In partial fulfillment of the requirements for the degree of 

Doctor of Philosophy in Biology

in the 

School of Biological Sciences

Shilpa Choudhury

will defend her dissertation

ANALYSIS OF POST-TRANSLATIONAL MODIFICATIONS

IN THE REGULATION OF CELL SIGNALING AND BEHAVIOR

Monday, July 2nd, 2018

1:00 PM

EBB Seminar Room (1005)

Thesis Advisor:

Dr. Matthew P. Torres

School of Biological Sciences

Georgia Institute of Technology

Committee members: 

Dr. Brian Hammer

School of Biological Sciences

Georgia Institute of Technology

Dr. Yury Chernoff

School of Biological Sciences

Georgia Institute of Technology

Dr. Amit Reddi

School of Chemistry and Biochemistry

Georgia Institute of Technology

Dr. John Hepler

Department of Pharmacology

Emory University

ROLE OF Gg SUBUNIT AS A NEGATIVE FEEDBACK PHOSPHO-REGULATOR OF G-PROTEIN SIGNALING IN YEAST

Summary

Heterotrimeric G proteins, composed of Ga, Gb, and Gg subunits, are essential for converting extracellular chemical stimuli into appropriate intracellular responses. The principal mechanism of G protein signal transduction is highly conserved throughout eukaryotes, including yeast. Binding of extracellular signal to cell surface receptors activates G proteins through a well characterized process involving GTP binding on the Ga subunit, dissociation of the heterotrimeric complex into Ga and Gbg subunits, and activation of downstream effectors that elicit a response to the stimulus. As mediators between transmembrane receptors and intracellular effectors, heterotrimeric G proteins are well positioned to serve as PTM-mediated regulators of G-protein signaling. Indeed, three primary PTMs are essential for G proteins to function: N-terminal myristoylation and palmitoylation of Ga subunits, and C-terminal prenylation of Gg subunits, all three of which function to anchor heterotrimeric G proteins to the plasma membrane of cells. In this context, while the signaling roles of Ga and Gb subunits and the PTMs which occur on these subunits are widely identified; Gg subunits are thought to serve the limited role of a membrane anchor for its obligate partner Gb and no other regulatory role was known.

This study highlights a previously unknown role of Gg subunit in the regulation of G-protein signaling mediated through phosphorylation of their N-terminal intrinsically disordered region. Using a yeast model organism, Saccharomyces cerevisiae, that harbors a single canonical G-protein signaling system to regulate a yeast mating pathway, we have identified a novel phosphorylation-dependent regulatory role of Gg subunit in yeast (Ste18). Several unique discoveries have ensued that provide a foundation for future studies in mammalian systems, where G protein signaling serves as a major drug target for the treatment of human disease. First, Ste18 is dynamically phosphorylated in response to GPCR activation. Second, this phosphorylation event is dependent on a MAPK (Fus3), which is the ortholog of human Erk2. Third, Ste18 phosphorylation, in conjunction with phosphorylation of a Gbg effector scaffold protein (Ste5), negatively regulates activation of the yeast mating pathway that is responsible for phosphorylation-based activation of Fus3. Fourth, negative regulation by phosphorylated Ste18/Ste5 is mechanistically achieved by reducing binding affinity between Gbg/(Ste4/18) and effector Ste5, which results in controlling the bulk rate of active signaling complex formation at the plasma membrane in response to a GPCR stimulus.  Fifth, Ste18/Ste5 phosphorylation regulates the sensitivity of the yeast mating process by altering its switch-like behavior. I go on to demonstrate that Ste18 phosphorylation can be promoted by signals other than GPCR stimulation. Specifically, I show that Ste18 phosphorylation is also sensitive to osmotic stress and cell-cycle progression, both of which may represent cross-talk mediated responses that funnel through the aforementioned Ste18/Ste5 regulatory mechanism. Together, these findings reveal that combinatorial phosphorylation of Ste18 and the Gbg effector protein (Ste5) constitute a dynamic regulatory module that mediates negative regulation of G protein signaling in yeast and provide a foundation for understanding similar mechanisms undergone in mammalian cells.