Localized feedback phosphorylation of Ste5p scaffold by associated MAPK cascade.
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 Gbeta 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.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Annette
A
Flotho
David M
DM
Simpson
Maosong
M
Qi
Elaine A
EA
Elion
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D015152:Blotting, Northern; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D020935:MAP Kinase Signaling System; D009154:Mutation; D010675:Pheromones / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D013997:Time Factors
D048868:Adaptor Proteins, Signal Transducing; D044387:GTP-Binding Protein beta Subunits; D010675:Pheromones; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C080321:STE5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C067576:Ste4 protein, S cerevisiae; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D017489:CDC28 Protein Kinase, S cerevisiae; D010744:Phosphoric Monoester Hydrolases