Volume 12, Issue 5, Pages 1261-1274 (November 2003) Hedgehog Signal Transduction via Smoothened Association with a Cytoplasmic Complex Scaffolded by the Atypical Kinesin, Costal-2  Lawrence Lum, Chi Zhang, Sekyung Oh, Randall K. Mann, Doris P. von Kessler, Jussi Taipale, Frances Weis-Garcia, Ruoyu Gong, Baolin Wang, Philip A. Beachy  Molecular Cell  Volume 12, Issue 5, Pages 1261-1274 (November 2003) DOI: 10.1016/S1097-2765(03)00426-X

Figure 1 Hh-Induced Protein Stabilization and Modification of Pathway Components (A) Western blot analysis of pathway components in cl-8 cells upon HhN stimulation. Proteins in the unstimulated state or modified in response to HhN are noted (black and blue arrows, respectively). Loading control: β-tubulin. (B) Su(fu) is phosphorylated in response to HhN. Lysates from HhN-treated cl-8 cells (24 hr) were incubated in the presence/absence of lambda phosphatase (λPPase, 200 U) and the phosphatase inhibitor Na3VO4 (1 mM), prior to Western blotting. (C) Su(fu) protein in Drosophila embryos. The pattern of slower migrating Su(fu) proteins is similar to that observed in HhN-stimulated cl-8 cells. (D) RNAi of Cos2 reduces levels of Fu expression, and of Hh-induced phosphorylation of Smo, Fu, and Su(fu). RNAi in S2R+ cells (2 days) was followed by incubation with HhN (24 hr) and Western blot analysis. Faster-migrating Smo in unstimulated cells can be detected upon longer exposure, and slower migration of Smo in stimulated cells is due to phosphorylation (Supplemental Figure S1A). (E) RNAi of Fu decreases Hh-dependent Su(fu) phosphorylation (lanes 6 and 2). Increased Su(fu) phosphorylation, resulting from RNAi of Ptc (lanes 3 and 4), is also decreased by RNAi of Fu (lanes 7 and 8). (F) Role of pathway components in biochemical events associated with pathway activity. The blue shading highlights functions of pathway components that, to the best of our knowledge, have not previously been reported. Molecular Cell 2003 12, 1261-1274DOI: (10.1016/S1097-2765(03)00426-X)

Figure 2 Cos2 Is Required for Fu Stabilization and Smo-Accumulation in Response to Hh (A) Levels of interacting Cos2 and Fu proteins in cl-8 and S2 cells remain constant with Hh stimulation. Cells treated with or without HhN medium (2 hr) were immunopurified (IP) and bound material analyzed by SDS-PAGE and silver staining. Ci and Cos2 in cl-8 cells comigrate. (B) Level of Fu (red) is decreased in wing imaginal disc clones lacking cos2 function (cos2W1 clones are marked by loss of α-Myc staining [green]). The anterior compartment of the disc is to the left and posterior to the right. (C) Levels of Smo (red) in cos2 wing imaginal disc clones (similarly marked) show loss of Smo accumulation in anterior cells near the compartment boundary (arrow) and no change in the posterior compartment (arrowhead). The posterior compartment is marked by a generally elevated level of Smo. Molecular Cell 2003 12, 1261-1274DOI: (10.1016/S1097-2765(03)00426-X)

Figure 3 Identification of a Complex that Includes Smo, Cos2, Fu, and Ci (A) Preparative scale isolation of native complexes from cl-8 cells using mAb affinity columns and identification of purified proteins by mass spectrometry. Lysates from cl-8 cells treated with control or HhN medium (12 hr) were incubated with mAb matrices and bound proteins analyzed by SDS-PAGE and subsequent silver-staining (lanes 1–4) or Western blotting (lanes 5–8). (B) Proteins and distinct peptides identified from the correspondingly numbered bands (see also Supplemental Table S2). Molecular Cell 2003 12, 1261-1274DOI: (10.1016/S1097-2765(03)00426-X)

Figure 4 Hh-Induced Accumulation of a Smo-Cos2-Fu-Ci Complex Lysates from cl-8 cells (A) treated with control or HhN medium (2 hr), and S2 parental or stable HhN-transfected cell lines (B), were incubated with mAb matrices and bound proteins analyzed by Western blotting. Boxed region indicates a shorter exposure. Negative control: kinesin heavy chain (Khc). (C) Su(fu) associates predominantly with Ci. Bound proteins from cl-8 cell lysates incubated with mAb matrices were analyzed by Western blotting. Little Fu, Cos2, and no Smo (not shown) were detectable in α-Su(fu) IP, and no Su(fu) was detected in α-Cos2 IP. Molecular Cell 2003 12, 1261-1274DOI: (10.1016/S1097-2765(03)00426-X)

