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    A modified tandem affinity purification technique identifies that 14-3-3 proteins interact with Tiam1, an interaction which controls Tiam1 stability.

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    Authors
    Woodcock, Simon A
    Jones, Richard C
    Edmondson, Ricky D
    Malliri, Angeliki
    Affiliation
    Cell Signalling Group, Cancer Research UK Paterson Institute for Cancer Research, University of Manchester, Manchester, M20 4BX, United Kingdom. amalliri@picr.man.ac.uk
    Issue Date
    2009-12
    
    Metadata
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    Abstract
    The Rac-specific GEF (guanine-nucleotide exchange factor) Tiam1 has important functions in multiple cellular processes including proliferation, apoptosis and adherens junction maintenance. Here we describe a modified tandem affinity purification (TAP) technique that we have applied to specifically enrich Tiam1-containing protein complexes from mammalian cells. Using this technique in conjunction with LC-MS/MS mass spectrometry, we have identified additional Tiam1-interacting proteins not seen with the standard technique, and have identified multiple 14-3-3 family members as Tiam1 interactors. We confirm the Tiam1/14-3-3 protein interaction by GST-pulldown and coimmunoprecipitation experiments, show that it is phosphorylation-dependent, and that they colocalize in cells. The interaction is largely dependent on the N-terminal region of Tiam1; within this region, there are four putative phospho-serine-containing 14-3-3 binding motifs, and we confirm that two of them (Ser172 and Ser231) are phosphorylated in cells using mass spectrometry. Moreover, we show that phosphorylation at three of these motifs (containing Ser60, Ser172 and Ser231) is required for the binding of 14-3-3 proteins to this region of Tiam1. We show that phosphorylation of these sites does not affect Tiam1 activity; significantly however, we demonstrate that phosphorylation of the Ser60-containing motif is required for the degradation of Tiam1. Thus, we have established and proven methodology that allows the identification of additional protein-protein interactions in mammalian cells, resulting in the discovery of a novel mechanism of regulating Tiam1 stability.
    Citation
    A modified tandem affinity purification technique identifies that 14-3-3 proteins interact with Tiam1, an interaction which controls Tiam1 stability. 2009, 8 (12):5629-41 J. Proteome Res.
    Journal
    Journal of Proteome Research
    URI
    http://hdl.handle.net/10541/109339
    DOI
    10.1021/pr900716e
    PubMed ID
    19899799
    Type
    Article
    Language
    en
    ISSN
    1535-3907
    ae974a485f413a2113503eed53cd6c53
    10.1021/pr900716e
    Scopus Count
    Collections
    All Paterson Institute for Cancer Research

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