FIGURE 2.

Identification of Aurora-A phosphorylation of AR at Thr²⁸² and Ser²⁹³. A, domain structure and location of three putative Aurora-A phosphorylation motifs of AR (top). In vitro Aurora-A kinase was carried out by incubation of recombinant Aurora-A with each GST-fused wild-type and Thr/Ser → Ala-mutated AR motif as substrate (middle). Panels 3 and 4 are Western blots showing the proteins used for the kinase assay. B and C, further definition of the phosphorylation sites with truncated and point-mutated AR. HEK293 cells were transfected with the indicated truncation (B) and point mutation (C) of full-length AR and then immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were used as substrates for in vitro Aurora-A kinase assay (top panels). Bottom panels are immunoblotting with anti-FLAG antibody showing expression of transfected plasmids. Note that the size of the nonspecific band (NS) is comparable with FLAG-AR-N3 (B). D, Aurora-A phosphorylates AR-Thr²⁸²/Ser²⁹³ in vivo. HEK293 cells were transfected with the indicated plasmids and labeled with [³²P]P_i (0.5 mCi/ml). FLAG-AR was immunoprecipitated, separated in SDS-PAGE, and exposed (top). Panels 2 and 3 show expression of the transfected AR and Aurora-A. E, knockdown of Aurora-A reduces phosphorylation of AR. PC3 cells were transfected with FLAG-AR and shRNA of Aurora-A, labeled with [³²P]P_i (0.5 mCi/ml), and analyzed as described in D (top). Panels 2–4 are Western blots hybridized with the indicated antibodies.