G protein signaling pathways are targeted by nearly half of all pharmaceuticals, and have revolutionized the treatment of cardiovascular disease and psychiatric disorders. However, G protein signaling must be carefully regulated to ensure proper responses. To identify new regulators of the G protein signaling pathway, we have systematically evaluated 870 essential genes in yeast, using a library of repressible-promoter strains. Knock- down expression revealed several dozen new proteins required for proper signaling. Included in this group are members of the SCF (Skp-Cullin-F-Box) ubiquitin ligase complex. We determined that SCF promotes degradation of the G protein ? subunit (Gpa1) in vivo and catalyzes Gpa1 ubiquitination in vitro. Further, we have shown that Gpa1 is phosphorylated by Elm1, that Gpa1 phosphorylation precedes ubiquitination, and that both modifications are dynamically regulated. In this proposal we will investigate additional targets of SCF ubiquitination and of additional kinase regulators of the G protein signaling pathway.
Aim 1 : Identify targets of the SCF ubiquitin ligase complex. Preliminary data indicate that SCF regulates the stability of at least four pathway components, in addition to Gpa1. We will test the hypothesis that SCF ubiquitinates multiple pathway components in a coordinated manner and in this way serves as a 'master regulator'of signal output.
Aim 2 : Elucidate G protein regulation by AMPK kinases. Elm1 phosphorylates Gpa1, thereby promoting its ubiquitination. Elm1, Sak1 and Tos3 are protein kinases that activate AMPK (AMP-activated protein kinase) in response to glucose depletion. We will now investigate the ability of all three AMPK kinases to regulate Gpa1 during glucose depletion. We will determine when and where Gpa1 is phosphoryated, and how phosphorylation alters G protein activity, both in vivo and in vitro.
Aim 3 : Identify pathway targets of Mps1, and identify new pathway regulators. Mps1 is a dual specificity protein kinase that we have shown dampens pheromone activation. We will identify pathway components targeted by Mps1, and determine how phosphorylation affects their activity. Finally we will establish how additional essential genes regulate cellular signaling function. Our efforts have revealed new components of the G protein signaling pathway in yeast. Mechanisms discovered in yeast are typically recapitulated in more complex organisms, so the activities elucidated here will likely apply as well to hormone and neurotransmitter function in humans.

Public Health Relevance

In this proposal we will define new ways that cells regulate their response to hormone signals. Detailed analysis of these processes will lay the foundation for understanding the causes and the most effective treatments of human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM059167-13A1
Application #
8296923
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Dunsmore, Sarah
Project Start
1999-06-01
Project End
2016-08-31
Budget Start
2012-09-15
Budget End
2013-08-31
Support Year
13
Fiscal Year
2012
Total Cost
$281,387
Indirect Cost
$91,387
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Hurst, Jillian H; Dohlman, Henrik G (2013) Dynamic ubiquitination of the mitogen-activated protein kinase kinase (MAPKK) Ste7 determines mitogen-activated protein kinase (MAPK) specificity. J Biol Chem 288:18660-71
Clement, Sarah T; Dixit, Gauri; Dohlman, Henrik G (2013) Regulation of yeast G protein signaling by the kinases that activate the AMPK homolog Snf1. Sci Signal 6:ra78
Isom, Daniel G; Sridharan, Vishwajith; Baker, Rachael et al. (2013) Protons as second messenger regulators of G protein signaling. Mol Cell 51:531-8
Nagiec, Michal J; Dohlman, Henrik G (2012) Checkpoints in a yeast differentiation pathway coordinate signaling during hyperosmotic stress. PLoS Genet 8:e1002437
Cappell, Steven D; Dohlman, Henrik G (2011) Selective regulation of MAP kinase signaling by an endomembrane phosphatidylinositol 4-kinase. J Biol Chem 286:14852-60
Torres, Matthew P; Clement, Sarah T; Cappell, Steven D et al. (2011) Cell cycle-dependent phosphorylation and ubiquitination of a G protein alpha subunit. J Biol Chem 286:20208-16
Cappell, Steven D; Baker, Rachael; Skowyra, Dorota et al. (2010) Systematic analysis of essential genes reveals important regulators of G protein signaling. Mol Cell 38:746-57
Wang, Yuqi; Dohlman, Henrik G (2006) Regulation of G protein and mitogen-activated protein kinase signaling by ubiquitination: insights from model organisms. Circ Res 99:1305-14
Wang, Xiao; Hao, Nan; Dohlman, Henrik G et al. (2006) Bistability, stochasticity, and oscillations in the mitogen-activated protein kinase cascade. Biophys J 90:1961-78
Wang, Yuqi; Dohlman, Henrik G (2006) Pheromone-regulated sumoylation of transcription factors that mediate the invasive to mating developmental switch in yeast. J Biol Chem 281:1964-9

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