characterized by six binding sites for PI3K. We identified STAT5 as a direct binding partner to EGFR and ErbB4 and discovered new recognition motifs for Shc and STAT5. The overall pattern of interaction partners of EGFR and ErbB4 suggests similar roles during signaling through their respective ligands. Phosphorylation kinetics of several tyrosine resides was measured by mass spectrometry and correlated with interaction partner preference. Our results demonstrate that system-wide mapping of peptide-protein interactions sites is possible, and suggest shared and unique roles of ErbB-receptor family members in downstream signaling

Reduced phosphorylation of brain insulin receptor substrate and Akt proteins in apolipoprotein-E4 targeted replacement mice

10.1038/srep03754Scientific Reports4

Numerical solutions of the compressible 3-D boundary-layer equations for aerospace configurations with emphasis on LFC

The application of stability theory in Laminar Flow Control (LFC) research requires that density and velocity profiles be specified throughout the viscous flow field of interest. These profile values must be as numerically accurate as possible and free of any numerically induced oscillations. Guidelines for the present research project are presented: develop an efficient and accurate procedure for solving the 3-D boundary layer equation for aerospace configurations; develop an interface program to couple selected 3-D inviscid programs that span the subsonic to hypersonic Mach number range; and document and release software to the LFC community. The interface program was found to be a dependable approach for developing a user friendly procedure for generating the boundary-layer grid and transforming an inviscid solution from a relatively coarse grid to a sufficiently fine boundary-layer grid. The boundary-layer program was shown to be fourth-order accurate in the direction normal to the wall boundary and second-order accurate in planes parallel to the boundary. The fourth-order accuracy allows accurate calculations with as few as one-fifth the number of grid points required for conventional second-order schemes

Natural enemies of rice leaf folder Cnaphalocrocis medinalis (Guenée) —a critical review

A chronological review of the biological control agents of rice leaf folder, Cnaphafalocrocis medinalis Guenée, including the stage of host attacked in different countries, their activity and suggestions for future. The rice leaf folder, Cnaphalocrocis medinalis Guenée, which was a pest of minor importance till the last decade, recently has assumed major importance in many rice ecosystem of Asia, the South Pacific Islands, Hawaii and Australia (Hirao, 1982). Increasingly frequent outbreaks of this pest (Table I) in recent years have been attributed to the introduction of modern rice technologies, namely: Continuous and overlapping cropping (Rajamma and Das, 1969); introduction of broad leaf dwarf, high tillering. fertilizer responsive susceptible rice varieties (Gargav and Katiyar, 1971; Dorge et al., 1972; Patel, 1975) and increased use of nitrogenous fertilizers (Michael Raj and Morachan, 1973; Chandragiri et al., 1974; Subraiah and Morachan, 1974; Dhaliwal et al., 1979; Chantaraprapha el al., 1980; Miynhara, 1981). In addition shading which causes the rice plants to grow tall with reduced silica content may make plants more susceptible (Chelliah, 1983). However, no detailed information is available as to how these factors bring about favourable conditions for larval growth and survival and or cause enhanced fecundity of the adults

A Three-Component Gene Expression System and Its Application for Inducible Flavonoid Overproduction in Transgenic Arabidopsis thaliana

Inducible gene expression is a powerful tool to study and engineer genes whose overexpression could be detrimental for the host organisms. However, only limited systems have been adopted in plant biotechnology. We have developed an osmotically inducible system using three components of plant origin, RD29a (Responsive to Dehydration 29A) promoter, CBF3 (C-repeat Binding Factor 3) transcription factor and cpl1-2 (CTD phosphatase-like 1) mutation. The osmotic stress responsible RD29a promoter contains the CBF3 binding sites and thus RD29A-CBF3 feedforward cassette enhances induction of RD29a promoter under stress. The cpl1-2 mutation in a host repressor CPL1 promotes stress responsible RD29a promoter expression. The efficacy of this system was tested using PAP1 (Production of Anthocyanin Pigment 1) transgene, a model transcription factor that regulates the anthocyanin pathway in Arabidopsis. While transgenic plants with only one or two of three components did not reproducibly accumulate anthocyanin pigments above the control level, transgenic cpl1 plants containing homozygous RD29a-PAP1 and RD29a-CBF3 transgenes produced 30-fold higher level of total anthocyanins than control plants upon cold treatment. Growth retardation and phytochemical production of transgenic plants were minimum under normal conditions. The flavonoid profile in cold-induced transgenic plants was determined by LC/MS/MS, which resembled that of previously reported pap1-D plants but enriched for kaempferol derivatives. These results establish the functionality of the inducible three-component gene expression system in plant metabolic engineering. Furthermore, we show that PAP1 and environmental signals synergistically regulate the flavonoid pathway to produce a unique flavonoid blend that has not been produced by PAP1 overexpression or cold treatment alone

Biological and clinical evidence for somatic mutations in BRCA1 and BRCA2 as predictive markers for olaparib response in high-grade serous ovarian cancers in the maintenance setting

To gain a better understanding of the role of somatic mutations in olaparib response, next-generation sequencing (NGS) of BRCA1 and BRCA2 was performed as part of a planned retrospective analysis of tumors from a randomized, double-blind, Phase II trial (Study 19; D0810C00019; NCT00753545) in 265 patients with platinum-sensitive high-grade serous ovarian cancer. BRCA1/2 loss-of-function mutations were found in 55% (114/209) of tumors, were mutually exclusive, and demonstrated high concordance with Sanger-sequenced germline mutations in matched blood samples, confirming the accuracy (97%) of tumor BRCA1/2 NGS testing. Additionally, NGS identified somatic mutations absent from germline testing in 10% (20/209) of the patients. Somatic mutations had >80% biallelic inactivation frequency and were predominantly clonal, suggesting that BRCA1/2 loss occurs early in the development of these cancers. Clinical outcomes between placebo- and olaparib-treated patients with somatic BRCA1/2 mutations were similar to those with germline BRCA1/2 mutations, indicating that patients with somatic BRCA1/2 mutations benefit from treatment with olaparib

Trapping DNA Replication Origins from the Human Genome

Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins’ structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using two different origin-trapping methods (i.e., UV- or chemical crosslinker-treatment and cell synchronization in early S phase using DNA replication inhibitors). Single-stranded DNAs (of 0.5–3 kb) that accumulated after such treatments were labeled with bromodeoxyuridine (BrdU). BrdU-labeled DNA was immunopurified after fractionation by alkaline sucrose density gradient centrifugation and cloned by complementary-strand synthesis and PCR amplification. Competitive PCR revealed an increased abundance of DNA derived from known replication origins (c-myc and lamin B2 genes) in the nascent DNA fractions from the UV-treated or crosslinked cells. Nucleotide sequences of 85 and 208 kb were obtained from the two libraries (I and II) prepared from the UV-treated log-phase cells and early S phase arrested cells, respectively. The libraries differed from each other in their G+C composition and replication-related motif contents, suggesting that differences existed between the origin fragments isolated by the two different origin-trapping methods. The replication activities for seven out of 12 putative origin loci from the early-S phase cells were shown by competitive PCR. We mapped 117 (library I) and 172 (library II) putative origin loci to the human genome; approximately 60% and 50% of these loci were assigned to the G-band and intragenic regions, respectively. Analyses of the flanking sequences of the mapped loci suggested that the putative origin loci tended to associate with genes (including conserved sites) and DNase I hypersensitive sites; however, poor correlations were found between such loci and the CpG islands, transcription start sites, and K27-acetylated histone H3 peaks