Hedgehog signaling pathway: an overview

Hh signaling cascade in Drosophila

Hh maturation, release and movement

Hh is first synthesized as a precursor. It undergoes autoproteolytic cleavage where a cholesterol molecule (Porter et al., 1996), and a palmitic acid molecule (Ingham and McMahon, 2001) are added to the final product. The primary role of these modifications is to direct the mature signal to interact with a set of cellular components that are responsible of the Hh secretion, movement and reception. In particular, cholesterol is involved in Hh trafficking and movement (Gallet et al., 2003), whereas palmitoylation in Hh signaling (Chamoun et al., 2001; Liu et al., 2007). 

Once Hh is modified, it is ready to be secreted from the cells (Burke et al., 1999). After secretion, Hh interacts with the extracellular matrix and has to find a way to move through it to reach the receiving cells, forming a concentration gradient.

Several models have been proposed to explain how Hh can move far from its source, such as its movement inside a special structures called lipoprotein particles (Bolanos-Garcia and Miguel, 2003; Olofsson et al., 1999) and through its interaction with heparan sulphate proteoglycans (HSPGs) (Jia et al., 2003; Nakato et al., 1995). 

At the plasma membrane

Hh signal transduction is initiated at the plasma membrane where Hh interacts with its 12 transmembrane protein receptor Patched (Ptc) (Ingham and McMahon,2001). The interaction between Hh and Ptc is facilitated by the Ihog/Cdo family of coreceptors (Zhang et al., 2010). The binding between Ptc and Hh has two main important roles:

1. Limiting the spreading of Hh: the binding between Hh and Ptc results in their internalization, targeting Hh to lysosomes for degradation (Gallet and Therond, 2005). 
2. Increase of Smoothened (Smo) expression and activation: (Chen and Struhl, 1996; Denef et al., 2000; Lum et al., 2003; Taipale et al., 2002) this gives rise to a cascade of signal transmission that function to regulate the transcription factor Cubitus interruputs (Ci) (Alexandre et al., 1996; Méthot and Basler, 1999).

Once Hh binds Ptc, the seven-pass transmembrane protein Smo undergoes several phosphorylation events (Hh dose-dependent) (Fan et al., 2012). Smo phosphorylation occurs at its cytoplasmic tail (C-tail) which contains several phosphorylation sites of PKA, CK1, GSK3 (Zhang et al., 2004). The main consequences of Smo phosphorylation are: 

1. Promoting Smo cell surface expression by inhibiting ubiquitation-mediated endocytosis and degradation (Fan et al., 2012).
2. Controlling Smo conformation, which occurs on the C-tail itself of the Smo dimer that lead to an INACTIVE (C-tails far from each other in the absense of Hh) or ACTIVE (C-tails opening and approach in the presence of Hh). This conformation change is exclusively due to the phosphorylation events (Zhao et al., 2007).

Within the cytoplasm

The activation or inhibition of the Hh pathway is regulated by a multi-protein complex (Hh signaling complex, HSC) downstream of Smo. The components of the HSC complex are:

• The transcription factor Ci
• The serine/threonine kinase Fused (Fu)
• ​The kinesin-like molecule Costal 2 (Cos2), which also binds to PKA, CK1 and GSK3, all implicated in the Hh signaling pathway (Aza-Blanc et al., 1997).
• Suppressor of fused (Sufu)

The HSC complex is associated with microtubules in the absense of Hh (Robbins et al., 1997; Sisson et al., 1997; Stegman et al., 2000). In the presence of Hh, the complex dissociates from the microtubule and the Cos-Fu-Ci complex interacts with the C-tail of Smo (Hooper, 2003; Ingham et al., 1991; Lum et al., 2003; Ogden et al., 2003; Ruel et al., 2003) whereas the Sufu-Ci complex remains cytoplasmic.

Both Cos-Fu-Ci and Sufu-Ci complexes regulate the status of the transcription factor Ci. Ci is a 155 kDa protein (Ci-FL, full length) that contains a zinc finger domain responsible for its DNA binding (Slusarski et al., 1995). Ci is converted to an ACTIVE FORM (Ci-A, 155 kDa) responsible for target gene activation in the presence of Hh, or to a REPRESSOR FORM (Ci-R, 75 kDa), that still bind DNA but inhibit the pathway in the absence of Hh. 

