Expanding the Interactome of TES by Exploiting TES Modules with Different Subcellular Localizations

The
multimodular nature of many eukaryotic proteins underlies their temporal
or spatial engagement in a range of protein cocomplexes. Using the
multimodule protein testin (TES), we here report a proteomics approach
to increase insight in cocomplex diversity. The LIM-domain containing
and tumor suppressor protein TES is present at different actin cytoskeleton
adhesion structures in cells and influences cell migration, adhesion
and spreading. TES module accessibility has been proposed to vary
due to conformational switching and variants of TES lacking specific
domains target to different subcellular locations. By applying iMixPro
AP-MS (“intelligent Mixing of Proteomes”–affinity
purification–mass spectrometry) to a set of tagged-TES modular
variants, we identified proteins residing in module-specific cocomplexes.
The obtained distinct module-specific interactomes combine to a global
TES interactome that becomes more extensive and richer in information.
Applying pathway analysis to the module interactomes revealed expected
actin-related canonical pathways and also less expected pathways.
We validated two new TES cocomplex partners: TGFB1I1 and a short form
of the glucocorticoid receptor. TES and TGFB1I1 are shown to oppositely
affect cell spreading providing biological validity for their copresence
in complexes since they act in similar processes.