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. 2011 Apr 13;6(4):e18638.
doi: 10.1371/journal.pone.0018638.

A novel signaling pathway mediated by the nuclear targeting of C-terminal fragments of mammalian Patched 1

Hiroki Kagawa  1 , Yuka Shino, Daigo Kobayashi, Syunsuke Demizu, Masumi Shimada, Hiroyoshi Ariga, Hiroyuki Kawahara
Affiliations

A novel signaling pathway mediated by the nuclear targeting of C-terminal fragments of mammalian Patched 1

Hiroki Kagawa et al. PLoS One. .

Abstract

Background: Patched 1 (Ptc1) is a polytopic receptor protein that is essential for growth and differentiation. Its extracellular domains accept its ligand, Sonic Hedgehog, while the function of its C-terminal intracellular domain is largely obscure.

Principal findings: In this study, we stably expressed human Ptc1 protein in HeLa cells and found that it is subjected to proteolytic cleavage at the C-terminus, resulting in the generation of soluble C-terminal fragments. These fragments accumulated in the nucleus, while the N-terminal region of Ptc1 remained in the cytoplasmic membrane fractions. Using an anti-Ptc1 C-terminal domain antibody, we provide conclusive evidence that C-terminal fragments of endogenous Ptc1 accumulate in the nucleus of C3H10T1/2 cells. Similar nuclear accumulation of endogenous C-terminal fragments was observed not only in C3H10T1/2 cells but also in mouse embryonic primary cells. Importantly, the C-terminal fragments of Ptc1 modulate transcriptional activity of Gli1.

