title: Combining genetic perturbations and proteomics to examine kinase-phosphatase networks in Drosophila embryos.
abstract: Connecting phosphorylation events to kinases and phosphatases is key to understanding the molecular organization and signaling dynamics of networks. We have generated a validated set of transgenic RNA-interference reagents for knockdown and characterization of all protein kinases and phosphatases present during early Drosophila melanogaster development. These genetic tools enable collection of sufficient quantities of embryos depleted of single gene products for proteomics. As a demonstration of an application of the collection, we have used multiplexed isobaric labeling for quantitative proteomics to derive global phosphorylation signatures associated with kinase-depleted embryos to systematically link phosphosites with relevant kinases. We demonstrate how this strategy uncovers kinase consensus motifs and prioritizes phosphoproteins for kinase target validation. We validate this approach by providing auxiliary evidence for Wee kinase-directed regulation of the chromatin regulator Stonewall. Further, we show how correlative phosphorylation at the site level can indicate function, as exemplified by Sterile20-like kinase-dependent regulation of Stat92E.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address: rsopko@genetics.med.harvard.edu.
authors.forename: Richelle
authors.initials: R
authors.lastname: Sopko
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Boston, MA 02115, USA.
authors.forename: Marianna
authors.initials: M
authors.lastname: Foos
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Arunachalam
authors.initials: A
authors.lastname: Vinayagam
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhai
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Boston, MA 02115, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Binari
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Boston, MA 02115, USA.
authors.forename: Sakara
authors.initials: S
authors.lastname: Randklev
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Lizabeth A
authors.initials: LA
authors.lastname: Perkins
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Boston, MA 02115, USA. Electronic address: perrimon@receptor.med.harvard.edu.
authors.forename: Norbert
authors.initials: N
authors.lastname: Perrimon
mesh_terms: D000818:Animals; D004330:Drosophila / Q000196:embryology / Q000201:enzymology / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D053263:Gene Regulatory Networks; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D020543:Proteome / Q000235:genetics* / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D020543:Proteome; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases