G protein-coupled receptors are major targets for the treatment of cardiovascular and psychiatric disorders. Receptors of this class transmit signals to a variety of effector enzymes including mitogen activated protein kinases (MAPKs). This proposal is focused on the regulation of two MAPKs in yeast, Fus3 and Kss1. Both of these MAPKs are activated by the same pheromone stimulus. However while activation of Fus3 requires a kinase scaffold (Ste5) Kss1 does not. Our investigations have revealed four additional proteins required for full activation of Fus3 but not Kss1;these include two phosphatidylinositol 4-kinases (Pik1 &Stt4) and two subunits of the phosphatidylinositol 3-kinase complex (Vps15 &Vps34). We postulate that these enzymes promote the selective activation of scaffolded MAP kinases. There are three aims that address the hypothesis:
Aim 1 : Determine the signaling function of Pik1 and Stt4. Pik1 and Stt4 are phosphatidylinositol 4- kinases, which produce phosphatidylinositol 4-P (PI4P). PI4P is known to bind to the MAPK scaffold Ste5, but the consequences for MAPK activation are not known. We will test the hypothesis that Ste5 binding to PI4P promotes the activation of Fus3 catalytic activity.
Aim 2 : Determine the signaling function of Vps34 and PI3P. Vps34 is a phosphatidylinositol 3-kinase and produces phosphatidylinositol 3-P (PI3P). PI3P binds to a defined set of proteins having PX or FYVE domains. We will test the hypothesis that one of these PI3P-binding proteins is specifically required for Fus3 catalytic activity.
Aim 3 : Determine the signaling function of Vps15. Vps15 is a protein kinase required for Vps34 function, as well as for activation of Fus3. We will test the hypothesis that autophosphorylation of Vps15 is required for PI3P production. In addition, we will identify other pathway components phosphorylated by Vps15, and determine how phosphorylation affects MAPK activity and substrate specificity. This proposal investigates new components of the G protein and MAPK signaling apparatus in yeast. Mechanisms discovered in yeast are typically recapitulated in more complex organisms, so the activities elucidated here will likely apply to hormone and neurotransmitter function in humans.

Public Health Relevance

In this proposal we will define new ways that cells respond to hormone signals. Detailed analysis of these new signaling proteins 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 #
3R01GM080739-07S1
Application #
8712857
Study Section
Program Officer
Dunsmore, Sarah
Project Start
2007-05-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
7
Fiscal Year
2014
Total Cost
$23,606
Indirect Cost
$7,517
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
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Lien, Evan C; Nagiec, Michal J; Dohlman, Henrik G (2013) Proper protein glycosylation promotes mitogen-activated protein kinase signal fidelity. Biochemistry 52:115-24
Jones, Janice C; Jones, Alan M; Temple, Brenda R S et al. (2012) Differences in intradomain and interdomain motion confer distinct activation properties to structurally similar Gýý proteins. Proc Natl Acad Sci U S A 109:7275-9
Jones, Janice C; Duffy, Jeffrey W; Machius, Mischa et al. (2011) The crystal structure of a self-activating G protein alpha subunit reveals its distinct mechanism of signal initiation. Sci Signal 4:ra8
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
Jones, Janice C; Temple, Brenda R S; Jones, Alan M et al. (2011) Functional reconstitution of an atypical G protein heterotrimer and regulator of G protein signaling protein (RGS1) from Arabidopsis thaliana. J Biol Chem 286:13143-50
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
Simpson, Peter J; Schwappach, Blanche; Dohlman, Henrik G et al. (2010) Structures of Get3, Get4, and Get5 provide new models for TA membrane protein targeting. Structure 18:897-902
Heenan, Erin J; Vanhooke, Janeen L; Temple, Brenda R et al. (2009) Structure and function of Vps15 in the endosomal G protein signaling pathway. Biochemistry 48:6390-401
Lee, Michael J; Dohlman, Henrik G (2008) Coactivation of G protein signaling by cell-surface receptors and an intracellular exchange factor. Curr Biol 18:211-5

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