Multisite interaction with Sufu regulates Ci/Gli activity through distinct mechanisms in Hh signal transduction

Abstract

The tumor suppressor protein Suppressor of fused (Sufu) plays a conserved role in the Hedgehog (Hh) signaling pathway by inhibiting Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) transcription factors, but the molecular mechanism by which Sufu inhibits Ci/Gli activity remains poorly understood. Here we show that Sufu can bind Ci/Gli through a C-terminal Sufu-interacting site (SIC) in addition to a previously identified N-terminal site (SIN), and that both SIC and SIN are required for optimal inhibition of Ci/Gli by Sufu. We show that Sufu can sequester Ci/Gli in the cytoplasm through binding to SIN while inhibiting Ci/Gli activity in the nucleus depending on SIC. We also find that binding of Sufu to SIC and the middle region of Ci can impede recruitment of the transcriptional coactivator CBP by masking its binding site in the C-terminal region of Ci. Indeed, moving the CBP-binding site to an "exposed" location can render Ci resistant to Sufu-mediated inhibition in the nucleus. Hence, our study identifies a previously unidentified and conserved Sufubinding motif in the C-terminal region of Ci/Gli and provides mechanistic insight into how Sufu inhibits Ci/Gli activity in the nucleus.

Suppressor of Fused Chaperones Gli Proteins To Generate Transcriptional Responses to Sonic Hedgehog Signaling

“…However, in Drosophila salivary glands and wing imaginal discs, ectopically expressed Sufu was shown to enter the nucleus with Ci (49), and mammalian Sufu was also shown to be capable of recruiting the transcriptional corepressor complex through an interaction with SAP18 (50). Sufu is known to form two contact points with Gli proteins (51,52); recent data indicate that Sufu impedes the nuclear trafficking of Ci by masking a proline-tyrosine nuclear localization signal (PY-NLS) in the N terminus and blocks the recruitment of transcriptional coactivator CBP to the C-terminal binding site (51,53). Moreover, Sufu was reported to interact with two nuclear proteins, p66␤ and MycBP (54), strongly suggesting that it possesses crucial nuclear functions.…”

Members of the Rusc protein family interact with Sufu and inhibit vertebrate Hedgehog signaling

“…One major difference is Sufu, which is dispensable for Drosophila Hh signaling (Préat, 1992) but functions as a major pathway inhibitor in vertebrates (Cooper et al, 2005;Svard et al, 2006;Min et al, 2011). Sufu physically interacts with Gli proteins and regulates their stability, localization and activities (Ding et al, 1999;Kogerman et al, 1999;Murone et al, 2000;Lin et al, 2014;Han et al, 2015). Loss of Sufu elevates vertebrate Hh signaling and induces severe patterning defects during development (Wolff et al, 2003;Cooper et al, 2005;Svard et al, 2006;Min et al, 2011).…”

“…As the major Gli inhibitor, Sufu forms complexes with Gli proteins and sequesters them in the cytoplasm (Ding et al, 1999;Kogerman et al, 1999;Pearse et al, 1999;Stone et al, 1999;Zhang et al, 2013;Han et al, 2015). In the nucleus, Sufu recruits p66β to the promoters of Hh target genes and represses Gli-dependent transcription (Lin et al, 2014).…”

“…At the molecular level, Sufu directly binds Gli proteins when the Hh pathway is quiescent (Ding et al, 1999;Kogerman et al, 1999;Pearse et al, 1999;Stone et al, 1999;Zhang et al, 2013;Han et al, 2015). Sufu can inhibit Gli-dependent transcription through sequestering Gli proteins in the cytoplasm (Ding et al, 1999;Kogerman et al, 1999;Murone et al, 2000;Han et al, 2015).…”

“…Sufu can inhibit Gli-dependent transcription through sequestering Gli proteins in the cytoplasm (Ding et al, 1999;Kogerman et al, 1999;Murone et al, 2000;Han et al, 2015). In the nucleus, Sufu recruits the NuRD repressor complex member p66β (Gatad2b) to the promoters of Hh target genes and suppresses Glidependent transcription (Lin et al, 2014).…”