Figure 1.
Phosphorylation of Sla1p and Pan1p by Ark1p. (A) In vitro phosphorylation of Sla1p by Ark1p. Wild-type Ark1p, the kinase-inactivated Ark1D158Yp, and wild-type Prk1p were expressed as HA-tagged proteins and quantified by immunoprecipitation and Western analysis. Equal amounts of the immunoprecipitated proteins were used in the kinase reactions (right panel). Phosphorylation results were shown as autoradiography and the input substrates were visualized by the Coomassie Blue staining (left panel). (B) Phosphorylation of Sla1p-SR, Pan1p-LR1, and Pan1p-LR2 by Ark1p and Prk1p in E. coli. GST fusion substrates, indicated below the panels, coexpressed with respective kinases, indicated above the panels, were purified after 1-h induction and analyzed by SDS-PAGE and Western analysis. Lane 4 (CIP) in each panel is same as lane 1 (Ark1) but treated with the phosphatase before loading. The band of added phosphatase is indicated by asterisk. (C) in vivo phosphorylation status of Sla1p and Pan1p in different kinase deletion mutants. Top, Myc-Sla1p was immunoprecipitated from cell lysate prepared from wild-type (YMC505) and five different kinase deletion strains: prk1Δ (YMC506), prk1Δ akl1Δ (YMC507), ark1Δ (YMC515), prk1Δ ark1Δ (YMC516), and prk1Δ ark1Δ akl1Δ (YMC504/508), at 30°C. The immunoprecipitates from wild type (YMC505) were incubated with 1 μl of CIP for 30 min at 37°C before loading. Bottom, Myc-tagged Pan1p was immunoprecipitated from cell lysate prepared from wild-type (YMC511) and three different kinase deletion strains: prk1Δ (YMC512), prk1Δ akl1Δ (YMC503), and prk1pΔ ark1Δ akl1Δ (YMC504), separated on SDS gels, and probed by the mouse anti-Myc antibody. The membrane was stripped and probed again with the rabbit anti-phosphothreonine antibody. The immunoprecipitates from wild type (YMC511) were incubated with 1 μl of CIP for 30 min at 37°C before loading.