Figure 3.

YpkA requires the last 20 aa for activation. (A) Schematic representation of YpkA constructs. Slashed lines denote the FLAG epitope tag. The C-terminal 20 aa are shown in black and white checkered boxes. Each of the deletion mutants is labeled, and the site of the C-terminal truncation is denoted with an asterisk. (B) YpkA requires the last 20 aa for kinase activity. HEK293 cells were transfected with YpkA, YpkA-K269A, YpkA-Δ450, YpkA-Δ660, YpkA-Δ710, or pCDNA3 (8 μg). All YpkA constructs were fused to an N-terminal FLAG epitope tag. YpkA was immunoprecipitated with anti-FLAG M2 affinity gel (Sigma) and subjected to in vitro kinase assays. Actin was detected in immunoprecipitates using an antibody against actin (Sigma). Levels of YpkA protein expression were assessed by immunoblot analysis with an antibody to the FLAG epitope (Sigma). (C) The C termini of YpkA and coronin are similar. Sequence alignment was performed by using the clustalw algorithm in the macvector program. Identical residues are highlighted in dark gray, whereas similar residues are highlighted in light gray.