Negative Feedback Phosphorylation of Gγ Subunit Ste18 and the Ste5 Scaffold Synergistically Regulates MAPK Activation in Yeast

Abstract

SUMMARYHeterotrimeric G proteins (Gαβγ) are essential transducers in G protein signaling systems in all eukaryotes. In yeast, G protein signaling differentially activates mitogen-activated protein kinases (MAPKs)—Fus3 and Kss1—a phenomenon controlled by plasma membrane (PM) association of the scaffold protein Ste5. Here, we show that phosphorylation of the yeast Gγ subunit (Ste18), together with Fus3 docking on Ste5, controls the rate and stability of Ste5/PM association. Disruption of either element alone by point mutation has mild but reciprocal effects on MAPK activation. Disabling both elements results in ultra-fast and stable bulk Ste5/PM localization and Fus3 activation that is 6 times faster and 4 times more amplified compared to wild-type cells. These results further resolve the mechanism by which MAPK negative feedback phosphorylation controls pathway activation and provides compelling evidence that Gγ subunits can serve as intrinsic regulators of G protein signaling.

Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5

“…This analysis identified over 40 Ste5 phosphorylation sites ( Figs. 2 A and S3 and Table S1), among them several previously described or predicted sites, confirming that Ste5 is a major hub for regulation ( Choudhury et al, 2018 ; Repetto et al, 2018 ). Interestingly, Ste5 was also phosphorylated on S185 close to the first pair of finger cysteine residues within its RING-H2 domain ( Figs.…”

“…Ste5 membrane association further requires cooperative effects of the pleckstrin homology (PH) domain and the plasma membrane binding motif (PM) domain to increase cell membrane affinity ( Winters et al, 2005 ). Pheromone signaling is down-regulated by negative feedback through the MAPK Fus3 ( Choudhury et al, 2018 ; Repetto et al, 2018 ) and CDK1-mediated phosphorylation of multiple sites flanking the PM domain ( Oehlen and Cross, 1994 ; Strickfaden et al, 2007 ) by electrostatic exclusion with the negatively charged head groups of the phospholipids ( Strickfaden et al, 2007 ).…”

Quantitative analysis of the yeast pheromone pathway

“…When binding to Ste5 is abrogated, Fus3 activation resembles that of Kss1, and the cell can no longer expand towards a pheromone gradient (Hao et al, ). Subsequent work using Phos‐tag has revealed a synergistic relationship between two important regulators of Fus3, Ste5 and the dual‐specificity phosphatase Msg5 (Nagiec et al, ) as well as cooperation between two targets of phosphorylation, Ste5 and the Gγ subunit Ste18 (Choudhury, Baradaran‐Mashinchi, & Torres, ).…”

Crossbreeding of Yeasts Domesticated for Fermentation: Infertility Challenges

“…MAT mating type locus; a or α allele [7,8] STE2 seven transmembrane receptor for α-factor pheromone [18] STE3 seven transmembrane receptor for a-factor pheromone [18] MFA1 mating pheromone a-factor [18] MFα1 mating pheromone α-factor [18] GPA1 GTP-binding α subunit of heterotrimeric G-protein [18,19] STE4 β subunit of heterotrimeric G-protein [18,19] STE18 γ subunit of heterotrimeric G-protein [18,19] IME1 master regulator of meiosis [20] RME1 zinc finger protein involved in control of meiosis [20] IRT1 long noncoding RNA located in the IME1 promoter [21] HMRa * silenced copy of a sequence [22] HMLα silenced copy of α sequence [22] HO site-specific endonuclease for the MAT locus [18] ASH1 zinc-finger inhibitor of HO transcription [23] SHE1 type V myosin motor involved in actin-based transport [24] SHE2 RNA-binding protein that binds specific mRNAs [24] SHE3 protein adaptor between She1 and the She2-mRNA complex [24] MSN2 stress responsive transcriptional activator [25,26] MSN4 stress responsive transcriptional activator [25,26] RIM15 protein kinase involved in cell proliferation [27] IME2 serine/threonine protein kinase involved in activation of meiosis [28,29] GIS1 histone demethylase and transcription factor [30] SPO11 meiosis-specific protein that initiates meiotic recombination [31]…”

“…Pheromone stimulation leads to the activation of heterotrimeric G-proteins comprising Gpa1 (Gα), Ste4 (Gβ) and Ste18 (Gγ) through the GPCR. The activated G-protein subsequently dissociates into Gα and Gβγ complex subunits, and the Gβγ complex induces activation of the mitogen-activated protein kinase (MAPK) cascade [19] followed by transcriptional activation for cell fusion (Figure 1). While the G-protein subunit genes GPA1, STE4, and STE18 are hsg, the pheromone and receptor genes are asg or αsg [18,34].…”