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Hedgehog signaling pathway: an overview

​​Proteins of the Hedgehog (Hh) family are powerful signaling molecules that act as morphogens during development in both vertebrates and invertebrates.  

Hh was first discovered in a genetic screen performed on cuticle embryo, that aimed to understand the body segmentation of Drosophila melanogaster (Nusslein-Volhard and Wieschaus, 1980). In this screen, mutant embryos for Hh developed as prickly little balls similar to a hedgehog (so the name of the protein).

The core components of the Hh pathway were initially identified in Drosophila​ and are conserved in vertebrates, where the pathway has maintained the same mechanisms of action through species (although with some exceptions). Most interesting, deregulation of the Hh pathway leads to developmental defects and cancer.

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.

References

  • Alexandre C, Jacinto A and Ingham PW (1996). Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins. Genes Dev 10, 2003–2013.
  • Aza-Blanc P1, Ramírez-Weber FA, Laget MP, Schwartz C and Kornberg TB (1997). Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell, 89, 1043–1053.
  • Bolanos-Garcia VM and Miguel RN (2003). On the structure and function of apolipoproteins: more than a family of lipid-binding proteins. Prog Biophys Mol Biol, 83, 47–68.
  • Burke R, Nellen D, Bellotto M, Hafen E, Senti KA, Dickson BJ and Basler K (1999). Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells. Cell, 99, 803–815.
  • Chamoun Z, Mann RK, Nellen D, von Kessler DP, Bellotto M, Beachy PA and Basler K (2001). Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal. Science, 293, 2080–2084.
  • Chen Y and Struhl G (1996). Dual roles for patched in sequestering and transducing Hedgehog. Cell, 1, 553–563.
  • Chen Y, Sasai N, Ma G, Yue T, Jia J, Briscoe J and Jiang J (2011). Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened. PLoS Biol, 9, e1001083. 
  • Chen Y and Jiang J (2013). Decoding the phosphorylation code in Hedgehog signal transduction. Cell Res, 23, 186–200.
  • Cheung HO, Zhang X, Ribeiro A, Mo R, Makino S, Puviindran V, Law KK, Briscoe J and Hui CC (2009). The kinesin protein Kif7 is a critical regulator of Gli transcription factors in mammalian hedgehog signaling. Sci Signal, 2, ra29.
  • Claret S, Sanial M and Plessis A (2007). Evidence for a novel feedback loop in the Hedgehog pathway involving Smoothened and Fused. Curr Biol, 17, 1326–33. 
  • Denef N, Neubüser D, Perez L and Cohen SM (2000). Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened. Cell, 18, 521–531.
  • Ding Q, Motoyama J, Gasca S, Mo R, Sasaki H, Rossant J and Hui CC (1998). Diminished Sonic hedgehog signaling and lack of floor plate differentiation in Gli2 mutant mice. Development, 125, 2533–2543.
  • Fan J, Liu Y and Jia J (2012). Hh-induced Smoothened conformational switch is mediated by differential phosphorylation at its C-terminal tail in a dose- and position-dependent manner. Dev Biol, 366, 172–184.
  • Gallet A, Rodriguez R, Ruel L and Therond, PP (2003). Cholesterol modification of hedgehog is required for trafficking and movement, revealing an asymmetric cellular response to hedgehog. Dev Cell, 4, 191–204.
  • Gallet A and Therond PP (2005). Temporal modulation of the Hedgehog morphogen gradient by a patched-dependent targeting to lysosomal compartment. Dev Biol, 277, 51–62.
  • Hooper JE (2003). Smoothened translates Hedgehog levels into distinct responses. Development, 130, 3951–3963.
  • Huangfu D, Liu A, Rakeman AS, Murcia NS, Niswander L and Anderson KV (2003). Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature, 426, 83–87.
