Cellular response to an external stimulus requires the appropriate cascade of signals from the cell surface to the nucleus. The integrated actions of both protein kinases and protein phosphatases govern the phosphorylation state of key intracellular proteins which are crucial to normal cellular signaling. In contrast to the protein serine/threonine kinases (PKs), which have been extensively studied in this context, much less attention has been given to the protein serine/threonine phosphatases (PPs). It is clear, however, that this group of proteins, especially nuclear PPs, play an integral role in the control of cell growth and differentiation. The catalytic subunits of two such phosphatases, PP2A and PPX, are highly homologous and presumably complexed with similar or related regulatory subunits. The subcellular distribution of the PP2A and PPX holoenzymes is, however, different since PP2A is predominantly cytosolic, whereas PPX is predominantly nuclear. The overall goals of this proposal are to determine the localization, regulation, and function of nuclear protein serine/threonine phosphatase 2A and X holoenzymes. A multidisciplinary approach employing biochemical, immunological, and molecular biology approaches will be used to accomplish these goals. A combination of independent experimental strategies, including tissue immunoblotting, immunofluorescence, subcellular fractionation and immunoblotting will be used to define the tissue distribution and subcellular localization of PP2A and PPX. Nuclear PP2A and PPX holoenzymes will be purified in order to establish the oligomeric composition of these enzymes. The mechanism(s) for regulation of PP2A and PPX will be elucidated by analyzing changes in the phosphorylation state of phosphatase subunits, changes in subcellular localization of phosphatase subunits, and/or changes in oligomeric structure of phosphatase holoenzymes. In parallel, three different, yet complementary, techniques are proposed for assaying phosphatase activity and function in intact cells. Finally, a combination of mutagenesis and molecular genetic strategies will be used to deduce the structural basis for regulation and nuclear localization of these enzymes. It is anticipated that the proposed studies, with their blend of predictably productive as well as high-risk efforts, will reveal the structure and regulation of nuclear phosphatases, their structural and regulatory relationship with cytosolic phosphatases, and their importance in nuclear events, including hormone-regulated transcription. Naturally, these studies will enhance our knowledge of the fundamental process of cell cycle control and nuclear transcription as well as the regulation of these processes by phosphorylation/dephosphorylation events. However, insights gained might also offer some molecular enlightenment of the defective nuclear events controlling perturbed development or oncogenesis. Finally, identification of the precise protein-protein interactions responsible for localization and regulation of phosphatase activity may suggest novel therapeutic strategies to foster or interfere with these processes in various pathological states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM051366-03S1
Application #
2648030
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1994-08-01
Project End
1998-07-31
Budget Start
1996-08-01
Budget End
1998-07-31
Support Year
3
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
LeNoue-Newton, Michele L; Wadzinski, Brian E; Spiller, Benjamin W (2016) The three Type 2A protein phosphatases, PP2Ac, PP4c and PP6c, are differentially regulated by Alpha4. Biochem Biophys Res Commun 475:64-9
Arora, Daleep K; Machhadieh, Baker; Matti, Andrea et al. (2014) High glucose exposure promotes activation of protein phosphatase 2A in rodent islets and INS-1 832/13 ?-cells by increasing the posttranslational carboxylmethylation of its catalytic subunit. Endocrinology 155:380-91
Mo, Shu-Ting; Chiang, Shang-Ju; Lai, Tai-Yu et al. (2014) Visualization of subunit interactions and ternary complexes of protein phosphatase 2A in mammalian cells. PLoS One 9:e116074
Mazalouskas, Matthew D; Godoy-Ruiz, Raquel; Weber, David J et al. (2014) Small G proteins Rac1 and Ras regulate serine/threonine protein phosphatase 5 (PP5)·extracellular signal-regulated kinase (ERK) complexes involved in the feedback regulation of Raf1. J Biol Chem 289:4219-32
Williams, Byron C; Filter, Joshua J; Blake-Hodek, Kristina A et al. (2014) Greatwall-phosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers. Elife 3:e01695
Jiang, Li; Stanevich, Vitali; Satyshur, Kenneth A et al. (2013) Structural basis of protein phosphatase 2A stable latency. Nat Commun 4:1699
Kamoun, Malek; Filali, Mohammed; Murray, Michael V et al. (2013) Protein phosphatase 2A family members (PP2A and PP6) associate with U1 snRNP and the spliceosome during pre-mRNA splicing. Biochem Biophys Res Commun 440:306-11
Watkins, Guy R; Wang, Ning; Mazalouskas, Matthew D et al. (2012) Monoubiquitination promotes calpain cleavage of the protein phosphatase 2A (PP2A) regulatory subunit ?4, altering PP2A stability and microtubule-associated protein phosphorylation. J Biol Chem 287:24207-15
Wang, Ning; Leung, Hung-Tat; Mazalouskas, Matthew D et al. (2012) Essential roles of the Tap42-regulated protein phosphatase 2A (PP2A) family in wing imaginal disc development of Drosophila melanogaster. PLoS One 7:e38569
LeNoue-Newton, Michele; Watkins, Guy R; Zou, Ping et al. (2011) The E3 ubiquitin ligase- and protein phosphatase 2A (PP2A)-binding domains of the Alpha4 protein are both required for Alpha4 to inhibit PP2A degradation. J Biol Chem 286:17665-71

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