Subcellular distribution of PP1 isoforms in holoenzyme complexes

Abstract

Unlike its counterpart Ser/Thr kinases, the predominant Ser/Thr protein phosphatase 1 (PP1) is a promiscuous enzyme that gains its subcellular localization and substrate specificity from a large panel of regulatory proteins with which it associates in predominantly dimeric complexes. Inhibition of specific PP1-mediated dephosphorylation events relies on targeting the regulatory rather than the catalytic subunit, which in turn relies on a comprehensive understanding of the holoenzyme complexes that underlie its distribution throughout the cell. Proteomic, bioinformatic and biochemical screens have assembled lists of putative regulatory proteins, which have been studied to varying degrees. We took a non-biased approach to link steady-state localization to complexes, using a combination of fluorescence imaging, cellular fractionation and quantitative affinity purification/mass spectrometry (AP/MS) to map interactomes for PP1[a]/{beta}/{gamma} in 3 human cell lines. Comparing the distribution of each isoform between the pool of identified regulatory subunits highlighted key signaling pathways and identified c20orf27 as a novel PP1 regulatory protein. Steady-state association of a large fraction of PP1 with the evolutionarily conserved SDS22 was demonstrated, as was redistribution at the entry to mitosis. This is consistent with recent work suggesting that SDS22 acts as a PP1 sink from which it can be recruited as needed. Moving forward, this approach can be used to assess the redistribution of PP1 during other cellular processes or in response to perturbations or disease states, facilitating identification of the relevant complexes and the design of strategies to target them therapeutically.