title: PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.
abstract: Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.
authors.affiliation: Department for General and Molecular Botany, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Ines
authors.initials: I
authors.lastname: Teichert
authors.affiliation: Department for General and Molecular Botany, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Eva Katharina
authors.initials: EK
authors.lastname: Steffens
authors.affiliation: Department for General and Molecular Botany, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Schnaß
authors.affiliation: Department of Analytical Chemistry, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Fränzel
authors.affiliation: Department of Analytical Chemistry, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Krisp
authors.affiliation: Department of Analytical Chemistry, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Dirk A
authors.initials: DA
authors.lastname: Wolters
authors.affiliation: Department for General and Molecular Botany, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Kück
mesh_terms: D002473:Cell Wall / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; D020929:Mitogen-Activated Protein Kinase Kinases