FIGURE 2.

Requirement for a MAGE-11 F-box in AR transactivation. A, schematic diagram of full-length MAGE-11 showing the MAGE homology domain (MHD) with F-box residues 329–369, phosphorylation site Thr-360 within the F-box, Lys-240 and Lys-245 monoubiquitinylation (Ub) sites outside the F-box, and the NH₂-terminal nuclear localization signal (NLS). B, sequence homology between MAGE-11 F-box residues 329–369, cyclin F F-box residues 35–73, and MAGE family members, with conserved acidic residues in blue and hydrophobic residues in red. C, alanine mutagenesis of the MAGE F-box at conserved hydrophobic residues. D, Ishikawa cells were transfected with pCMV-AR (10 ng/well), 0.1 μg/well of PSA-Enh-Luc, and 0.1 μg of pSG5 empty vector (—), pSG5-MAGE wild-type (WT), or the indicated F-box mutant. Cells were incubated in the absence and presence of 1 nm DHT with or without 0.1 μg/ml of EGF. Bottom panel, Ishikawa cells were transfected with 2 μg of pSG5 (—), pSG5-MAGE WT, and the indicated F-box mutant. Cells were extracted in IB lysis buffer and 30 μg of protein/lane analyzed on immunoblots probed with antibody raised against FLAG-tagged human MAGE-11 (2 μg/ml). E, FLAG-b empty vector (—) or FLAG-AR (4 μg) were expressed in COS cells with pSG5-HA-MAGE-(112–429) WT or Δ329–369 F-box deletion (0.5 μg of DNA/10-cm dish). Cells were treated in the absence and presence of 10 nm DHT and 0.1 μg/ml EGF, lysed in IP lysis buffer, and incubated with FLAG resin overnight at 4 °C. Immunoprecipitates and cell extracts (25 μg of protein/lane) were analyzed by immunoblot using FLAG and HA antibodies.