Investigating the Protein Kinase C Polarized Growth‐Related Protein Complex in Aspergillus nidulans

Abstract

The long‐term goal of our research is to advance understanding of the mechanisms involved in hyphal growth, as well as maintenance of cell walls in filamentous fungi. Recently our work has focused on proteins that localize to sites of cell wall synthesis, namely growing cell apices (hyphal tips) and forming septa. This project focusses on Protein kinase C (PkcA). In previous work, we identified a mutation in the gene that encodes an orthologue of protein kinase C in our model organism Aspergillus nidulans, which results in reduced cell wall integrity. We showed that wild type PkcA localizes to growing hyphal apices and the contractile actomyosin ring at forming septa, and we identified amino acid sequences within PkcA that are required for PkcA to localize to these sites of cell wall synthesis. This project seeks to uncover the protein‐protein interactions involving PkcA and determine the PkcA‐complexed proteins it phosphorylates and those that it does not. Previously we reported that PkcA forms a complex with the formin SepA, and that this interaction requires the FH2 domain of SepA. Published results from another group suggested that the Rho‐type GTPase Rho4 plays a role in SepA function and that Bud3 acts as a GEF for Rho4. Here we show that the putative Rho binding domain of PkcA (HR1A) is sufficient for its interaction with SepA. Fluorescence microscopy of a dually labeled A. nidulans strain illustrate that although PkcA‐mRFP and Rho4‐GFP often reside at the contractile actomyosin concurrently, PkcA precedes Rho4 in ring constriction and yeast two‐hybrid assays failed to show an interaction between PkcA and Rho4. Further, neither of 4 other Rho homologues tested interacted with PkcA in yeast two‐hybrid experiments. Sequence analysis of the FH2 domain of SepA revealed a Rho‐like motif encompassing amino acids 377–408 having 43.3% sequence identity with RhoA. This result suggests that the sufficiency of the HR1A domain for PkcA’s interaction with SepA is due to its affinity for the Rho‐like motif within FH2. Co‐immunoprecipitation of GFP‐PkcA followed by tandem mass spectrometry and proteomics analysis of complexed proteins identified chitin synthase B and the glucan synthase FksA within the PkcA complex. Ongoing studies will seek to determine if these proteins are substrates for PkcA.Support or Funding InformationNSF RUI 1615192