Yak1 sequences distant from the sites of phosphorylation were required for efficient recognition by PKA. (A) Mapping the Tpk1-recognition site (RS-Y) in Yak1. The relative binding affinity to Tpk1KH in a two-hybrid assay is indicated, along with the relative phosphorylation signal observed in an in vitro reaction with Tpk1. The positions of the four consensus PKA phosphorylation sites are indicated with asterisks, and the RS-Y domain by the solid area. (B) The RS-Y domain of Yak1 was sufficient for the interaction with the Tpk1KH variant. A two-hybrid assay with Tpk1KH and the indicated fragments of Yak1 are shown. The Yak1, Yak1-ΔRS, and RS-Y constructs included residues 193–362, 193–311, and 312–362, respectively. (C) The RS-Y domain interacted with Tpk1KH in an immunoprecipitation assay. The Tpk1 proteins were precipitated with an anti-HA affinity resin, and the amount of associated RS-Y fragment was assessed by Western blotting. (D) Deletion of the RS-Y domain resulted in decreased phosphorylation by Tpk1 in vitro. The indicated Yak1 fragments were precipitated from cell extracts and incubated with Tpk1 and [γ-32P]ATP, as described in materials and methods. The relative level of phosphorylation following phosphorimager quantification of the data is shown. (E) The absence of the RS-Y domain resulted in decreased phosphorylation of Yak1 by bPKA in vitro. The Yak1 constructs were precipitated from yeast cell extracts, treated with phosphatase, and incubated with 0.02 units of bPKA and 3 mm ATP. The level of PKA phosphorylation was then assessed by Western blotting with the anti-PKA substrate antibody (α-Subs). (F) The RS-Y domain was required for efficient Yak1 phosphorylation by PKA in vivo. The indicated Yak1 constructs were precipitated from yeast cell extracts, and the level of PKA phosphorylation was assessed by Western blotting with the anti-PKA substrate antibody (see materials and methods). The band marked with the asterisk is the heavy chain of the antibody used for the immunoprecipitation, and the dotted oval indicates the position of the Yak1-ΔRS fragment.