Feedback phosphorylation of an RGS protein by MAP kinase in yeast

Regulators of G protein signaling (RGS proteins) are well. known to acceler ate G protein GTPase activity in vitro and to promote G protein desensitiza tion in vivo. Less is known about how RGS proteins are themselves regulated . To address this question we purified the RGS in yeast, Sst2, and used ele ctrospray ionization mass spectrometry to identify post-translational modif ications. This analysis revealed that Sst2 is phosphorylated at Ser-539 and that phosphorylation occurs in response to pheromone stimulation. Ser-539 lies within a consensus mitogen-activated protein (MAP) kinase phosphorylat ion site, Pro-X-Ser-Pro. Phosphorylation is blocked by mutations in the MAP kinase genes (FUS3, KSS1), as well as by mutations in components needed fo r MAP kinase activation (STE11, STE7, STE4, STE18). Phosphorylation is also blocked by replacing Ser-539 with Ala, Asp, or Glu (but not Thr). These po int mutations do not alter pheromone sensitivity, as determined by growth a rrest and reporter transcription assays. However, phosphorylation appears t o slow the rate of Sst2 degradation. These findings indicate that the G pro tein-regulated MAP kinase in yeast can act as a feedback regulator of Sst2, itself a regulator of G protein signaling.