Figure 5
From: A new negative feedback mechanism for MAPK pathway inactivation through Srk1 MAPKAP kinase

Wis1 is phosphorylated by Srk1 (a) Recombinant Srk1, GST-tagged Wis1-KR (catalytically inactive) Wis1-4A-KR, and Wis1-5A-KR were incubated separately or together with γ32P-ATP in the absence or presence of recombinant Srk1. The autoradiogram (top) and Coomassie blue (bottom) results are shown. (b) Schematic representation of Wis1 protein domains and the different mutations performed to obtain Wis1 phospho-mutants (see text for details). (c) In vitro phosphorylation assays with recombinant GST-Wis1-KR without (blue planels) or with recombinant GST-Srk1 (red panels) and processed for LC–MS phosphopeptide analysis. Two peptides are shown, peptide SMPVVNK (159–166 AA) (first two panels) and peptide NPLLNRPTSFNR (218–229 AA) (last two panels).