Localized Feedback Phosphorylation of Ste5p Scaffold by Associated MAPK Cascade

• Published in: The Journal of biological chemistry 2004-11, Vol.279 (45), p.47391-47401
• Main Authors: Flotho, Annette, Simpson, David M, Qi, Maosong, Elion, Elaine A
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing - metabolism; Adaptor Proteins, Signal Transducing - physiology; Binding Sites; Blotting, Northern; CDC28 Protein Kinase, S cerevisiae - metabolism; Cell Membrane - metabolism; Enzyme Activation; Genes, Reporter; Glutathione Transferase - metabolism; Green Fluorescent Proteins - metabolism; GTP-Binding Protein beta Subunits - metabolism; MAP Kinase Signaling System; Mutation; Pheromones - metabolism; Phosphoric Monoester Hydrolases - metabolism; Phosphorylation; Plasmids - metabolism; Protein Processing, Post-Translational; Recombinant Fusion Proteins - metabolism; RNA, Messenger - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - metabolism; Saccharomyces cerevisiae Proteins - physiology; Signal Transduction; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M405681200

Scaffold proteins play pivotal roles during signal transduction. In Saccharomyces cerevisiae , the Ste5p scaffold protein is required for activation of the mating MAPK cascade in response to mating pheromone and assembles a G protein-MAPK cascade complex at the plasma membrane. To serve this function, Ste5p undergoes a regulated localization event involving nuclear shuttling and recruitment to the cell cortex. Here, we show that Ste5p is also subject to two types of phosphorylation and increases in abundance as a result of MAPK activation. During vegetative growth, Ste5p is basally phosphorylated through a process regulated by the CDK Cdc28p. During mating pheromone signaling, Ste5p undergoes increased phosphorylation by the mating MAPK cascade. Multiple kinases of the mating MAPK cascade contribute to pheromone-induced phosphorylation of Ste5p, with the mating MAPKs contributing the most. Pheromone induction or overexpression of the Ste4p Gβ subunit increases the abundance of Ste5p at a post-translational step, as long as the mating MAPKs are present. Increasing the level of MAPK activation increases the amount of Ste5p at the cell cortex. Analysis of Ste5p localization mutants reveals a strict requirement for Ste5p recruitment to the plasma membrane for the pheromone-induced phosphorylation. These results suggest that the pool of Ste5p that is recruited to the plasma membrane selectively undergoes feedback phosphorylation by the associated MAPKs, leading to an increased pool of Ste5p at the site of polarized growth. These findings provide evidence of a spatially regulated mechanism for post-activation control of a signaling scaffold that potentiates pathway activation.