Coordinated Translocation of Mammalian Gli Proteins and Suppressor of Fused to the Primary Cilium
Figure 4
Protein kinase A negatively regulates the ciliary localization of Gli and Sufu proteins.
(A) The ciliary localization of GFP-tagged Gli1, Gli2, Gli3 and Sufu are inhibited by a treatment (4 hours for Gli proteins and 18 hours for Sufu) with 40 µM forskolin, but not the solvent (DMSO) alone. The reduction of Gli1 ciliary localization is relatively moderate compared to Gli2 and Gli3 and weak ciliary signal is shown in the image. (B) Peptide sequence of mouse Gli2 protein in the region of residues 785–855, as well as its alignment with Gli1 and Gli3. In SD1-3 and SD1-4, the first three or all four Serine residues targeted by PKA are mutated to Aspartic acids. In P1-4, C1-4 and G2-4, Serine-to-Alanine mutations are created for all PKA, CK1 and GSK3 target sites, respectively. (C) Variants of GFP-Gli2 with mutations in their kinase sites are localized to the cilia in the presence of the solvent (DMSO), but not 40 µM forskolin (FSK) (4 hours of treatment for SD1-3, SD1-4 and P1-4; 18 hours of treatment for C1-4 and G2-4). Immunofluorescent images of MEFs transfected with GFP-tagged Gli proteins are shown. Cilia are labeled with acetylated tubulin and nuclei are stained with DAPI. Filled arrowheads indicate GFP signal at the tips of the cilia, unfilled arrowheads indicate the tips of cilia without GFP signal. Numbers at the lower-right corners of each image indicate numbers of cells with ciliary localization of GFP-tagged proteins over total numbers of transfected cells.