Control of the active/inactive form of Ci is mediated by phosphorylation events that are mainly under the control of Cos2. In the absense of Hh, Cos2-Fu-Ci and Sufu-Ci complexes promote Ci-R formation preventing its activation (Robbins et al., 1997; Sisson et al., 1997; Wang et al., 2000; Wang and Holmgren, 2000; Wang and Jiang, 2004; Zhang et al., 2004). In the presence of Hh, the Cos2-Fu-Ci complex interacts with the C-tail of Smo domains, which is regulated by Cos2 phosphorylation (Liu et al., 2007; Nybakken et al., 2002; Ranieri et al., 2012; Ranieri et al., 2014; Ruel et al., 2007), promoting Ci-A formation and consequent pathway activation.

Figure 1. Drosophila Hh signal transduction pathway (Chen and Jiang, 2013). The mature Hh molecule reaches Hh receiving cells by binding with HSPGs, such as Dally and Dally-like (Dlp). In the absense of Hh, Ptc inhibits Smo allowing Ci to be phosphorylated by PKA, CK1 and GSK3. These phosphorylation events target Ci to a partial proteolytic cleavage (mediated by Slimb/β​TRCP) to generate the repressor form (Ci-R). Binding of Hh to its receptor Ptc and co-receptor Ihog releases Ptc inhibition on Smo, which undergoes phosphorylation mainly by PKA and CK1. Consequently, Smo accumulates at the cell surface recruiting the Cos2-Fu-Ci complex. Here, according to the amount of Hh received by the cell, phosphorylation events on Cos2 and Fu regulate the activation of Ci and therefore of the pathway itself.

Hh signaling orthologues in vertebrates

In mammals, there are three paralogous Hh genes: Sonic hedgehog (Shh, the most broadly expressed and best studied Hh molecule), Indian hedgehog (Ihh, primarily involved in bone differentiation) and Desert hedgehog (Dhh, involved in gonad differentiation).

The main difference between Hh signaling in Drosophila and vertebrates is the requirement for the vertebrate intraflaggular transport (IFT), which consists of large multisubunits complexes that are responsible for the bidirectional transport of proteins between the base and the tip cilia (Huangfu et al., 2003).

Both Ptc and Smo can localize to primary cilia in a mutually exclusive way, where the binding of Shh to Ptc allows Smo to move into the cilium, promoting pathway activation through the Gli transcription factors (Rohatgi et al., 2007).

Main similarities and differences between Drosophila and vertebrate Hh signaling are:

• The Smo structure is highly conserved between Drosophila and vertebrates. Interestingly, the phospho-sites on the Smo C-tail and their dimerization mechanism is conserved as well, though the kinases involved are slightly different (Chen et al., 2011).
• There are three Ci homologues known as Gli1, Gli2 and Gli3. Gli1 and Gli2 are transcriptional activators, whereas Gli3 functions as a transcriptional repressor (Ding et al., 1998; Matise et al., 1998; Park et al., 2000; Tempé et al., 2006). 
• Unlike Drosophila Sufu, vertebrate Sufu has a central and very important role in the Shh pathway (Svä​rd et al., 2006). However, the two proteins share high sequence homology (Merchant et al., 2004; Stone et al., 1999).
• The Cos2 homologues, kif7 and kif27, have conserved their negative role within the pathway by controlling Gli's function and abundance (Cheung et al., 2009; Tay et al., 2005; Wilson et al., 2009).
• Mammalian Fu can associate to kif27 and being involved in ciliogenesis, while a compensatory Fu kinase, associated with kif7, is necessary for Hh signaling (Wilson et al., 2009).

These suggest an evolutionary conservation in the Shh intracellular cascade, though further studies are necessary to better understand the molecular functions of the protein involved.

Figure 2. Mammal Hh signal transduction pathway (Chen and Jiang, 2013). The mature Hh molecule reaches Hh receiving cells by binding with HSPGs (such as GPC3, GPC4 and GPC6). In the absence of Hh, Ptc inhibits Smo allowing Gli to be phosphorylated by PKA, CK1 and GSK3. These phosphorylation events target Gli to a partial proteolytic cleavage (mediated by β​-TRCP) to generate the repressor form (Gli-R). In the presence of Hh, binding of Hh to its receptor Ptc and co-receptor Cdo releases Ptc inhibition on Smo, which undergoes phosphorylation by mainly CK1 and GRK2. Consequently, Smo accumulates at the cell surface (within the cilia). Sufu is the major negative regulator of the pathway (kif7 is a minor one). In the presence of Hh, Sufu destabilization and degradation allows the release of its repression on Gli, with consequent pathway activation.