Multisite interaction with Sufu regulates Ci/Gli activity through distinct mechanisms in Hh signal transduction Yuhong Hana,1, Qing Shia,1, and Jin Jianga,b,2 Departments of aDevelopmental Biology and bPharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 Edited by Gary Struhl, Columbia University College of Physicians and Surgeons, New York, NY, and approved April 16, 2015 (received for review November 11, 2014) The tumor suppressor protein Suppressor of fused (Sufu) plays a respectively. Furthermore, we provide evidence that binding conserved role in the Hedgehog (Hh) signaling pathway by inhibiting of Sufu to Ci impedes the recruitment of the transcriptional Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) coactivator CBP. transcription factors, but the molecular mechanism by which Sufu inhibits Ci/Gli activity remains poorly understood. Here we show that Results Sufu can bind Ci/Gli through a C-terminal Sufu-interacting site (SIC) in Sufu Can Inhibit a Full-Length Ci Lacking the N-Terminal Sufu-Binding addition to a previously identified N-terminal site (SIN), and that both Site. Previous studies indicated that Ci/Gli binds Sufu through its SIC and SIN are required for optimal inhibition of Ci/Gli by Sufu. N-terminal domain containing an SYGH core motif (Fig. 1A) We show that Sufu can sequester Ci/Gli in the cytoplasm through (11, 12, 23, 24), which we named SIN (Sufu-interacting site in the binding to SIN while inhibiting Ci/Gli activity in the nucleus depend- N-terminal region). We were surprised to observe that the tran- Δ ing on SIC. We also find that binding of Sufu to SIC and the middle scriptional activity of a Ci variant (Ci-PKA N) lacking SIN was still region of Ci can impede recruitment of the transcriptional coactiva- inhibited by Sufu (Fig. S1). Of note, Ci-PKA has three PKA sites tor CBP by masking its binding site in the C-terminal region of Ci. mutated to Ala and is no longer processed into a truncated re- Indeed, moving the CBP-binding site to an “exposed” location can pressor (CiR) (25). By using Ci-PKA as a backbone for structure– render Ci resistant to Sufu-mediated inhibition in the nucleus. Hence, function study, we could focus on the regulation of the activator our study identifies a previously unidentified and conserved Sufu- form of Ci (CiA) by Sufu. When SIN was deleted in CiGA1, in binding motif in the C-terminal region of Ci/Gli and provides mech- which the Ci sequence C-terminal to its Zn-finger DNA-binding anistic insight into how Sufu inhibits Ci/Gli activity in the nucleus. domain was replaced by the Gal4 activation domain (GA), the resulting CiGA1ΔN was less inhibited by Sufu (Fig. S1), sug- Hedgehog | Sufu | Ci | Gli | CBP gesting that Sufu can inhibit Ci through a region C-terminal to its Zn-finger DNA-binding domain. he Hedgehog (Hh) signaling pathway controls embryogenesis Tand adult tissue homeostasis by regulating the Cubitus Sufu Interacts with a Conserved C-Terminal Site in Ci/Gli. To deter- interruptus (Ci)/Glioma-associated oncogene homolog (Gli) mine whether Sufu could interact with Ci through a domain(s) family of zinc-finger transcription factors (1, 2). Initially identi- other than SIN, we divided Ci into three fragments—Ci1–439 fied as a suppressor of the segmentation defects caused by the (amino acids 1–439), Ci440–1160 (amino acids 440–1160), and Drosophila loss of the serine/threonine kinase Fused (Fu) in (3), Ci1161–1397 (amino acids 1161–1397)—and examined their inter- Suppressor of fused (Sufu) plays a conserved negative role in Hh action with Sufu by coimmunoprecipitation (CoIP) assay. When signal transduction by inhibiting the Ci/Gli transcription factors coexpressed with a Flag-tagged Sufu (Fg-Sufu) in S2 cells, all (4–6). Moreover, mutations in human Sufu predispose to me- dulloblastoma and meningioma (7, 8). Much of the attention in Significance BIOLOGY the past has been given to the role of Sufu in the cytoplasmic DEVELOPMENTAL – sequestration of Ci/Gli (5, 9 14). In addition, Sufu is also re- Hedgehog (Hh) signaling controls embryonic development and quired for the production of the repressor form of Gli in mam- adult tissue homeostasis by regulating the Cubitus inter- mals (15–17), a function carried out by the kinesin-like protein Drosophila ruptus (Ci)/Glioma-associated oncogene homolog (Gli) fam- Costal2 (Cos2) in (18). However, several studies have ily of transcription factors. Abnormal Hh pathway activity suggested that Sufu may also function in the nucleus to inhibit causes congenital disease and cancer. As a conserved negative Drosophila cos2 the activator form of Ci/Gli. For example, in regulator of the Hh signaling pathway, the tumor suppressor mutant wing discs, full-length Ci accumulated in the nucleus in a protein Suppressor of fused (Sufu) binds and inhibits Ci/Gli, but latent form inhibited by Sufu (18, 19). In cultured mammalian