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.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
3R01GM098482-04S1
Application #
8899933
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Gerratana, Barbara
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Brown University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Providence
State
RI
Country
United States
Zip Code
02912
Choy, Meng S; Hieke, Martina; Kumar, Ganesan Senthil et al. (2014) Understanding the antagonism of retinoblastoma protein dephosphorylation by PNUTS provides insights into the PP1 regulatory code. Proc Natl Acad Sci U S A 111:4097-102
Krishnan, Navasona; Koveal, Dorothy; Miller, Daniel H et al. (2014) Targeting the disordered C terminus of PTP1B with an allosteric inhibitor. Nat Chem Biol 10:558-66
Peti, Wolfgang; Nairn, Angus C; Page, Rebecca (2013) Structural basis for protein phosphatase 1 regulation and specificity. FEBS J 280:596-611
Kumar, Ganesan Senthil; Zettl, Heiko; Page, Rebecca et al. (2013) Structural basis for the regulation of the mitogen-activated protein (MAP) kinase p38* by the dual specificity phosphatase 16 MAP kinase binding domain in solution. J Biol Chem 288:28347-56
Peti, Wolfgang; Page, Rebecca (2013) Molecular basis of MAP kinase regulation. Protein Sci 22:1698-710
Minnebo, Nikki; Gornemann, Janina; O'Connell, Nichole et al. (2013) NIPP1 maintains EZH2 phosphorylation and promoter occupancy at proliferation-related target genes. Nucleic Acids Res 41:842-54