Figure 1.

YpkA requires a protein cofactor for activation. (A) Recombinant YpkA requires an activator for kinase activity. Recombinant YpkA or YpkA-K269A was expressed in E. coli and purified. YpkA was incubated with either buffer or HeLa cell extract protein (3 μg) and subjected to in vitro kinase assays. (B) HEK293 cells were transfected with either wild-type YpkA, YpkA-K269A, or vector (8 μg). Both wild-type and mutant YpkA were N-terminal FLAG epitope tagged. YpkA was immunoprecipitated with anti-FLAG M2 affinity gel (Sigma) and was subjected to in vitro kinase assays. Similar levels of YpkA are present in all of the immunoprecipitation extracts as determined by immunoblot analysis with an antibody to the FLAG epitope (Sigma). (C) HEK293 cells were transfected with wild-type YpkA (8 μg). YpkA was immunoprecipitated with anti-FLAG M2 affinity gel (Sigma) and was subjected to in vitro kinase assays in the presence of the artificial substrates shown, as described in Materials and Methods.