| Title: | Quantitative proteomics reveals a Galpha/MAPK signaling hub that controls pheromone-induced cellular polarization in yeast |
| Author(s): | Waszczak N; Deflorio R; Ismael A; Cheng N; Stone DE; Metodiev MV; |
| Address: | "Department of Biological Sciences, University of Illinois at Chicago, 900 South Ashland Ave, Chicago, USA. Department of Biological Sciences, University of Illinois at Chicago, 900 South Ashland Ave, Chicago, USA. Electronic address: dstone@uic.edu. School of Life Sciences, University of Essex, Wivenhoe Park, Colchester, UK. Electronic address: mmetod@essex.ac.uk" |
| DOI: | 10.1016/j.jprot.2019.103467 |
| ISSN/ISBN: | 1876-7737 (Electronic) 1874-3919 (Linking) |
| Abstract: | "The mating-specific yeast Galpha controls pheromone signaling by sequestering Gbetagamma and by regulating the Fus3 MAP kinase. Disrupting Galpha-Fus3 interaction leads to severe defects in chemotropism. Because Galpha concentrates at the chemotropic growth site where Fus3 is required for the phosphorylation of two known targets, we screened for additional proteins whose phosphorylation depends on pheromone stimulation and Galpha-Fus3 interaction. Using a mutant form of Galpha severely defective in Fus3-binding, Galpha(DSD), and quantitative mass spectrometry, fourteen proteins were identified as potential targets of Galpha-recruited Fus3, ten of which were previously implicated in cell polarity and morphogenesis. To explore the biological relevance of these findings, we focused on the Spa2 polarisome protein, which was hypophosphorylated on multiple serine residues in pheromone-treated Galpha(DSD) cells. Six sites were mutagenized to create the Spa2(6XSA) mutant protein. Spa2(6XSA) exhibited increased affinity for Fus3, consistent with a kinase-substrate interaction, and Spa2(6XSA) cells exhibited dramatic defects in gradient sensing and zygote formation. These results suggest that Galpha promotes the phosphorylation of Spa2 by Fus3 at the cortex of pheromone-stimulated cells, and that this mechanism plays a role in chemotropism. How the Galpha-Fus3 signaling hub affects the other putative targets identified here has yet to be determined. SIGNIFICANCE: Previously, interaction between the G alpha protein, Gpa1, and the MAPK of the pheromone response pathway, Fus3, was shown to be important for efficient sensing of the pheromone gradient and for the maintenance of cell polarity during mating. Here we show that the underlying molecular mechanisms involve the phosphorylation of specific cortical targets of Gpa1/Fus3. These have been identified by quantitative phosphoproteomics using a mutant of Gpa1, which is defective in interacting with Fus3. One of these targets is the polarisome protein Spa2. Alanine substitution of the Spa2 phosphorylation sites targeted by Gpa1/Fus3 lead to a dramatic defect in pheromone gradient sensing and zygote formation. These results reveal how the G alpha protein and the MAPK control cell polarity in a prototypical model system. Our results have wider significance as similar mechanisms exist in higher eukaryotes and are involved in important biological such as neuron development, immunity, and cancer cell metastasis" |
| Keywords: | "Amino Acid Substitution Cytoskeletal Proteins/genetics/metabolism GTP-Binding Protein alpha Subunits/genetics/*metabolism *MAP Kinase Signaling System Mating Factor/genetics/*metabolism Mitogen-Activated Protein Kinases/genetics/*metabolism Mutation, Miss;" |
| Notes: | "MedlineWaszczak, Nicholaz DeFlorio, Reagan Ismael, Amber Cheng, Naiyuan Stone, David E Metodiev, Metodi V eng Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Netherlands 2019/07/28 J Proteomics. 2019 Sep 15; 207:103467. doi: 10.1016/j.jprot.2019.103467. Epub 2019 Jul 24" |
