Fig 1: Fu phosphorylates the C-terminal Sufu-binding domain of Ci.(A) Schematic diagram of Ci. Blue boxes and red bars indicate the Sufu-binding domains and Fu/CK1 phosphorylation clusters, respectively. ZF, Zinc-Finger DNA-binding domain; CBP, CBP-binding domain. The primary sequences of three phosphorylation clusters are shown with Fu and CK1 sites color-coded in red and green, respectively. (B) The primary sequence of the Ci C-terminal region in GST-CiC. (C, D) In vitro kinase assay using purified CC-FuEE or CC-FuKR as kinase and the indicated GST-CiC fusion proteins as substrates. Phosphorylation was detected by pIMAGO (C) or pS1382 antibody (D). (E) Western blot analysis of Ci phosphorylation in S2R+ cells transfected with the indicated Ci and Fu constructs. Two phospho-specific antibodies (E11888p and E11889p) were used to detect S1382 phosphorylation. (F) Western blot analysis of Ci phosphorylation on the indicated sites in S2R+ cells transfected with CC-FuEE and the indicated Myc-Ci-PKA constructs. (G) Western blot analysis of Ci phosphorylation on S1382 in S2R+ cells transfected with Myc-Ci-PKA and treated with or without Hh-conditioned medium. (H) Western blot analysis of endogenous Ci phosphorylation on S1382 in Cl8 cells treated with or without Hh-conditioned medium. (I) Western blot analysis of endogenous Ci phosphorylation on S1382 in Cl8 cells treated with the indicated dsRNA in the absence or presence of Hh. (J) Western blot analysis of HA-Sufu pulled down by the indicated GST fusion proteins. 5 µg of GST or GST-CiC fusion proteins were incubated with equal amounts of cell lysates from S2R+ cells transfected with HA-Sufu construct.
Fig 2: Hh activates Ci/Gli through differential phosphorylation.Graded Hh signals gradually increase the overall levels of Ci/Gli phosphorylation on multiple Fu/Ulk3/CK1 sites, leading to gradual release of Sufu repression and progressive increase in CiA/GliA activity. See text for details.
Fig 3: Phosphorylation of Ci N-terminal Fu/CK1 sites increased Ci activity in vivo.(A, B) Adult wings expressing UAS-Myc-Ci-PKA_WT (A) or UAS-Myc-Ci-PKA_SD1 (B) under the control of the C765 Gal4 driver at 30°C. (C, C’, C”, D, D”) Wing discs expressing UAS-Myc-Ci-PKA_WT (C, C’, C”) or UAS-Myc-Ci-PKA_SD1 (D, D’, D”) under the control of the C765 Gal4 driver at 30°C were immunostained for Ci (red) and Ptc (blue).
Fig 4: The C-terminal phosphorylation cluster is conserved in Gli2 and Gli3.Alignment of the C-terminal sequence from Ci (aa1371-1397), mouse Gli2 (aa1517-1544), and mouse Gli3 (aa1556-1583). Ulk3 and CK1 sites are color-coded in “red” and “green,” respectively. The acidic residues at +6/7 position of Ulk3 site is indicated in magenta.
Fig 5: Multisite phosphorylation of Gli2 promotes its activation.(A) Schematic diagram of mouse Gli2 with primary sequences of the three phosphorylation clusters shown underneath. Ulk3 and CK1 sites are color-coded in “red” and “green,” respectively. Amino acid substitutions for individual Gli2 constructs are indicated. (B) In vitro kinase assay using immunopurified HA-Ulk3WT or HA-Ulk3KR as kinase and the indicated GST-fusion proteins as substrates. Phosphorylation was detected by pIMAGO. (C, C’, F, F’) Relative Gli1 mRNA expression (C, F) or Gli2 protein levels (C’ and F’) in NIH3T3 cells expressing the indicated shRNA and Myc-Gli2WT or its variants with or without SAG. (D, D’, D”, E, E’, E”) Western blot analysis of Myc-Gli2 phosphorylation on S230/232 in NIH3T3 cells infected with lentivirus expressing shRNA targeting the 3' UTR of Gli2 and lentivirus expressing Myc-Gli2WT (NIH3T3Gli2-shRNA/Myc-Gli2) in the presence of increasing amounts of Shh-N for 24 h (D) or exposed to a fixed amount of Shh-N (1 ng/ml) for increasing amounts of time (E). Quantification of pS230/232 signals (D’, E’) or Ptch1 mRNA (D”, E”) under these conditions. (G, J) Gli-luc reporter activity in NIH3T3 cells transfected with increasing amounts of the indicated Gli2 constructs (G) or fixed amount of the indicated Gli2 constructs and increasing amounts of Sufu constructs (J). (H) Western blot analysis of Fg-Sufu pulled down by the indicated GST fusion proteins from HEK293T cells expressing a Fg-Sufu construct. (I) Western blot analysis of Flag-Sufu coimmunoprecipitated with Myc-Gli2WT, Myc-Gli2SD1, and Myc-Gli2SD12. Data are mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 (t test).
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