Harnessing the activity of protein kinases and phosphatases is crucial for signal transduction by hormones and growth factors. Inhibitor-1 (I-1) represents the first mechanism identified for hormonal control of protein phosphatases. PKA phosphorylates and activates I-1 to suppress the activity of PP1, a major cellular protein serine/threonine phosphatase and amplify the cellular responses to the second messenger, cyclic AMP. Though identified as a regulator of glycogen metabolism in skeletal muscle, I-1 is widely expressed and has been implicated in many physiological processes. Our studies identified the growth arrest and DNA damage-inducible gene product, GADD34, as a novel protein scaffold that recruits PP1 and I-1 to regulate protein translation. The PP1-binding domain of GADD34 shares structural homology to the protein product of the Herpes Simplex Virus HSV-1 gamma-34.5 gene that generates a phosphatase that dephosphorylates the eukaryotic translation initiation factor, eIF2alpha, and facilitates protein synthesis. Our studies suggest that the PP1/GADD34/I-1 complex is a major eIF2alpha phosphatase in many mammalian tissues and is disassembled in response to cell stress, such as hypoxia and nutrient deprivation. This results in increased eIF2alpha phosphorylation and shut-down of protein synthesis. Persistent phosphorylation of eIF2alpha has also been linked to programmed cell death. The discovery of a mutation in the gene encoding the major human pancreatic eIF2alpha kinase in Wolcott-Rollins (WR) syndrome suggests that eIF2alpha phosphorylation-dephosphorylation controls insulin synthesis and the survival of pancreatic beta-cells. Thus, WR individuals develop early insulin-dependent diabetes and other serious disorders. These and other studies suggest that eIF2alpha phosphorylation functions as an important physiological mechanism for sensing glucose and nutrient availability. Thus, understanding the structure-function and regulation of the eIF2alpha phosphatase should provide new avenues for the treatment of viral infections, diabetes and other human diseases.
The Specific Aims of this project are: 1) Utilize genetic and biochemical assays to define the molecular basis for I-1 as a regulator of PP1 function, specifically the dephosphorylation of eIF2alpha and initiation of protein translation. 2) Define the role of GADD34 in the assembly of the mammalian eIF2alpha phosphatase and the transduction of physiological signals that regulate protein synthesis and dictate the switch between cell death and survival.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK052054-09
Application #
6819722
Study Section
Metabolism Study Section (MET)
Program Officer
Blondel, Olivier
Project Start
1997-01-01
Project End
2007-11-30
Budget Start
2004-12-01
Budget End
2007-11-30
Support Year
9
Fiscal Year
2005
Total Cost
$220,990
Indirect Cost
Name
Duke University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Brush, Matthew H; Shenolikar, Shirish (2008) Control of cellular GADD34 levels by the 26S proteasome. Mol Cell Biol 28:6989-7000
Gibbons, Jennifer A; Kozubowski, Lukasz; Tatchell, Kelly et al. (2007) Expression of human protein phosphatase-1 in Saccharomyces cerevisiae highlights the role of phosphatase isoforms in regulating eukaryotic functions. J Biol Chem 282:21838-47
Roadcap, David W; Brush, Matthew H; Shenolikar, Shirish (2007) Identification of cellular protein phosphatase-1 regulators. Methods Mol Biol 365:181-96
Gibbons, Jennifer A; Weiser, Douglas C; Shenolikar, Shirish (2005) Importance of a surface hydrophobic pocket on protein phosphatase-1 catalytic subunit in recognizing cellular regulators. J Biol Chem 280:15903-11
Brush, Matthew H; Guardiola, Amaris; Connor, John H et al. (2004) Deactylase inhibitors disrupt cellular complexes containing protein phosphatases and deacetylases. J Biol Chem 279:7685-91
Weiser, Douglas C; Sikes, Suzanne; Li, Shi et al. (2004) The inhibitor-1 C terminus facilitates hormonal regulation of cellular protein phosphatase-1: functional implications for inhibitor-1 isoforms. J Biol Chem 279:48904-14
Brush, Matthew H; Weiser, Douglas C; Shenolikar, Shirish (2003) Growth arrest and DNA damage-inducible protein GADD34 targets protein phosphatase 1 alpha to the endoplasmic reticulum and promotes dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. Mol Cell Biol 23:1292-303
Margolis, Seth S; Walsh, Susan; Weiser, Douglas C et al. (2003) PP1 control of M phase entry exerted through 14-3-3-regulated Cdc25 dephosphorylation. EMBO J 22:5734-45
Leach, Craig; Shenolikar, Shirish; Brautigan, David L (2003) Phosphorylation of phosphatase inhibitor-2 at centrosomes during mitosis. J Biol Chem 278:26015-20
Kang-Park, Maeng-Hee; Sarda, Meredith A; Jones, Katherine H et al. (2003) Protein phosphatases mediate depotentiation induced by high-intensity theta-burst stimulation. J Neurophysiol 89:684-90

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