Hedgehog signaling downregulates Suppressor of Fused through the HIB/SPOP-Crn axis in Drosophila

Abstract

Hedgehog (Hh) signaling plays vital roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and several types of cancer. Suppressor of Fused (Su(fu)) is a conserved inhibitory component of the Hh signaling pathway, but how it is regulated remains poorly understood. Here we demonstrate that in Drosophila Hh signaling promotes downregulation of Su(fu) through its target protein HIB (Hh-induced BTB protein). Interestingly, although HIB-mediated downregulation of Su(fu) depends on the E3 ubiquitin ligase Cul3, HIB does not directly regulate Su(fu) protein stability. Through an RNAi-based candidate gene screen, we identify the spliceosome factor Crooked neck (Crn) as a regulator of Su(fu) level. Epistasis analysis indicates that HIB downregulates Su(fu) through Crn. Furthermore, we provide evidence that HIB retains Crn in the nucleus, leading to reduced Su(fu) protein level. Finally, we show that SPOP, the mammalian homologue of HIB, can substitute HIB to downregulate Su(fu) level in Drosophila. Our study suggests that Hh regulates both Ci and Su(fu) levels through its target HIB, thus uncovering a novel feedback mechanism that regulates Hh signal transduction. The dual function of HIB may provide a buffering mechanism to fine-tune Hh pathway activity.

Hedgehog-induced phosphorylation by CK1 sustains the activity of Ci/Gli activator

“…Interestingly, HIB is up-regulated in response to Hh in both embryos and imaginal discs (31,32), and HIB also down-regulates Sufu through Crn (33), thus forming feedback loops to fine-tune Ci A activity. However, it is not clear how HIB-mediated degradation of Ci A is kept in check to prevent premature loss of Hh signaling activity.…”

Regulation of Hedgehog signaling by ubiquitination

“…Disrupting the Cul3-HIB complex, Cul3 E3 ligase activity, proteosomal activity, or polyubiquitination stabilized Sufu protein. However, in contrast to the direct ubiquitination of Sufu observed in mammalian cells (Yue et al 2009), HIB did not target Sufu itself for degradation in Drosophila , nor did it affect nuclear export or levels of Sufu mRNA when overexpressed (Liu et al 2014a). …”

“…Hh signaling induced downregulation of Sufu was also observed in flies and requires HIB, the substrate recognition and binding component of Cul3-HIB E3 ligase complex (Liu et al 2014a). Either activating Hh signaling or overexpressing HIB in the wing imaginal disc promoted degradation of Sufu (Zhang et al 2006a; Liu et al 2014a).…”

“…Either activating Hh signaling or overexpressing HIB in the wing imaginal disc promoted degradation of Sufu (Zhang et al 2006a; Liu et al 2014a). HIB itself is upregulated by the Hh signal (Zhang et al 2006a), and mutant clones that lack HIB had elevated Sufu protein levels, while re-expression of HIB along with Hh restored Sufu downregulation (Liu et al 2014a). These findings could be recapitulated when HIB was replaced by its mammalian homolog SPOP, indicating that HIB regulation of Sufu protein levels through Hh signaling may be conserved from flies to mammals (Liu et al 2014a).…”

“…HIB itself is upregulated by the Hh signal (Zhang et al 2006a), and mutant clones that lack HIB had elevated Sufu protein levels, while re-expression of HIB along with Hh restored Sufu downregulation (Liu et al 2014a). These findings could be recapitulated when HIB was replaced by its mammalian homolog SPOP, indicating that HIB regulation of Sufu protein levels through Hh signaling may be conserved from flies to mammals (Liu et al 2014a). Disrupting the Cul3-HIB complex, Cul3 E3 ligase activity, proteosomal activity, or polyubiquitination stabilized Sufu protein.…”