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).
Highlights
Identification of protein-protein interactions is crucial for unraveling cellular processes and biochemical mechanisms of signal transduction
The protein A moiety of the tandem affinity purification (TAP) tag might interact with mouse circulating IgGs, no side effects such as toxicity or autoimmune reactions were observed in the transgenic lines expressing TAP-tagged 14-3-3
The expression level of TAP-14-3-3 is in the same range as the endogenous brain expression of 14-3-3 proteins avoiding problems typically associated with overexpression that can lead to non-physiological interactions (Fig. 1D)
Summary
CDNA Cloning and Transgenic Vector Engineering—Expression vectors were generated by site-specific recombination (Gateway system, Invitrogen) of PCR-amplified ORFs into TAP-, hemagglutinin (HA) epitope-, myelocytomatosis (Myc) oncogene epitope-, yellow fluorescent protein (YFP)-, or cyan fluorescent protein (CFP)-tagged versions of the Moloney murine leukemia virus-based vector pZome (Cellzome; available from Euroscarf GmbH). Supernatants containing cytoplasmic proteins were stored, and the pellets, containing the unbroken nuclei, were incubated in LBA buffer (50 mM Tris-HCl (pH 7.5), 0.5% Nonidet P-40, 150 mM NaCl, 10% glycerol, 10 mM NaPPi, 20 mM NaF, 1 mM sodium orthovanadate, 1ϫ Complete) for 10 min at 4 °C. Rac and Cdc Activated Pulldown Assays—Transfected HeLa cells were starved for 48 h, washed two times with chilled TBS buffer, and lysed in RPL buffer (50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 200 mM NaCl, 5 mM MgCl2, 1 mM DTT, 10% glycerol, 20 mM NaF, 1 mM sodium orthovanadate, 1ϫ Complete EDTA-free). Immunoblotting and Immunoprecipitation—For immunoprecipitation, 20 l of protein A-Sepharose beads (Amersham Biosciences) were washed with PBS and incubated with 10 l of anti-HA (12CA5; Roche Applied Science) or 4 l of anti-Myc (9E10; Santa Cruz Biotechnologies, Santa Cruz, CA) antibodies for 1 h at room temperature. Quired using an epifluorescence microscope (Zeiss, Goettingen, Germany) equipped with a digital camera (SpotRT; Diagnostic Instruments, Sterling Heights, MI) and analyzed with MetaMorph software (Visitron, Munich, Germany)
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