Characterization of the Physical Interaction of Gli Proteins with SUFU Proteins

Abstract

The Hedgehog signaling pathway is involved in both development and cancer induction in a wide range of organisms. The end point of the Hedgehog signal-transduction cascade is the Gli/Ci, zinc-finger transcription factors. Proteins such as Fused, Suppressor of fused (SUFU), Costal-2, and protein kinase A are essential for regulation of Gli/Ci processing, activity, and localization. Coimmunoprecipitation and Far Western assays, coupled with truncation analysis and mutagenesis have been used to define the region of interaction between Gli proteins and SUFU. We identify a novel motif SYGH in Gli/Ci family proteins, which is required for the interaction with SUFU. Mutational studies revealed that Gly 122 and His 123 are crucial for binding to SUFU, suggesting the importance of hydrophobicity for the correct binding conformation. Functional analysis revealed that the activity of GLI transcription factors with mutations in this motif is no longer suppressed by coexpression of SUFU. Moreover, we have found that a C-terminal 19-amino acid deletion in SUFU (⌬465) is sufficient to abrogate interaction with GLI1. Interestingly, this SUFU mutant localizes in the nucleus, most probably because it is not efficiently sequestered in the cytoplasm. Taken together, we identified a novel motif in the Gli/Ci family of proteins that is essential both for protein-protein interaction with SUFU and for functional repression of GLI1 by SUFU.

Modulation of the Transactivation Function and Stability of Krüppel-like Zinc Finger Protein Gli-similar 3 (Glis3) by Suppressor of Fused

“…4). SUFU-DsRed was observed predominantly in the cytoplasm and to a lesser degree in the nucleus when expressed alone, in agreement with previous reports (22,26). However, SUFU was largely (Ͼ80% of cells) localized to the nucleus in cells in which SUFU was co-expressed with Glis3 (Fig.…”

“…A previous report showed that protein-protein interactions by SUFU involve recognition of an SYGH motif (22). Interestingly, Glis3 contains a similar motif, VYGH, located between amino acids 348 and 351 within the conserved region described above ( Fig.…”

“…The plasmids p3xFlag-CMV-Glis3⌬N34, -65, -155, -288, -295, -319, -333, -355, and -388 and p3xFlag-CMV-Glis3⌬C389 were generated by PCR amplification of the respective fragments and inserting them into the EcoRI and BamHI restriction sites of the p3xFlag-CMV10 expression vector (Sigma). pCMV-Myc-SUFU was kindly provided by Rune Toftgard (Karolinska Institutet, Huddinge, Sweden) (22). The mIP(Ϫ696)-Luc luciferase reporter construct was described previously (6).…”

Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate

“…Su(fu) tethers both the activator and repressor forms of Gli in the cytoplasm, resulting in downregulation of both activities 6,16 . Inhibition of Su(fu) by miR-214 in adaxial cells, which experience high amounts of Hedgehog signaling, allows maximal activation of Gli-mediated transcription (Fig.…”

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development

“…Sufu (Suppressor of fused) acts as a central negative regulator of Hh signaling by sequestering the Gli transcription factors in an inactive complex (Kogerman et al , 1999; Dunaeva et al , 2003; Merchant et al , 2004; Humke et al , 2010). Sufu deletion in mice leads to the continuous activation of Hh signal and embryonic lethality at day 9.5 (Cooper et al , 2005; Svard et al , 2006), underscoring the essential role of Sufu for proper development.…”