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. 2009 Aug 18;284(42):28762–28774. doi: 10.1074/jbc.M109.034165

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© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 4. — Competition between SMILE and coactivators. Reporter assays in A–C (upper panels) were performed as described under “Experimental Procedures.” The means ± S.D. (n = 3) of a representative experiment are shown. HepG2 cells were cotransfected with 0.1 μg of sft4-Luc reporter plasmids, together with the indicated expression vectors for FLAG-ERRγ (0.2 μg), FLAG-SMILE, HA-PGC-1α, HA-PGC-1β, and HA-GRIP1. A–C, lower panels, in vitro competition of SMILE with PGC-1α, HA-PGC-1β, or HA-GRIP1. ³⁵S-Radiolabeled ERRγ were incubated with the indicated GST or GST-SMILE fusion proteins together with increasing amounts of unlabeled in vitro-translated HA-PGC-1α, HA-PGC-1β, or HA-GRIP1 (0, 3, 6, or 12 μl) proteins. The protein interactions were detected via autoradiography. D, schematic representation of SMILE and LXXLL motif mutants of SMILE. Upper arrows indicate the locations of four LXXLL motifs in SMILE and lower arrows indicate the mutation of LXXLL motifs to AXXAL. m1, SMILE-m1 (1st LXXLL mutated to AXXAL); m2, SMILE-m2 (2nd LXXLL mutated to AXXAL); m3, SMILE-m3 (3rd LXXLL mutated to AXXAL); m4, SMILE-m4 (4th LXXLL mutated to AXXAL); m5, SMILE-m5 (all of the four LXXLL mutated to AXXAL). E, Western blot analysis using specific antibodies for SMILE and tubulin, with whole-cell extracts from HepG2 cells transfected with expression vectors encoding wild-type (wt) FLAG-SMILE or FLAG-SMILE-m1, -m2, -m3, -m4, and -m5. F, effects of SMILE LXXLL mutants on ERRγ-mediated transcriptional activity. HepG2 cells were cotransfected with reporter vector sft4-Luc, together with indicated expression vector for ERRγ, wild-type (wt) FLAG-SMILE or FLAG-SMILE LXXLL mutants. Luciferase activity was measured after 48 h of transfection. The means ± S.D. (n = 3) of a representative experiment are shown. G, in vivo interactions of SMILE LXXLL mutants with ERRγ. HepG2 cells were cotransfected with expression vectors for FLAG-ERRγ and wild-type pEBG-SMILE (GST-SMILE), or indicated GST-SMILE mutants, or pEBG alone (GST). Protein interactions were examined via in vivo GST pulldown. The top and middle panels (GST puri) show GST bead-precipitated FLAG-ERRγ and GST fusions, respectively. The bottom panel shows the protein expression levels of FLAG-ERRγ in cell lysates. The data shown are representative of at least three independent experiments with similar results.