Reversible protein phosphorylation, the best studied post-translational modification, regulates most cellular processes, including signaling, migration, cell cycle progression, DNA damage repair, stress response and modulaton of the activities of metabolic enzymes. Therefore, it has emerged as a key therapeutic target in diseases in which these processes are deregulated. Unlike kinases, protein phosphatase 1 (PP1) is a promiscuous enzyme that gains its substrate specificity from a large group of “regulatory subunits” with which it associates to form a range of holoenzyme complexes targeted to specific subcellular localizations and substrates. Inhibition of a specific dephosphorylation event therefore relies on targeting the regulatory rather than the catalytic subunit. The present study uses GFP as a molecular reporter to assess the localization of PP1c and identify the underlying binding events that govern it via a combination of fluorescence imaging, cellular fractionation, affinity purification and quantitative mass spectrometry.
While there is some overlap in their targeting and intracellular roles, the three PP1 isoforms show distinct localizations based on relative preferences for particular regulatory subunits. In this study we assembled a comprehensive map of isoform- and compartment- specific phosphatase complexes in three different cultured human cell lines, using the data to extrapolate, with confidence, the distribution of each PP1 isoform between a large pool of known/predicted and novel regulatory subunits. Network analysis also highlighted key multiprotein complexes to which PP1 is targeted by these regulatory subunits, and identified a novel regulatory subunit that links phosphatase activity to regulation of protein degradation.
Our work confirmed that Mypt1, the regulatory subunit that targets PP1 activity to the myosin light chain, preferentially associates with the beta isoform of PP1c. We further demonstrated that they are in complex in both the cytoplasm and nucleus, and represent ~30% of the total PP1β holoenzyme complexes in both interaphase and mitotic cells. Further investigation of these complexes led to the discovery of Specc1 and Specc1L, which associate with Mypt1/PP1β via direct binding to Mypt1. Specc1/1L are microtubule binding proteins that can also associate with actin filaments, and we demonstrated that they mediate the distribution of Mypt1/PP1β complexes between these two cytoskeletal networks. Given that disruption of this balance has been implicated in disease states including cancer and hypertension, the Specc1/L family represents a novel therapeutic target for the regulation of Mypt1/PP1 activity.
With PP1 now emerging as a promising therapeutic target and the first PP1-targeted therapeutic drug, Sephin 1, in clinical trials, a better understanding of PP1’s in vivo distribution between holoenzyme complexes is essential. Our work establishes an initial “snapshot” of this distribution against which changes can be assessed, as we demonstrated here by showing its re-distribution in mitotic cells. Dynamic redistributions during specific cell processes such as differentiation or in response to perturbations or disease states can be assessed in a similar fashion in future, facilitating both identification of the relevant complexes and the design of specific strategies to target them therapeutically.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/36465 |
| Date | January 2017 |
| Creators | Mehta, Virja |
| Contributors | Trinkle-Mulcahy, Laura |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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