2: Sonic Hedgehog Signaling Pathway

Hedgehog Pathway

• Discovered through Drosophila melanogaster larvae screens
• Fifteen loci were identified through the Drosophila screens as being involved in segmental patterning during development
• Hedgehog vertebrate homologs include Desert, Indian, and Sonic

Pathway Discovery

• Drosophila melanogaster Heidelberg screens led to its discovery
• Nobel Prize in medicine/physiology was awarded for it in 1995

Cyclopamine and Cyclopia

• Holoprosencephaly (cyclopia) is a common phenotype found in livestock who ate corn lily in Idaho from 1957-1968
• In 1957, the US Department of Agriculture started an eleven-year investigation that identified cyclopamine as the cause of the birth defect

Sonic Hedgehog (Shh) Signaling Pathway

• Sonic hedgehog (Shh) signaling is essential for proper embryonic development
• Full-length Shh autocleaves into an active 20-kDa N-terminal fragment (ShhN)
• ShhN binds to Patched (Ptc1), a 12-pass transmembrane receptor
• This binding reverses an inhibitory effect on Smoothened (Smo)
• This de-repression activates Gli transcription factors
• These factors regulate the transcription of target genes including Gli1 and Ptch1
• Uncontrolled activation of Hh-Gli pathway is linked to medulloblastoma (BBB), basal cell carcinoma, lung, liver, stomach, prostate and pancreatic cancer
• Selective targeting of pathway in these cells could serve as a potential chemotherapy
• Ptc inhibits Smo in the absence of a ligand
• Hedgehog is activated by autocatalytic cleavage, which yields a cholesterol modified amino terminal signaling domain
• Subsequent palmitoylation leads to an active ligand
• Hedgehog (Shh) relieves Ptch inhibition of Smo
• Downstream activation of GLi zinc finger transcription factors

Developmental Impact

• Important for brain, lungs, skeletal, musculature, and GI tract development
• Dysregulation of pathway has serious developmental implications like cyclopia

Hedgehog Signal Transduction Network

• Hedgehog (Hh) proteins regulate the Gli family of transcription factors, part of the "canonical" Hh signaling pathway
• Numerous studies have shown that Hh proteins also signal through Gli-independent mechanisms that are collectively referred to as “noncanonical” signaling pathways
• Noncanonical Hh signaling is subdivided into two distinct signaling modules:
  • Those not requiring Smoothened (Smo)
  • Those downstream of Smo that do not require Gli transcription factors

Gli and Ci Regulation

• In the absence of signal:
  • Drosophila melanogaster Cubitus interruptus (Ci) and vertebrate GLI proteins convert into a transcriptional repressor (CiR and GLIR, respectively)
  • The sequential phosphorylation of Ci and GLI proteins by protein kinase A (PKA), CKI and glycogen synthase kinase 3β (GSK3β)
    which is in part facilitated by Costal 2 (Cos2) and KIF7 in D. melanogaster and vertebrates respectively.
  • Modified protein gets partially proteolytically processed by the proteasome
  • Truncated protein translocates to the nucleus to repress target gene expression
• Activation of Smoothened (SMO) promotes the formation of a transcriptional activator (CiA and GLIA) by blocking Ci and GLI processing
• Additional post-translational modifications, might generate transcriptionally active forms of Ci and GLI proteins (CiA and GliA)
• Cellular metabolic processes, such as axon guidance, can also be influenced by the pathways.
• Structural features for GLI2 and GLI3 include:
  • Five C2H2 zinc-fingers which makeup a DNA-binding domain
  • An amino-terminal transcriptional repressor domain
  • Clusters of phosphorylation sites that are essential for proteolytic process to yield a transcriptional repressor

Additional points

• In vertibrates, Ptc is considered a negative regulator of Smo
• Prevents Smo membrane localization and activation so that Smo is retained on intracellular vesicles
• Full-length Gli is held in a microtubule-associated complex containing the kinase Kif7 and Suppressor of Fused (Sufu)
• Sufu promotes phosphorylation of Gli by PKA, GSK3, and CK1 and signalling is thought to take place on primary cilia.
• Smo translocates to both the plasma membrane and to the primary cilium, where it regulates Gli processing and activation when Hh ligand binds