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van Drogen, F, Stucke, VM, Jorritsma, G and Peter, M (2001) MAP kinase dynamics in response to pheromones in budding yeast. Nat. Cell Biol. 3:1051-9
Although scaffolding is a major regulator of mitogen-activated protein kinase (MAPK) pathways, scaffolding proteins are poorly understood. During yeast mating, MAPK Fus3p is phosphorylated by MAPKK Ste7p, which is activated by MAPKKK Ste11p. This MAPK module interacts with the scaffold molecule Ste5p. Here we show that Ste11p and Ste7p were predominantly cytoplasmic proteins, while Ste5p and Fus3p were found in the nucleus and the cytoplasm. Ste5p, Ste7p and Fus3p also localized to tips of mating projections in pheromone-treated cells. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that Fus3p rapidly shuttles between the nucleus and the cytoplasm independently of pheromones, Fus3p phosphorylation and Ste5p. Membrane-bound Ste5p can specifically recruit Fus3p and Ste7p to the cell cortex. Ste5p remains stably bound at the plasma membrane, unlike activated Fus3p, which dissociates from Ste5p and translocates to the nucleus.
Adaptor Proteins, Signal Transducing; Carrier Proteins; Cell Membrane/metabolism; Cell Nucleus/metabolism; Cytoplasm/metabolism; Enzyme Activation/drug effects; Enzyme Activation/physiology; Fungal Proteins/metabolism; Green Fluorescent Proteins; Indicators and Reagents/metabolism; Luminescent Proteins/metabolism; MAP Kinase Kinase Kinases/metabolism; MAP Kinase Signaling System/drug effects; MAP Kinase Signaling System/physiology; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases/metabolism; Pheromones/pharmacology; Phosphorylation; Protein Binding/physiology; Protein Kinases/metabolism; Saccharomyces cerevisiae Proteins/metabolism; Saccharomycetales/enzymology; Transcription Factors/metabolism
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