1/3 of all dephosphorylation reactions are controlled by ser/thr protein phosphatase 1 (PP1), which is present in all eukaryotic cells. PP1 is a single domain metal-binding protein, which lacks any intrinsic specificity. Rather, it is closely regulated by its interaction with >200 confirmed targeting proteins, which localize PP1 to distinct regions of the cell and modulate its substrate specificity. While 1000's of cell biology and biochemical reports describe key biological roles for PP1, only very few structural efforts have so far been successful. Here we describe a complete research plan to understand the regulation of PP1 in the nucleus. The presented research project uses a powerful integrated approach that combines NMR spectroscopy, X-ray crystallography and SAXS with biochemical and in vivo experiments to obtain novel insights into the molecular mechanisms that regulate PP1. Specifically we are focusing on two PP1-targeting proteins: 1) the nuclear inhibitor of PP1 (NIPP1) &2) the PP1 nuclear targeting subunit (PNUTS). More than 1/3 of the nuclear pool of PP1 forms a holoenzyme with NIPP1. Mice lacking NIPP1 are embryonic lethal and several substrate of NIPP1:PP1 are oncogenes. The NIPP1:PP1 holoenzyme regulates cell cycle progression, epigenetic silencing through chromatin remodeling and pre-mRNA splicing, among other essential biological functions. Thus it is of no surprise that deregulation of the NIPP1:PP1 holoenzyme leads to disease. PNUTS associates with chromatin and promotes chromosome de-condensation. It also controls cell death in response to cellular stresses through the post-translational modification of p53 and MDM2 and it plays essential role in the regulation of the retinoblastoma protein in response to cellular stress, critical processes for the regulation of cancer. In our combined efforts, we will: 1) determine the structures of the free form of these biologically critical PP1 regulators, 2) determine the structures of the PP1 holoenzymes and 3) determine how these complexes direct and regulate PP1 activity. Furthermore, we will leverage these protein and protein complex structures to elucidate, at a molecular level, the biological functions and modes of action of these key nuclear PP1 holoenzymes. The research described in this proposal leverages the extensive expertise of the investigators in the PP1 research field, as well as takes advantage of the best possible national and international collaborators. Furthermore, it has the preliminary data that demonstrates that this work will provide unique, novel insights into the molecular regulation of PP1.

Public Health Relevance

PP1 is a ubiquitous ser/thr protein phosphatase which regulates ~1/3 of all dephosphorylation reactions in higher eukaryotes. Despite 1000's of cell biological efforts, a detailed molecular picture of PP1's regulation is still missing. Therefore, our molecular insights into the regulation of PP1 in the nucleus are essential to establish PP1 into a powerful drug target.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function E Study Section (MSFE)
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Gerratana, Barbara
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Brown University
Schools of Medicine
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Machado, Luciana E S F; Page, Rebecca; Peti, Wolfgang (2018) 1H, 15N and 13C sequence specific backbone assignment of the vanadate inhibited hematopoietic tyrosine phosphatase. Biomol NMR Assign 12:5-9
Peti, Wolfgang; Page, Rebecca; Boura, Evzen et al. (2018) Structures of Dynamic Protein Complexes: Hybrid Techniques to Study MAP Kinase Complexes and the ESCRT System. Methods Mol Biol 1688:375-389
Machado, Luciana E S F; Critton, David A; Page, Rebecca et al. (2017) Redox Regulation of a Gain-of-Function Mutation (N308D) in SHP2 Noonan Syndrome. ACS Omega 2:8313-8318
Machado, Luciana E S F; Shen, Tun-Li; Page, Rebecca et al. (2017) The KIM-family protein-tyrosine phosphatases use distinct reversible oxidation intermediates: Intramolecular or intermolecular disulfide bond formation. J Biol Chem 292:8786-8796
Choy, Meng S; Swingle, Mark; D'Arcy, Brandon et al. (2017) PP1:Tautomycetin Complex Reveals a Path toward the Development of PP1-Specific Inhibitors. J Am Chem Soc 139:17703-17706
Choy, Meng S; Li, Yang; Machado, Luciana E S F et al. (2017) Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery. Mol Cell 65:644-658.e5
Wang, Xinru; Bajaj, Rakhi; Bollen, Mathieu et al. (2016) Expanding the PP2A Interactome by Defining a B56-Specific SLiM. Structure 24:2174-2181
Chen, Emily; Choy, Meng S; Petrényi, Katalin et al. (2016) Molecular Insights into the Fungus-Specific Serine/Threonine Protein Phosphatase Z1 in Candida albicans. MBio 7:
Peti, Wolfgang; Page, Rebecca (2016) NMR Spectroscopy to Study MAP Kinase Binding to MAP Kinase Phosphatases. Methods Mol Biol 1447:181-96
Sheftic, Sarah R; Page, Rebecca; Peti, Wolfgang (2016) Investigating the human Calcineurin Interaction Network using the ??LxVP SLiM. Sci Rep 6:38920

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