how Sufu contacts Ci/Gli and how Sufu–Ci/Gli interaction in- cells, overexpression of a truncated Sufu could inhibit Gli activity hibits Hh signaling activity remain poorly understood. Here we without sequestering it in the cytoplasm (20). Sufu can interact identified a conserved Sufu-binding site in the C-terminal with several nuclear proteins, including the Drosophila myelodys- region of Ci/Gli. Further characterization of this Sufu-binding plasia/myeloid leukemia factor and transcriptional corepressor site provided insight into how Sufu blocks Ci/Gli activation in the complex Sin3–SAP18 (21, 22); however, a role for these nuclear nucleus. Understanding the mechanism by which Sufu regulates factors in the regulation of Ci/Gli activity has not been demon- Ci/Gli activity is important for developing therapeutic treat- strated by a loss-of-function study (17). ment of cancers caused by abnormal Hh pathway activation. In this study, we observed that Sufu could still inhibit a full- length Ci lacking the previously identified N-terminal Sufu-binding Author contributions: Y.H., Q.S., and J.J. designed research; Y.H. and Q.S. performed motif. Following up this unexpected observation, we identified a research; Y.H., Q.S., and J.J. analyzed data; and Q.S. and J.J. wrote the paper. previously unidentified and conserved Sufu-binding motif located The authors declare no conflict of interest. at the C terminus of Ci/Gli. We show that both the N- and C-ter- This article is a PNAS Direct Submission. minal Sufu-interacting sites are required for optimal binding of 1Y.H. and Q.S. contributed equally to this work. Ci/Gli to Sufu as well as for effective inhibition of Ci/Gli by Sufu. 2To whom correspondence should be addressed. Email: [email protected]. We find that the N- and C-terminal sites can mediate cyto- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. plasmic retention and nuclear inhibition of Ci/Gli by Sufu, 1073/pnas.1421628112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1421628112 PNAS | May 19, 2015 | vol. 112 | no. 20 | 6383–6388 Downloaded by guest on September 25, 2021 Fig. 1. Sufu binds Ci through both the N- and C-terminal domains. (A) A diagram of Ci domain structure and sequence alignment of SIN (blue bar) and SIC (green bar) within Ci and human (h) Gli proteins. ZF and CBP indicate the positions of the zinc-finger DNA-binding domain and CBP-binding domain, re- spectively. The SYGH motif is highlighted by a dashed box, and conserved residues in SIN and SIC are colored in blue and green, respectively. (B) Western blots (Left) and quantification (Right) of coimmunoprecipitation experiments from lysates of S2 cells coexpressing Fg-Sufu and various Myc-tagged Ci fragments (asterisks). The arrow indicates IgG. Data are means ± SD from two independent experiments. IB, immunoblotting; IP, immunoprecipitation. (C) Western blots of GST pull-down experiments. One or 5 μg of GST or GST fusion proteins was used as bait and coincubated with equal amounts of cell lysates from S2 cells expressing Fg-Sufu. (D) Western blots (Top) and quantification (Bottom) of coimmunoprecipitation experiments from lysates of S2 cells coexpressing Fg-Sufu and the indicated Myc-tagged Ci proteins. TCL, total cell lysates. Data are means ± SD from two independent experiments. (E) Western blots of a competition experiment. Equal amounts of immunopurified Fg-Sufu were incubated with cell extracts from S2 cells expressing a fixed amount of Myc-Ci1161–1397 and increasing amounts of Myc-Ci1–439. The arrow indicates IgG. (F) Western blots of coimmunoprecipitates from lysates of S2 cells coexpressing Fg-Sufu or D154R Fg-Sufu with Myc-Ci1–439 or Myc-Ci1161–1397. three Ci fragments coimmunoprecipitated Fg-Sufu, with Ci1–439 suggest that Sufu may simultaneously interact with both SIN and exhibiting 10-fold higher affinity than Ci440–1160 or Ci1161–1397 SIC, and that this multisite interaction greatly increases the overall (Fig. 1B). Sequence alignment revealed a C-terminally conserved binding affinity. sequence motif present in Ci (amino acids 1370–1397), Gli2, and Gli3 (Fig. 1A). To determine whether this conserved sequence Both SIN and SIC Contribute to Sufu-Mediated Inhibition of Ci. Having mediates the interaction between Ci and Sufu, we deleted it from established that Sufu can bind Ci through both SIN and SIC, we Ci1161–1397 to generate Ci1160–1370, and found that Ci1160–1370 next explored the functional significance of these Sufu-binding failed to bind Sufu (Fig. 1B). GST pull-down experiments domains in mediating Ci inhibition. We first compared the activity -PKA -PKAΔN -PKAΔC -PKAΔNΔC revealed that GST-Ci1370–1397 pulled down Fg-Sufu derived from of Ci ,Ci ,Ci ,andCi in the absence or S2 cell extracts but 10-fold less effectively compared with GST- presence of coexpressed Fg-Sufu using the ptc-luc reporter assay in -PKA – C – S2 cells.