Targeting Hedgehog Signalling through the Ubiquitylation Process: The Multiple Roles of the HECT-E3 Ligase Itch

Abstract

Hedgehog signalling (Hh) is a developmental conserved pathway strongly involved in cancers when deregulated. This important pathway is orchestrated by numerous regulators, transduces through distinct routes and is finely tuned at multiple levels. In this regard, ubiquitylation processes stand as essential for controlling Hh pathway output. Although this post-translational modification governs proteins turnover, it is also implicated in non-proteolytic events, thereby regulating the most important cellular functions. The HECT E3 ligase Itch, well known to control immune response, is emerging to have a pivotal role in tumorigenesis. By illustrating Itch specificities on Hh signalling key components, here we review the role of this HECT E3 ubiquitin ligase in suppressing Hh-dependent tumours and explore its potential as promising target for innovative therapeutic approaches.

Keywords: GLI1; Hedgehog; Itch; Numb; Patched1; SuFu; cancer; ubiquitylation; β-arrestin2.

Figure 1

Ubiquitylation process. Ubiquitin (Ub) is attached to substrates by a cascade of reactions involving three enzymes. An E1 enzyme activates Ub in an ATP-dependent way. An E2 enzyme binds and then transfers the activated Ub to a substrate specifically bound to an E3 ligase. E3 enzyme can be divided into two main families: the RING and the HECT E3 ligases. Although, the ubiquitylation process usually leads to the degradation of the substrate, it can also drive regulative events.

Figure 2

Schematic representation of the modular structure of Itch and its involvement in Hh pathway regulation. (A) Itch protein consists of an N-terminal Ca²⁺/lipid-binding (C2) domain (green hexagon), a proline rich motif (orange rectangle) displaying regulatory functions, a central region containing four WW domains (yellow bars) involved in protein-protein interaction and a catalytic HECT domain (blue rectangle). (B) The scheme shows the substrates of Itch involved in the regulation of Hh pathway (SuFu, GLI1 and PTCH1). Itch mediates regulatory events on SuFu and proteasome degradation on GLI1 and PTCH1 by the interaction with the adaptor proteins β-arrestin2 and Numb, respectively.

Figure 3

Role of Itch in canonical Hh pathway. (A) Model of the Numb/Itch-dependent regulation of GLI1 activity. Numb binds WW2 domain of Itch, releases Itch from its close-inactive conformation, thus allowing the recruitment of GLI1 and its degradation. In cancer, loss of Numb impairs this process enhancing GLI1 activity and promoting cell proliferation and tumorigenesis. (B) Model of the β-arrestin2/Itch-dependent regulation of the SuFu function. Itch, in complex with β-arrestin2, binds and ubiquitylates SuFu. This event does not lead to SuFu degradation but increases the association between SuFu and GLI3 promoting the generation of GLI3 repressor form (GLI3R) and inhibiting Hh signalling. Alterations in this mechanism, caused by SuFu mutations that make it insensitive to Itch-dependent ubiquitylation, are responsible for MB development.

Figure 4

Role of Itch in Type I non-canonical Hh pathway. (A) In the absence of Hh, PTCH1 recruits a pro-apoptotic complex, including DRAL, TUCAN1 and Caspase 9, to its C-Terminal Domain (CTD). The activation of the Caspase 9 promotes the cleavage of the PTCH1 CTD by the Caspase 3, leading to irreversible commitment to cell death. (B) In the presence of Itch, the Lys1413 at the PTCH1 CTD is ubiquitylated, leading to the internalization and degradation of the PTCH1 and to the impairment of its pro-apoptotic activity.