Sck1 Negatively Regulates Gpa2-Mediated Glucose Signaling in Schizosaccharomyces pombe

Schizosaccharomyces pombe detects extracellular glucose via a G protein-mediated cyclic AMP (cAMP)-signaling pathway activating protein kinase A (PKA) and regulating transcription of genes involved in metabolism and sexual development. In this pathway, Gpa2 Gα binds to and activates adenylyl cyclase in response to glucose detection by the Git3 G protein-coupled receptor. Using a two-hybrid screen to identify extrinsic regulators of Gpa2, we isolated a clone that expresses codons 471 to 696 of the Sck1 kinase, which appears to display a higher affinity for Gpa2K270E -activated Gα relative to Gpa2+ Gα. Deletion ofsck1 + or mutational inactivation of the Sck1 kinase produces phenotypes reflecting increased PKA activity in strains expressing Gpa2+ or Gpa2K270E , suggesting that Sck1 negatively regulates PKA activation through Gpa2. In contrast to the Gpa2K270E GDP-GTP exchange rate mutant, GTPase-defective Gpa2R176H weakly binds Sck1 in the two-hybrid screen and a deletion ofsck1 + in a Gpa2R176H strain confers phenotypes consistent with a slight reduction in PKA activity. Finally, deletingsck1 + in agpa2 Δ strain results in phenotypes consistent with a second role for Sck1 acting in parallel with PKA. In addition to this parallel role with PKA, our data suggest that Sck1 negatively regulates Gpa2, possibly targeting the nucleotide-free form of the protein that may expose the one and only AKT/PKB consensus site in Gpa2 for Sck1 to bind. This dual role for Sck1 may allowS. pombe to produce distinct biological responses to glucose and nitrogen starvation signals that both activate the Wis1-Spc1/StyI stress-activated protein kinase (SAPK) pathway.