Reversible protein phosphorylation is a fundamental posttranslational control mechanism in eukaryotes. Protein phosphatase 2A (PP2A) is one of the major classes of Ser/Thr phosphatases with likely thousands of phospho-protein substrates. The predominant form of PP2A is a heterotrimer of catalytic, scaffolding, and variable regulatory subunits. The identity of the regulatory subunit defines substrate specificity, subcellular localization, and regulation of PP2A by second messengers and reversible phosphorylation. The recent crystal structure of one of the several dozen possible PP2A heterotrimers offered a first glimpse into the molecular architecture and regulation of this fascinating enzyme. Still, substrates and physiological functions of specific PP2A heterotrimers remain poorly defined. Here, we propose to characterize two brain-specific PP2A regulatory subunits with relevance to neurodegenerative disorders, B'? and B?2. B'? acts as a gatekeeper in the regulation of tyrosine hydroxylase (TH) activity and catecholamine synthesis. Inhibitors of PP2A enzymes containing this subunit may therefore prove useful for the treatment of Parkinson's disease (PD). B?2 is derived from a gene mutated in spinocerebellar ataxia type 12 (SCA12). B?2 expression is pro-apoptotic, while B'? silencing protects neurons against a variety of insults. PP2A/B?2 inhibitors may thus form the basis for novel neuroprotective therapies.
The first aim of this application is exploratory in nature, while the remaining two are hypothesis-driven.
Aim 1 uses quantitative proteomics to identify interactors and substrates of PP2A/B'? and PP2A/B?2 in stable PC12 cell lines that inducibly express or silence these neuronal regulatory subunits.
Aims 2 and 3 investigate how these two neuron-specific PP2A holoenzymes are regulated and how they mediate disease-related substrate dephosphorylation in vitro, in cell culture, and in animal models. Based on recent PP2A hetereotrimer crystal structures, aim 2 examines structural determinants of TH dephosphorylation by B'?, regulation of PP2A/B'? by kinases, and regulation of dopamine synthesis by B'? in vivo. Lastly, aim 3 seeks to define the mechanisms by which B?2 promotes apoptosis. We hypothesize that PP2A/B?2 dephosphorylates and inactivates mitofusin-2, a protein mutated in a common peripheral neuropathy, Charcot-Marie-Tooth disease type 2A. These studies are significant in that they advance our understanding of the critical regulatory enzyme PP2A with the ultimate goal of better therapies for several neurodegenerative disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS043254-09
Application #
8010164
Study Section
Cellular and Molecular Biology of Neurodegeneration Study Section (CMND)
Program Officer
Mamounas, Laura
Project Start
2002-12-01
Project End
2012-11-30
Budget Start
2010-12-01
Budget End
2012-11-30
Support Year
9
Fiscal Year
2011
Total Cost
$303,629
Indirect Cost
Name
University of Iowa
Department
Pharmacology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Jin, Zhigang; Chung, Jin Wei; Mei, Wenyan et al. (2015) Regulation of nuclear-cytoplasmic shuttling and function of Family with sequence similarity 13, member A (Fam13a), by B56-containing PP2As and Akt. Mol Biol Cell 26:1160-73
Wilson, Theodore J; Slupe, Andrew M; Strack, Stefan (2013) Cell signaling and mitochondrial dynamics: Implications for neuronal function and neurodegenerative disease. Neurobiol Dis 51:13-26
Merrill, Ronald A; Slupe, Andrew M; Strack, Stefan (2013) N-terminal phosphorylation of protein phosphatase 2A/Bβ2 regulates translocation to mitochondria, dynamin-related protein 1 dephosphorylation, and neuronal survival. FEBS J 280:662-73
Oberg, Elizabeth A; Nifoussi, Shanna K; Gingras, Anne-Claude et al. (2012) Selective proteasomal degradation of the B'β subunit of protein phosphatase 2A by the E3 ubiquitin ligase adaptor Kelch-like 15. J Biol Chem 287:43378-89
Ulrich, Jason D; Kim, Man-Su; Houlihan, Patrick R et al. (2012) Distinct activation properties of the nuclear factor of activated T-cells (NFAT) isoforms NFATc3 and NFATc4 in neurons. J Biol Chem 287:37594-609
Strack, Stefan; Cribbs, J Thomas (2012) Allosteric modulation of Drp1 mechanoenzyme assembly and mitochondrial fission by the variable domain. J Biol Chem 287:10990-1001
Slupe, Andrew M; Merrill, Ronald A; Strack, Stefan (2011) Determinants for Substrate Specificity of Protein Phosphatase 2A. Enzyme Res 2011:398751
Dickey, Audrey S; Strack, Stefan (2011) PKA/AKAP1 and PP2A/Bβ2 regulate neuronal morphogenesis via Drp1 phosphorylation and mitochondrial bioenergetics. J Neurosci 31:15716-26
Lu, Yuan; Zha, Xiang-ming; Kim, Eun Young et al. (2011) A kinase anchor protein 150 (AKAP150)-associated protein kinase A limits dendritic spine density. J Biol Chem 286:26496-506
Merrill, Ronald A; Dagda, Ruben K; Dickey, Audrey S et al. (2011) Mechanism of neuroprotective mitochondrial remodeling by PKA/AKAP1. PLoS Biol 9:e1000612

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