FIG. 4.

Hog1 phosphorylates Ser519 at the regulatory domain of Rck2. (A) Phosphorylation of different fragments of Rck2 by Hog1. Various Rck2 fragments were tested for their ability to be phosphorylated by an in vitro-activated Hog1 (as described in Materials and Methods). After the in vitro kinase assay, phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography. Positions of the Rck2 fragments included in the constructs are indicated in parentheses. Proteins were His tagged and contained a Lys201→Met mutation which results in catalytically inactive enzymes, to avoid autophosphorylation. (B) Mutation of Rck2 Ser519→Ala abolishes Hog1 phosphorylation. The full-length RCK2(KD) and its Ser519 mutant form were tested for Hog1 phosphorylation as described for panel A. After phosphorylation, proteins were resolved by SDS-PAGE and transferred to a nylon membrane. Phosphorylated proteins were detected by autoradiography (upper panel). His-tagged Rck2 proteins were detected by immunoblotting by using the anti-His monoclonal antibody BMG-His-1 (lower panel).