Quantitative phosphoproteomics detected activity of two key protein kinases in hyphal induction.

(A) Experimental scheme of parallel proteomic and phosphoproteomic analyses. Cells were grown at 37°C in liquid galactose medium and then 50 mM GlcNAc was added for 2 h to induce hyphae. For phosphorylation analysis, phosphopeptides were enriched by polymer-based metal-ion affinity capture (PolyMAC). There were 2 biologically independent replicates for each condition. (B) Representative null comparisons of the biological replicates show high reproducibility. We compared the replicate data of WT+GlcNAc_1 and WT+GlcNAc_2 from both whole proteome and phosphoproteome. (C) Representative results of kinase-substrate enrichment analysis. Gene set enrichment analysis (GSEA) algorithm [50] was used to identify significantly enriched or depleted groups of phosphorylated substrates. The potential substrates of both Cdc28 and Yck2 kinases were more phosphorylated (up-regulated) during the hyphal induction in the WT and PR13 cells. (D) The color-coded diagram illustrates changes of the selected kinase activities indicated by the scale bar at the bottom (2 fold change > 0.5). Note that 605 phosphopeptides were upregulated in both WT and PR13 strains. (F) Overrepresented motifs were extracted from the 605 phosphopeptides, using the motif-x algorithm [88] (p −6). 44 phosphopeptides had the Cdc28 consensus motif ([S/T]-P-x-K/R) indicating they were potential substrates. (G) Venn diagrams of the upregulated Cdc28 substrates ([S/T]-P-x-K/R) or Yck2 substrates (S/T-x-x-[S/T]). 11 phosphopeptides were potential substrates of both Cdc28 and Yck2 kinases.