Cell Cycle-Independent Phospho-Regulation of Fkh2 during Hyphal Growth Regulates Candida albicans Pathogenesis
Fig 2
Fkh2 is phosphorylated by Cdc28.
A) Schematic showing Cdc28 minimal (circles) and full (diamond) consensus target sites on Fkh2, with those detected by phospho-peptide mapping to be phosphorylated on hyphal induction indicated in blue. B) Phosphorylation of Fkh2 on hyphal induction detected using an antibody that recognises phosphorylated residues in Cdc28 target sites (αPSER(CDK) (Cell-Signalling 2324S). C) In vitro kinase assay with Cdc28-HA purified from a hyphal lysate and recombinant GST-Fkh2(CT) (aa419–687 intron removed) and GST-Fkh2-A-CT (as GST-Fkh2(CT) fragment with the serine/threonine in the five C-terminal Cdc28 consensus sites mutated to alanine). GST-Fkh2(CT) but not GST-Fkh2-A-CT is phosphorylated in vitro by Cdc28-HA. The parental strain BWP17, in which Cdc28 is not HA-tagged, provided the mock lysate to demonstrate that the activity was not due to a co-purifying kinase. D) Removal of Fkh2’s C-terminus containing the Cdc28 target site cluster abolishes the double band upon hyphal induction. fkh2(1–426)-GFP and fkh2/FKH2-YFP were grown as yeast or hyphae as previously, samples were treated with/without phosphatase and then resolved by SDS-PAGE. E) Autoradiograms from 2D gels and quantitative intensity profiles of the indicated Fkh2 phosphosite mutants at the indicated times and in the indicated culture conditions. The grey dashed line represents the parental Fkh2-YFP profile grown in the corresponding condition as shown in Fig. 1.