Phosphorylation of the Gα protein Gpa2 promotes protein kinase A signaling in yeast

Abstract

Heterotrimeric G proteins are important molecular switches that facilitate transmission of a variety of signals from the outside to the inside of cells. G proteins are highly conserved, enabling study of their regulatory mechanisms in model organisms such as the budding yeast Saccharomyces cerevisiae. Gpa2 is a yeast Gα protein that functions in the nutrient signaling pathway. Using Phos-tag, a highly specific phosphate binding tag for separating phosphorylated proteins, we found that Gpa2 undergoes phosphorylation and that its level of phosphorylation is markedly increased upon nitrogen starvation. We also observed that phosphorylation of Gpa2 depends on glycogen synthase kinase (GSK). Disrupting GSK activity diminishes Gpa2 phosphorylation levels in vivo, and the purified GSK isoforms Mck1 and Ygk3 are capable of phosphorylating Gpa2 in vitro. Functionally, phosphorylation enhanced plasma membrane localization of Gpa2 and promoted nitrogen starvation-induced activation of protein kinase A. Together, the findings of our study revealamechanismbywhichGSK-andnutrient-dependentphosphorylation regulates subcellular localization of Gpa2 and its ability to activate downstream signaling.