A modified tandem affinity purification technique identifies that 14-3-3 proteins interact with Tiam1, an interaction which controls Tiam1 stability.
Affiliation
Cell Signalling Group, Cancer Research UK Paterson Institute for Cancer Research, University of Manchester, Manchester, M20 4BX, United Kingdom. amalliri@picr.man.ac.ukIssue Date
2009-12
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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 ResearchDOI
10.1021/pr900716ePubMed ID
19899799Type
ArticleLanguage
enISSN
1535-3907ae974a485f413a2113503eed53cd6c53
10.1021/pr900716e
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