Figure 2.

The activator of YpkA kinase activity is actin. (A) Partial purification of the YpkA activator. Bovine brain cytosol proteins were separated by column chromatography. Proteins contained within the peak fractions collected from the Sephacryl 200 gel filtration column were analyzed by SDS/PAGE and detected by silver staining. Eluted actin was detected by immunoblot analysis using an antibody to actin (Sigma). (B) Dose-dependent activation of YpkA by actin. Actin was added in increasing concentrations to recombinant YpkA (1.5 μg). Actin is also phosphorylated by YpkA in vitro. (C) Actin was depleted from the Sephacryl 200 gel filtration peak fraction by using successive incubations with DNase I-Sepharose as previously described. Fractions were assayed for their ability to stimulate YpkA kinase activity.