This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. PP2A and the related PP4 and PP6 proteins are evolutionary conserved and widely expressed serine/threonine phosphatases, involved in a broad array of cellular processes. PP2A is believed to generally function as a trimer, and substrate specificity is thought to be mediated by a number of different regulatory subunits. Although two regulatory subunits for mammalian PP4 have been identified, whether the PP4 and PP6 phosphatases function as trimeric complexes, and the identity of the regulatory and adaptor proteins taking part in these complexes, remained unknown. We set out to characterize the protein-protein interaction network surrounding PP2A, PP4 and PP6 in mammalian cells, using a combination of tandem affinity purification (TAP-tagging) and mass spectrometric analysis. Fusion proteins were stably expressed in HEK293 cells, and isolated via IgG-sepharose (to recover the ProteinA tag) and calmodulin-sepharose (for the calmodulin-binding domain). Recovered complexes were digested with trypsin, and directly analyzed by LC-MS. Identified binding partners were cloned in turn, and the process was repeated in an iterative manner, until a high density network of interactions was obtained. A high density (> 150 interactions) protein-protein interaction map was generated for the PP2A, PP4 and PP6 phosphatases; the map now contains 50 proteins. This analysis led to the high confidence identification of 16 new phosphatase-associated proteins (falling into 9 families), and to the discovery of many previously unknown interactions amongst characterized proteins. The amount and quality of data collected in this analysis has also allowed us to analyze the supramolecular assembly of the phosphatases. Interestingly, while the catalytic subunits for PP2A, PP4 and PP6 share a number of common interactors, they also sequester into highly specific modules. Novel PP4 and PP6-specific trimeric and multimeric complexes were uncovered, and higher order complexes for PP2A also became apparent. We have now cloned most of the components of this mammalian network and confirmed all of the interactions detected by TAP-tagging. In addition, we have carefully compared our mammalian network with an equivalent yeast network, initially extracted from the literature, and later confirmed by TAP-tag analysis in our laboratory. Like PP2A, the range of interactions and supramolecular structure of the PP4 isoenzyme and to some extent PP6 have been largely conserved throughout eukaryotic evolution.
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