FIGURE 2.

PIM-1 kinases phosphorylate AR at Ser-213 and Thr-850. A, putative PIM-1 phosphorylation sites in AR protein. B, PIM-1 kinases induce AR phosphorylation at Ser-213. FLAG-tagged PIM-1 kinases were transfected into COS-1 (left) or infected with lentiviruses in LNCaP cells (right). The level of AR Ser-213 phosphorylation in total cell lysates was detected by immunoblotting with phosphospecific antibody for Ser-213 of AR (pARS213). HA-tagged AKT1 served as a positive control for Ser-213 phosphorylation in COS-1 cells. AR, FLAG, tubulin, and HA blots were used as controls. C, COS-1 cells were cotransfected with wild-type AR (AR-WT) or the AR-S213A mutant with FLAG-tagged PIM-1 kinases. Total cell lysates were used for Western blot analysis as in B. D, PIM-1L induces AR phosphorylation at Thr-850. COS-1 cells were transfected with FLAG-tagged PIM-1 kinases and AR (left panel). Lysates were immunoprecipitated with anti-AR followed by immunoblotting with a phospho-threonine (pThr) antibody (right panel). AR and FLAG blots were used as controls. E, a similar IP experiment was performed along with the AR-T850A mutant to confirm specificity of pThr signal PIM-1L-induced AR Thr-850 phosphorylation was detected by an antibody specific for phosphorylated Thr-850 (pT850). F, in vitro kinase assays using purified GST-PIM-1S and -PIM-1L, AR phosphorylation was detected by using pARS213 and pART850 antibodies.