  • Ingham PW, Taylor AM and Nakano Y. (1991). Role of the Drosophila patched gene in positional signalling. Nature, 353, 184–187.
  • Ingham PW and McMahon AP (2001). Hedgehog signalling in animal development: paradigms and principles. Genes Dev, 15, 3059–3087.
  • Jia J, Tong C and Jiang J (2003). Smoothened transduces Hedgehog signal by physically interacting with Costal2/Fused complex through its C-terminal tail. Genes Dev, 17, 2709–2720.
  • Khare N and Baumgartner S (2000). Dally-like protein, a new Drosophila glypican with expression overlapping with wingless. Mech Dev, 99, 199–202.
  • Lee JD and Treisman JE (2001). Sightless has homology to transmembrane acyltransferases and is required to generate active Hedgehog protein. Curr Biol, 11, 1147–1152.
  • Liu Y, Cao X, Jiang J and Jia J (2007). Fused-Costal2 protein complex regulates Hedgehog-induced Smo phosphorylation and cell-surface accumulation. Genes Dev, 21, 1949–1963.
  • Lum L, Zhang C, Oh S, Mann RK, von Kessler DP, Taipale J, Weis-Garcia F, Gong R, Wang B and Beachy PA (2003). Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2. Mol Cell, 12, 1261–1274.
  • Matise MP, Epstein DJ, Park HL, Platt KA and Joyner AL (1998). Gli2 is required for induction of floor plate and adjacent cells, but not most ventral neurons in the mouse central nervous system. Development, 125, 2759–2770.
  • Merchant M, Vajdos FF, Ultsch M, Maun HR, Wendt U, Cannon J, Desmarais W, Lazarus RA, de Vos AM and de Sauvage FJ. (2004). Suppressor of fused regulates Gli activity through a dual binding mechanism. Mol Cell Biol, 24, 8627–8641.
  • Méthot N and Basler K (1999). Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Cell, 96, 819–831.
  • Nakato H, Futch TA and Selleck SB (1995). The division abnormally delayed (dally) gene: a putative integral membrane proteoglycan required for cell division patterning during postembryonic development of the nervous system in Drosophila. Development, 121, 3687–3702.
  • Nusslein-Volhard C and Wieschaus E (1980). Mutation affecting segment number and polarity in Drosophila. Nature, 287, 795–801.
  • Nybakken KE, Turck CW, Robbins DJ and Bishop JM (2002). Hedgehog-stimulated phosphorylation of the kinesin-related protein Costal2 is mediated by the serine/threonine kinase fused. J Biol Chem, 277, 24638–24647.
  • Ogden SK, Ascano M Jr, Stegman MA, Suber LM, Hooper JE and Robbins DJ (2003). Identification of a functional interaction between the transmembrane protein Smoothened and the kinesin-related protein Costal2. Curr Biol, 13, 1998–2003.
  • Olofsson SO, Asp L and Borén J (1999). The assembly and secretion of apolipoprotein B-containing lipoproteins. Curr Opin Lipidol, 10, 341–346. 
  • Park HL, Bai C, Platt KA, Matise MP, Beeghly A, Hui CC, Nakashima M and Joyner AL (2000). Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation. Development, 127, 1593–1605.
  • Pepinsky RB, Zeng C, Wen D, Rayhorn P, Baker DP, Williams KP, Bixler SA, Ambrose CM, Garber EA, Miatkowski K, Taylor FR, Wang EA and Galdes A (1998). Identification of a palmitic acid modified form of human Sonic hedgehog. J Biol Chem, 273, 14037–14045.
  • Porter JA, Young KE and Beachy PA (1996). Cholesterol modification of hedgehog signaling proteins in animal development. Science, 274, 255–259.
  • Ranieri N, Ruel L, Gallet A, Raisin S and Thérond PP (2012). Distinct phosphorylations on kinesin costal-2 mediate differential hedgehog signaling strength. Dev Cell, 22, 279–294.
  • Ranieri N, Thérond PP and Ruel L (2014). Switch of PKA substrates from Cubitus interruptus to Smoothened in the Hedgehog signalosome complex. Nat Commun, 5, 5034.
