PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway, Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C

<div><p>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 <i>Sordaria macrospora</i>. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated <i>mik1</i> 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.</p></div

PRO40 is required for correct signaling via the CWI pathway.

<p>(A) Time course of MAK1 phosphorylation in <i>pro40</i> deletion (Δ; S69656) and overexpression strains (OE; T184.2NS11) in comparison to wildtype. Strains were grown for three to six days, and phosphorylated MAK1 was detected in a Western blot using an anti-phospho-p44/42 antibody. The signal for tubulin was used as internal standard. Representative immunoblots of two to four independent experiments with three technical replicates are shown. (B) Stress-induced MAK1 phosphorylation in <i>pro40</i> deletion (Δ; S69656) and overexpression strains (OE; T184.2NS11) in comparison to wildtype. Strains were grown for three days and subjected to 0.01% H₂O₂ for 0, 15, 30, and 45 minutes prior to harvesting. Phosphorylated MAK1 was detected using an anti-phospho-p44/42 antibody, and the signal for tubulin was used as internal standard. Representative immunoblots of three independent experiments with three technical replicates are shown. (C) Model of the scaffolding function of PRO40 for the CWI pathway. Details are discussed in the text.</p

Localization of MIK1, MEK1, and MAK1.

<p>(A) GFP-labeled MIK1 and MEK1 are present in the cytoplasm and absent from nuclei. GFP-labeled MAK1 localizes to the cytoplasm, but is also targeted to the nucleus. (B) Co-localization experiments with MEK1-GFP and H2B-tdTomato show that spherical organelles devoid of MEK1 labeling are nuclei (arrowheads). Scale bar, 10 µm.</p

Shared interaction network of MEK1 and PRO40.

<p>Venn diagram comparing the three datasets generated from affinity purification and mass spectrometry with strains Δpro40::PRO40-FLAG (PRO40), Δmek1::NTAP-MEK1 (MEK1), and Δpro40::NTAP-MEK1 (MEK1(Δpro40)). The 12 proteins found in all three datasets and the 17 proteins found only in the PRO40 and MEK1 datasets are represented by boxes with <i>S. macrospora</i> locus tag numbers or protein designations. The magenta and blue box color indicates that the transcript of the encoding gene belongs to the top500 transcripts (with respect to read counts) in protoperithecia and vegetative hyphae, respectively (data taken from a previous transcriptomics analysis <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004582#pgen.1004582-Teichert1" target="_blank">[41]</a>).</p

Phenotypic characterization of kinase deletion strains.

<p>(A) Sexual development was assayed after 7 days of growth on BMM slides. Mature perithecia are only generated in wildtype (wt) and complemented deletion strains (Δmik1::<i>mik1</i>, Δmek1::<i>mek1</i>, and Δmak1::<i>mak1</i>). Δmik1, Δmek1, and Δmak1 generate only protoperithecia. White scale bar, 100 µm; black scale bar, 20 µm. (B) DIC microscopy of hyphal fusion in subperipheral regions. Strains were grown on solid MMS with a cellophane layer for 2 days. Hyphal fusion bridges are indicated by black arrowheads, whereas hyphae that grow in close contact without fusion are indicated by white arrowheads. Scale bar, 10 µm.</p

Shared interaction partners of MEK1 and PRO40.

<p>#s, number of spectral counts; #p, number of peptide counts; cov, coverage (%). <i>Sm</i>, <i>S. macrospora</i>.</p><p>Shared interaction partners of MEK1 and PRO40.</p

The Δmek1/pro40 double mutants shares phenotypic characteristics with Δmek1 and Δpro40.

<p>(A) Sexual development was assayed after 7 days of growth on BMM slides. Δpro40 and the Δmek1/pro40 double mutant generate only protoperithecia. White scale bar, 100 µm; black scale bar, 20 µm. (B) Δpro40 and the Δmek1/pro40 double mutant are unable to undergo hyphal fusion, although hyphae often grow in close contact (white arrowheads). Scale bar, 10 µm. (C) Localization of GFP-tagged MIK1, MEK1, and MAK1 in vegetative hyphae of the pro40 mutant and Δpro40. Scale bar, 10 µm. (D) Localization of GFP-tagged MIK1, MEK1, and MAK1 in three days old protoperithecia of the wildtype, the pro40 mutant, and the <i>pro40</i> deletion strain Δpro40. Scale bar, 20 µm.</p

Interactions of PRO40 with components of the CWI pathway.

<p>(A) Structures of proteins used for yeast two-hybrid analysis. Derivatives generated in addition to full-length constructs are shown below the protein structures. (B) Yeast two-hybrid analysis of CWI pathway components and PRO40. Yeast cells were drop-plated on SD medium lacking leucine, tryptophan, histidine, and adenine. Empty squares indicate that interactions were not tested. (C) Schematic overview of signal transduction and protein-protein interactions within the PRO40-CWI complex. Signaling through the pathway is depicted by gray arrows; interactions are depicted by black arrows. (D) Interaction sites between PRO40, PKC1, MIK1, MEK1, and MAK1. Black bars represent interaction sites tested in yeast two-hybrid analyses (A, B). For reasons of clarity, only PRO40 is depicted as homodimer.</p

Effects of resveratrol and piceatannol on the FdL-peptide deacetylation activities of Sirt3 and Sirt5.

<p><b>A</b> Deacetylation activity of Sirt3 against FdL2 (○) and Sirt5 against FdL1 (•) determined at increasing resveratrol concentrations. Activities are given relative to the value in absence of resveratrol. <b>B</b> Dose-response experiment for the piceatannol-dependent stimulation of FdL1 deacetylation by Sirt5. <b>C</b> Inhibition of Sirt3 FDL2 deacetylation activity by piceatannol. Error bars represent standard deviations.</p

Model for Sirtuin regulation by resveratrol-like compounds.

<p><b>A</b> Superimposition of Sirt5/FdL1/resveratrol and Sirt5/succinylated H3 peptide/NAD⁺. The movement of the β8/α13 loop is indicated by an arrow. The succinylated H3 peptide and the NAD⁺ molecule are omitted for clarity. <b>B</b> Model for the regulation of Sirtuins by resveratrol-like compounds. After binding of the substrate polypeptide the small molecule attaches to a “docking patch” (DP). It induces ordering of an “adaptable loop” (AL), leading to closure of the peptide exit and stabilization of the enzyme/substrate complex. Depending on the fit between substrate and small-molecule, the substrate is properly oriented in the active site (AS; e.g. Sirt5/Cytochrome c/resveratrol) or adopts a non-productive conformation (e.g. Sirt3/GDH/resveratrol), leading to stimulation or inhibition of turnover, respectively. After deacetylation, the activator dissociates, opening the peptide exit for product release.</p