Figure 5 A Positive Role for Cos2 in High-Level Hh Signal Response (A) Cos2 is required for highest levels of Hh pathway activity in cl-8 cells. Activity of a ptc-luciferase reporter (normalized to control Renilla luciferase) was used to measure Hh pathway response. RNAi of Ptc maximizes response to HhN medium (dashed line; see text) in a Smo-dependent manner. Combined RNAi of Cos2 and Ptc dramatically reduces reporter activity, indicating a requirement for Cos2 function in maximal Smo-mediated pathway activity. RNAi of Ptc alone does not induce constitutive reporter activity due to a compensatory increase in ptc mRNA expression (Lum et al., 2003). Cotransfection of full-length Hh with reporter construct also results in maximal levels of pathway activity. Fold induction by HhN is noted above. (B) Relative levels of Smo and Cos2 determine effect on Hh pathway activity. In cells cotransfected with Hh expression construct to produce maximal pathway activity, reporter activity at fixed levels of Smo increases at lower levels of added Cos2 but decreases at highest levels. Dashed lines indicate levels of reporter activity with 0.1 μg of Smo alone (top), Cos2 alone (bottom), or YFP control (middle). Inset: 0.1 μg of Smo- or Cos2-Renilla luciferase fusion proteins (Smo-R and Cos2-R, respectively) were transfected into cl-8 cells followed by incubation with control or HhN medium. Similar levels of each protein are noted, and HhN stimulation does not cause accumulation of Smo-R, likely due to high constitutive levels of expression. Molecular Cell 2003 12, 1261-1274DOI: (10.1016/S1097-2765(03)00426-X)

Figure 6 The Smo Cytotail Physically and Functionally Interacts with Cos2 (A) Alignment of Drosophila, Anopheles, and human Smo cytotail sequences. Conserved sequences extending from transmembrane domain 7 (TM7) to Ser707 of Drosophila Smo are shown (a complete alignment is shown in Supplemental Figure S5). Blue letters denote the last three amino acids of C-terminal truncation mutants and the circles indicate Ala substitutions. Ile586 (boxed) is important in Smo protein stability as Ala substitution results in loss of Smo expression and function (data not shown). (B) Effects of deleting Smo cytoplasmic tail sequences on Smo function. Endogenous Smo mRNA was targeted using dsRNA corresponding to the 5′ untranslated region (UTR; schematic diagram). Expression constructs encoding Smo but lacking the 5′ UTR sequence were used to test for rescue of Smo function. Graph: ptc-luciferase reporter activity in cells transfected with Smo 5′ UTR dsRNA with control YFP, wild-type (wt) Smo, or various Smo C-terminal deletion and point mutation expression constructs (see above). Dashed line: control level of signaling. Luciferase activities were normalized to Renilla luciferase activities. A representative assay is shown. (C) Effects of Smo C-terminal deletions on binding to Cos2. Cos2-R was expressed with either YFP, wt Smo, or mutant Smo proteins in S2 cells stimulated with HhN. Levels of Cos2-R binding to Smo were measured by Renilla luciferase activity associated with precipitated proteins using the α-Smo mAb matrix (see diagram). Coprecipitations were performed in triplicate. (D) Cos2 stalk and cargo domains interact with Smo. Myc epitope-tagged domains of Cos2 (left diagram) coexpressed with Smo-R in S2 cells were isolated by IP of protein complexes (in triplicate) using α-Myc mAb (9E10) and associated Renilla luciferase activity was measured. Smo-R were coprecipitated with full-length Cos2, as well as stalk and cargo domain proteins. Deletion mutants were expressed at comparable levels (data not shown). (E) Smo cytotail interacts directly with Cos2 in vitro. Glutathione agarose beads alone, preloaded with GST, or with GST-SmoC-RRN fusion protein (Smo cytotail from Ser557 to Asn686), were incubated with lysates from COS1 cells expressing Cos2, HA-Ci, Cos2 cargo-Fc, Cos2 stalk-Fc, or Fc protein alone. Purified GST proteins were visualized by Coommassie staining. Cos2 and Ci were detected using an α-Cos2 polyclonal or α-HA epitope monoclonal (for HA-Ci). Fc proteins were detected using an α-huIgG Fc polyclonal antibody. Input: 3% (Cos2 and HA-Ci) or 10% (Fc proteins) of material used for IP. (F) Cos2 cargo domain is required for Smo response. The effects of transfecting different levels of wt Cos2 or Δcargo construct with and without 10 ng Smo construct on pathway activity were measured using the ptc-luciferase reporter assay in cl-8 cells. Dashed line: control level of signaling. Molecular Cell 2003 12, 1261-1274DOI: (10.1016/S1097-2765(03)00426-X)

Figure 7 Accumulation of Smo Is Critical for Strong Hh Signaling (A) Cycloheximide inhibits new protein synthesis in cl-8 cells. Protein synthesis was monitored by incubation of cl-8 cells in S35-Met- and S35-Cys-containing medium for 2 hr. Medium contained cycloheximide (CHX; 50 μg/ml) or vehicle throughout the labeling period and for a 1.5 hr pretreatment period. Resulting lysates were run on SDS-PAGE and dried gel exposed to film. (B) New protein synthesis is required for strong Hh response in cl-8 cells. Cells pretreated as in (A) were incubated with control or HhN medium for 2 hr in the continuing presence of CHX or vehicle. Pathway component response was determined by Western blotting. Note that phosphorylation of cytoplasmic components in the presence of CHX occurs but is dramatically reduced. (C) New protein synthesis is required for an increase in Hh-induced Smo-Cos2 association. Levels of Cos2 that co-IP with Smo were measured by Western blotting of lysates prepared from control or CHX- or HhN-treated cl-8 cells. (D) Model for response of cytoplasmic complex to Smo at three different stages of Hh stimulation. Pathway activity is also partially activated in the absence of Cos2 (see Discussion). Molecular Cell 2003 12, 1261-1274DOI: (10.1016/S1097-2765(03)00426-X)