Figure 2. Serine 185 within the RING-H2 domain of Ste5 is phosphorylated by Pkc1. (A) Schematic of Ste5 functional domains. MAPK, MAPK docking site; VWA, von Willebrand factor type A domain. Phosphorylated sites identified by MS analysis (localization probability >0.75 according to MaxQuant) in cells exposed to α-factor after phosphopeptide enrichment on a TiO2 column are indicated by tick marks. The RING domain sequence is aligned with those of different yeast Ste5 proteins and the RING domain of S. cerevisiae Far1. Ser185 of S. cerevisiae is indicated in bold red and conserved in other yeast species. An analogous serine residue (Ser210) located within the Far1 RING domain (bold red) is also phosphorylated, together with two lower confidence sites (red; Ser208 and Ser211). (B) Recombinant 6His-tagged Ste5- and Far1-RING-H2 fragments were incubated for 30 min at 30°C with GST-Pkc1 purified from yeast extracts containing γ32P-ATP with (+) or without (−) 30 µM cercosporamide (cerc.). Phosphorylated proteins were visualized by autoradiography. An aliquot of purified Ste5- and Far1-RING-H2 fragments were separated by SDS-PAGE and stained with Coomassie blue. The bar points to the 35-kD size marker. (C–F) In vitro kinase assays using either WT or the nonphosphorylatable S185A mutant RING-H2 domain with amino acids 149–238 of Ste5 (Ste5149−238) as a substrate and incubated as indicated with yeast extracts in the absence (DMSO) or presence of cercosporamide were analyzed by 600 MHz 1H-15N correlation NMR spectra (SOFAST HMQC; Schanda et al., 2005). The resonance position of the phosphorylated Ser185 amide group is magnified in C and highlighted in the dashed circle in D–F. The spectra in C are an overlay of the Ste5-RING-H2 fragment incubated with (black) or without (red) Pkc1. (G) M-track protein–protein proximity assays using extracts of cells expressing HKMT-myc–tagged Ste5 (bait) and protA-H3–tagged Pkc1 (prey). Cells were treated with α-factor for the times indicated (hours). Proximity signals were detected by Western blotting using an antibody against triple-methylated lysine of histone H3 (α-me). Loading was controlled via a hemagglutinin epitope embedded in the protA-H3 tag (α-HA). The asterisk marks an unspecific band.
Figure 2.

Serine 185 within the RING-H2 domain of Ste5 is phosphorylated by Pkc1. (A) Schematic of Ste5 functional domains. MAPK, MAPK docking site; VWA, von Willebrand factor type A domain. Phosphorylated sites identified by MS analysis (localization probability >0.75 according to MaxQuant) in cells exposed to α-factor after phosphopeptide enrichment on a TiO2 column are indicated by tick marks. The RING domain sequence is aligned with those of different yeast Ste5 proteins and the RING domain of S. cerevisiae Far1. Ser185 of S. cerevisiae is indicated in bold red and conserved in other yeast species. An analogous serine residue (Ser210) located within the Far1 RING domain (bold red) is also phosphorylated, together with two lower confidence sites (red; Ser208 and Ser211). (B) Recombinant 6His-tagged Ste5- and Far1-RING-H2 fragments were incubated for 30 min at 30°C with GST-Pkc1 purified from yeast extracts containing γ32P-ATP with (+) or without (−) 30 µM cercosporamide (cerc.). Phosphorylated proteins were visualized by autoradiography. An aliquot of purified Ste5- and Far1-RING-H2 fragments were separated by SDS-PAGE and stained with Coomassie blue. The bar points to the 35-kD size marker. (C–F) In vitro kinase assays using either WT or the nonphosphorylatable S185A mutant RING-H2 domain with amino acids 149–238 of Ste5 (Ste5149−238) as a substrate and incubated as indicated with yeast extracts in the absence (DMSO) or presence of cercosporamide were analyzed by 600 MHz 1H-15N correlation NMR spectra (SOFAST HMQC; Schanda et al., 2005). The resonance position of the phosphorylated Ser185 amide group is magnified in C and highlighted in the dashed circle in D–F. The spectra in C are an overlay of the Ste5-RING-H2 fragment incubated with (black) or without (red) Pkc1. (G) M-track protein–protein proximity assays using extracts of cells expressing HKMT-myc–tagged Ste5 (bait) and protA-H3–tagged Pkc1 (prey). Cells were treated with α-factor for the times indicated (hours). Proximity signals were detected by Western blotting using an antibody against triple-methylated lysine of histone H3 (α-me). Loading was controlled via a hemagglutinin epitope embedded in the protA-H3 tag (α-HA). The asterisk marks an unspecific band.

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