Transgenic mouse proteomics identifies new 14-3-3-associated proteins involved in cytoskeletal rearrangements and cell signaling

Mol Cell Proteomics. 2006 Dec;5(12):2211-27. doi: 10.1074/mcp.M600147-MCP200. Epub 2006 Sep 6.

Abstract

Identification of protein-protein interactions is crucial for unraveling cellular processes and biochemical mechanisms of signal transduction. Here we describe, for the first time, the application of the tandem affinity purification (TAP) and LC-MS method to the characterization of protein complexes from transgenic mice. The TAP strategy developed in transgenic mice allows the emplacement of complexes in their physiological environment in contact with proteins that might only be specifically expressed in certain tissues while simultaneously ensuring the right stoichiometry of the TAP protein versus their binding partners and represents a novelty in proteomics approaches used so far. Mouse lines expressing TAP-tagged 14-3-3zeta protein were generated, and protein interactions were determined. 14-3-3 proteins are general regulators of cell signaling and represent up to 1% of the total brain protein. This study allowed the identification of almost 40 novel 14-3-3zeta-binding proteins. Biochemical and functional characterization of some of these interactions revealed new mechanisms of action of 14-3-3zeta in several signaling pathways, such as glutamate receptor signaling via binding to homer homolog 3 (Homer 3) and in cytoskeletal rearrangements and spine morphogenesis by binding and regulating the activity of the signaling complex formed by G protein-coupled receptor kinase-interactor 1 (GIT1) and p21-activated kinase-interacting exchange factor beta (betaPIX).

MeSH terms

  • 14-3-3 Proteins / metabolism*
  • Animals
  • Carrier Proteins / metabolism
  • Cell Cycle Proteins / metabolism
  • Cells, Cultured
  • Cytoskeleton / metabolism*
  • HeLa Cells
  • Homer Scaffolding Proteins
  • Humans
  • Immunoprecipitation
  • Mice
  • Mice, Inbred Strains
  • Mice, Transgenic
  • Models, Biological
  • Multiprotein Complexes / isolation & purification
  • Nerve Tissue Proteins / isolation & purification
  • Nerve Tissue Proteins / metabolism*
  • Protein Binding
  • Protein Isoforms / metabolism
  • Protein-Serine-Threonine Kinases / metabolism
  • Proteomics / methods*
  • Signal Transduction

Substances

  • 14-3-3 Proteins
  • Carrier Proteins
  • Cell Cycle Proteins
  • Homer Scaffolding Proteins
  • Multiprotein Complexes
  • Nerve Tissue Proteins
  • Protein Isoforms
  • MARK3 protein, human
  • Mark2 protein, mouse
  • Protein-Serine-Threonine Kinases