Conclusions: Although Ptc1 protein was originally thought to be restricted to cell membrane fractions, our findings suggest that its C-terminal fragments can function as an alternative signal transducer that is directly transported to the cell nucleus.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Modest expression of Ptc-ICD7 fragment leads to its nuclear accumulation.
C-terminally-FLAG-tagged Ptc-ICD7 cDNA in pCI-neo expression vector was transfected into HeLa cells (Ptc-ICD7-FLAG TF) and immunostained with anti-FLAG antibody. At the time of cDNA transfection, protein synthesis inhibitor cyclohexamide (CHX) was added at indicated concentrations. “No CHX” indicates cell culture without CHX addition (A, B). Cells were harvested 24 hr after transfections. Addition of 12.5 µg/mL CHX completely suppressed protein expression from transfected expression plasmid (E, I), while the addition of 0.4 µg/mL CHX reduced expression of FLAG-tagged Ptc-ICD7 to less than half compared with “No CHX” (I). “Mock-TF” indicates empty vector transfection without CHX addition as a negative control (G, H). Anti-FLAG immunostain in green (A, C, E, G), and DAPI DNA stain in blue (B, D, F, H). Scale bar, 20 µm.
Figure 2
Figure 2. Subcellular localization of stably expressed Ptc1.
(A) Schematic representation of stably transfected full-length human Ptc1 protein with possess 12 transmembrane domains. Red box at N-terminus indicates 3×T7-tag, green box at end of intracellular domain indicates C-terminal 3×FLAG-tag, respectively. (B) Western blot analyses of Ptc1 protein stably expressed in HeLa cells. Ptc1 immunoprecipitated from cell extracts with C-terminal FLAG-tag and immunoblotted with anti-N-terminal T7 tag antibody (left panel) or anti-C-terminal FLAG tag antibody (right panel). Anti-T7 blot detects doublet bands corresponding to full-length Ptc1 (Ptc1 FL), while anti-FLAG blot reveals small C-terminal fragments representing the intracellular domain of Ptc1 (ICD7 fragments) as well as Ptc1 FL. (C) Multiple staining of Ptc1-stably expressing cells by anti-T7 tag immunostain (a: Ptc1 N-terminus), anti-FLAG-tag immunostain (b: Ptc1 C-terminus), DAPI DNA stain (c: nucleus) and their merged image (d). Scale bar, 20 µm. (D) Cell fractionation analysis of Ptc1-stably expressing cells. BIP and Histone H2B used as cytoplasmic and nuclear marker, respectively. Ptc1 FL (+), cells stably expressing full-length Ptc1; Ptc1 FL (−), control HeLa cells.
Figure 3
Figure 3. Endogenous Ptc1 ICD7 fragment in C3H10T1/2 cell.
(A) Schematic diagram of human Ptc1 protein (isoform L). Twelve transmembrane (TM) domains are indicated by black boxes. Antigenic regions of SSD (500–600) and ICD7 (1420–1434) are indicated by underlines. (B) Amino acid alignment of the C-terminus of Ptc1. Antigenic sequence of anti-Ptc-ICD7 (1420–1434) antibody is indicated by a box. Identical residues in humans and mice are indicated by dots. (C) Western blot analysis with mouse C3H10T1/2 total cell lysate. Endogenous Ptc1 protein in cells was detected with either anti-Ptc-ICD7 (1420–1434) antibody or anti-Ptc SSD (500–600) antibody. Inclusion of competitive antigenic peptide is indicated as (−) or (+). Asterisk indicates unidentified signal. (D) Cell fractionation assay of C3H10T1/2 cells. Each fraction was subjected to immunoblot analysis using anti-Ptc-ICD7 (1420–1434) antibody. TCL: total cell lysate. Cyto.: cytoplasmic fraction. Nuc.: nuclear fraction. Antigenic peptide competition abolished the 37 kDa immunosignal in the nuclear fraction.
Figure 4
Figure 4. Nuclear localization of endogenous Ptc1 ICD7 fragments in C3H10T1/2 cells.
Immunocytochemical analyses of endogenous Ptc1 protein in mouse C3H10T1/2 cells with a series of anti-Ptc1 antibody. Antibodies used are as follows: anti- Ptc-ICD7 (1420–1434) antibody (A), anti-Ptc1 antibody that recognizes SSD (500–600) region (C). Anti-α-tubulin DM1A antibody used as positive control for cytoplasmic immunostaining (E), and “Pre-immune serum” staining used as negative control (K). (G–J) Nuclear immunosignals of endogenous ICD7 fragments were competitively suppressed by corresponding antigenic peptide. 5.5 nM anti-Ptc-ICD7 (1420–1434) primary antibody was absorbed with (I) or without (G) excessive amounts of antigenic peptide. Antigenic peptide concentrations for competition were 1.2 µM. Hoechst DNA stain, blue (B, D, F, H, J, L). Scale bar, 20 µm.
Figure 5
Figure 5. Nuclear accumulation of endogenous Ptc1 ICD7 fragments in mouse embryonic primary cells.
Immunocytochemical analyses of endogenous Ptc1 ICD7 fragments in mouse embryonic primary cells. (A) Anti-Ptc-ICD7 (1420–1434) antibody staining. (C) Anti-α-tubulin DM1A antibody as positive control for cytoplasmic immunostaining. (E) Pre-immune serum staining was used as a negative control. 5.5 nM anti-Ptc-ICD7 (1420–1434) primary antibody was absorbed with (I) or without (G) excessive amounts of antigenic peptide. Antigenic peptide concentrations for competition were 0.6 µM. Hoechst DNA stain, blue (B, D, F, H, J). Scale bar, 20 µm.
Figure 6
Figure 6. Gli1-based luciferase-reporter assay reveals Shh response in Ptc1 transfected cells.
(A) Dual-luciferase reporter assay performed with Gli1 expression vector (Gli1) and a reporter construct consisting of eight copies of the Gli-binding site (8×3′Gli-BS) or its mutated version (8×m3′Gli-BS) as a negative control. Normalization of transfection efficiencies was carried out using Renilla luciferase activities as an internal control. Relative luciferase activity was monitored in cells stably expressing full-length Ptc1 (Ptc1 FL Stable exp.) and its negative control (Mock exp.) with (+) or without (−) Shh-N-conditioned media. This medium contains 19 kDa form of Shh-N fragment of Myc-tagged-Shh-N that was processed and secreted from in HeLa cells expressing full-length Shh. Addition of the Shh-N fragment stimulates transcriptional activity of Gli1 in cells stably expressing full-length Ptc1. All reporter assay experiments were repeated at least three times, and transfection was done in duplicate. (B) Stably expressed full-length Ptc1 binds with secreted Shh-N. HeLa cells that were stably expressed N-terminally T7-tagged full-length Ptc1 was exposed to Myc-Shh-N-conditioned medium. After cells were harvested, full-length Ptc1 was immunoprecipitated with N-terminal T7-tag from solubilized cell lysates (IP:anti-T7) and precipitates were probed with anti-Myc antibody (IB: anti-Myc). (C) Shh-N stimulates the production of Ptc-ICD7 fragments. Cells stably expressing full-length Ptc1 (with C-terminal FLAG-tag) were cultured with (+) or without (−) Shh-N-conditioned media. Solubilized cell lysates were subjected to FLAG-immunoprecipitation and subsequently probed with anti-FLAG antibody. (D) Relative luciferase activity as in (A) monitored in cells over-expressing Ptc-ICD7 fragment (pCI-Ptc1-ICD7), full-length Ptc1 (pCI-Ptc1 FL), and mock control (pCI-empty).
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