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Dev Cell. 2014 Oct 13;31(1):114-27. doi: 10.1016/j.devcel.2014.07.027. Epub 2014 Oct 2.

Combining genetic perturbations and proteomics to examine kinase-phosphatase networks in Drosophila embryos.

Author information

1
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address: rsopko@genetics.med.harvard.edu.
2
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Boston, MA 02115, USA.
3
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
4
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
5
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.

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.

PMID:
25284370
PMCID:
PMC4208667
DOI:
10.1016/j.devcel.2014.07.027
[Indexed for MEDLINE]
Free PMC Article

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