Fig. 2. The protein kinase Sak1 and the phosphatase regulatory subunit Reg1 act on Gpa1.

(A) Coimmunoprecipitation of Gpa1 and Sak1. WT cells were transformed with plasmids encoding the indicated proteins and were cultured under high-glucose (H) or low-glucose (L) conditions. Cells were subjected to immunoprecipitation (IP) with an anti-FLAG antibody (αFLAG), eluted in SDS-PAGE sample buffer, and then analyzed by Western blotting (IB) to detect coimmunoprecipitated Sak1-TAP with antibody against protein A (αProtein A). Cell lysates (input) were also analyzed by Western blotting with the indicated antibodies. (B) In vitro kinase assays. Purified TAP fusion proteins of WT Sak1 (Sak1-TAP) or a kinase-deficient mutant Sak1 (Sak1^D277A-TAP) were incubated with or without purified recombinant Gpa1 protein in the presence of [γ-³²P]ATP. The Sak1-TAP fusion proteins were purified from a sak1Δsnf1Δ strain to avoid potential copurification of Snf1. Left: Autoradiogram showing the incorporation of radioactive phosphate into the indicated proteins. Right: The Sak1-TAP input was detected by Western blotting analysis with antibody against protein A, whereas the Gpa1 input was detected by Coomassie gel staining. (C) Coimmunoprecipitation of Reg1 and Gpa1. WT cells were transformed with plasmids encoding the indicated constructs and were cultured under high- or low-glucose conditions. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody, eluted in SDS-PAGE sample buffer, and then analyzed by Western blotting with an anti-hemagglutinin (HA) antibody to detect coimmunoprecipitated Reg1-HA. Cell lysates (input) were also analyzed by Western blotting with the indicated antibodies. (D) Purified recombinant 6×His-Gpa1 and Reg1-MBP (maltose-binding protein) proteins were combined in vitro and resolved by steric exclusion chromatography. Proteins were detected by Western blotting analysis with antibodies specific for Gpa1 or MBP. All data are representative of two independent experiments.