  • Robbins DJ, Nybakken KE, Kobayashi R, Sisson JC, Bishop JM and Thérond PP (1997). Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein costal2. Cell, 90, 225–234.
  • Rohatgi R, Milenkovic L and Scott MP (2007). Patched1 regulates hedgehog signaling at the primary cilium. Science, 317, 372–376.
  • Ruel L, Rodriguez R, Gallet A, Lavenant-Staccini L and Thérond PP (2003). Stability and association of Smoothened, Costal2 and Fused with Cubitus interruptus are regulated by Hedgehog. Nat Cell Biol, 5, 907–913.
  • Ruel L, Gallet A, Raisin S, Truchi A, Staccini-Lavenant L, Cervantes A and Thérond PP (2007). Phosphorylation of the atypical kinesin Costal2 by the kinase Fused induces the partial disassembly of the Smoothened-Fused-Costal2-Cubitus interruptus complex in Hedgehog signalling. Development, 134, 3677–3689.
  • Sisson JC, Ho KS, Suyama K and Scott MP (1997). Costal2, a novel kinesin-related protein in the Hedgehog signaling pathway. Cell, 90, 235–245.
  • Slusarski DC, Motzny CK and Holmgren R (1995). Mutations that alter the timing and pattern of cubitus interruptus gene expression in Drosophila melanogaster. Genetics, 139, 229–240.
  • Stegman MA, Vallance JE, Elangovan G, Sosinski J, Cheng Y and Robbins DJ (2000). Identification of a tetrameric hedgehog signaling complex. J Biol Chem, 275, 21809–21812.
  • Stone DM, Murone M, Luoh S, Ye W, Armanini MP, Gurney A, Phillips H, Brush J, Goddard A, de Sauvage FJ and Rosenthal A (1999). Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli. J Cell Sci, 112, 4437–4448.
  • Svärd J, Heby-Henricson K, Persson-Lek M, Rozell B, Lauth M, Bergström A, Ericson J, Toftgård R and Teglund S (2006). Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway. Dev Cell, 10, 187–197. 
  • Tay SY, Ingham PW and Roy S (2005). A homologue of the Drosophila kinesin-like protein Costal2 regulates Hedgehog signal transduction in the vertebrate embryo. Development, 132, 625–634.
  • Tempé D, Casas M, Karaz S, Blanchet-Tournier MF and Concordet JP (2006). Multisite protein kinase A and glycogen synthase kinase 3beta phosphorylation leads to Gli3 ubiquitination by SCFbetaTrCP. Mol Cell Biol, 26, 4316–4326.
  • Wang G, Amanai K, Wang B and Jiang J (2000). Interactions with Costal2 and suppressor of fused regulate nuclear translocation and activity of cubitus interruptus. Genes Dev, 14, 2893–2905.
  • Wang G and Jiang J (2004). Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Dev Biol, 268, 493–505.
  • Wang QT and Holmgren RA (2000). Nuclear import of cubitus interruptus is regulated by hedgehog via a mechanism distinct from Ci stabilization and Ci activation. Development, 127, 3131–3139.
  • Wilson CW, Nguyen CT, Chen MH, Yang JH, Gacayan R, Huang J, Chen JN and Chuang PT (2009). Fused has evolved divergent roles in vertebrate Hedgehog signalling and motile ciliogenesis. Nature, 459, 98–102.
  • Zhang C, Williams EH, Guo Y, Lum L and Beachy PA (2004). Extensive phosphorylation of Smoothened in Hedgehog pathway activation. Proc Natl Acad Sci USA, 101, 17900–17907. 
  • Zhang W, Zhao Y, Tong C, Wang G, Wang B, Jia J and Jiang J (2005). Hedgehog-regulated Costal2-kinase complexes control phosphorylation and proteolytic processing of Cubitus interruptus. Dev Cell, 8, 267–278.
  • Zhao Y, Tong C and Jiang J (2007). Hedgehog regulates smoothened activity by inducing a conformational switch. Nature, 450, 252–258. 
  • Zheng X, Mann RK, Sever N and Beachy PA. (2010). Genetic and biochemical definition of the Hedgehog receptor. Genes Dev, 24, 57–71. 
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