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. 2009 Jun 5;106(25):10183–10188. doi: 10.1073/pnas.0812105106

Fig. 2.

Fig. 2.

GRK2 blocks PTCH1-mediated inhibition of cyclin B1 nuclear translocation by disrupting the interaction between PTCH1 and cyclin B1. (A) GRK2 is essential for ShhN-induced cyclin B1 nuclear translocation. HEK293 cells were transfected with the cyclin B1 derivative cyc-C (NLS-CRSGlu-HA) alone (Left), cyc-C and PTCH1-myc (Middle), or cyc-C, PTCH1-myc, and miR-GRK2-EGFP (Right). At 48 h posttransfection, the cells were treated with vehicle (con, control) or 5 nM ShhN for 2 h and then analyzed by immunofluorescence microscopy. (B) GRK2 promotes cyclin B1 nuclear translocation. Cells were cotransfected with cyc-C and PTCH1-myc, in combination with GRK2 or its mutants. (C) Effects of GRK2 BP-Flag and Δ312–379-Flag on the interaction between PTCH1 and cyclin B1. Cells were transfected with PTCH1-myc alone or in combination with cyclin B1-HA, or cyclin B1-HA and BP-Flag, or Δ312–379-Flag. Lysates were immunoprecipitated with anti-myc antibody. (D) ShhN stimulation induces changes in the PTCH1/GRK2/cyclin B1 complex. Cells were treated with ShhN for indicated times 48 h after transfection with PTCH1-myc and GRK2 plasmids. Immunoprecipitation was performed by using anti-myc antibody and proteins were detected in Western blots by using the indicated antibodies. (E) GRK2 is essential for the dissociation of cyclin B1 and PTCH1 under ShhN stimulation. Cells transfected with PTCH1-myc, cyclin B1-HA, and miR-GRK2 or control siRNA (con) were treated with ShhN for 2 h, and interactions were detected as described above.