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Hedgehog signaling pathway

Hedgehog signaling pathway. Indian hedgehog : expressed in gut and chondrocytes Desert hedgehog : expressed in sertoli cells of the testes Sonic hedgehog : Involved in many developmental processes. Best characterized. Gilbert, Figure 6.24. Shh and Cholesterol.

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Hedgehog signaling pathway

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  1. Hedgehog signaling pathway • Indian hedgehog: expressed in gut and chondrocytes • Desert hedgehog: expressed in sertoli cells of the testes • Sonic hedgehog: Involved in many developmental processes. Best characterized

  2. Gilbert, Figure 6.24

  3. Shh and Cholesterol • Amino-terminal portion is secreted and functional (also palmitoylated) • Cholesterol is required for the cleavage of Shh (N-terminal is active peptide) • Patched protein requires cholesterol in order to function (sterol sensing domain) • Mutations or chemicals that interfere with cholesterol biosynthesis cause abnormalities akin to SHH knockout animals

  4. Patched protein (Ptc) • Is the Shh “receptor” • 12 Transmembrane domains • Negative regulator of Shh signaling • 2 Vertebrate homologs Ptc-1 and Ptc-2 • Ptc-1 is upregulated in response to Shh • Ptc has a sterol sensing domain (SSD)

  5. Smoothened protein (Smo) • 7 transmembrane domains (like G-protein coupled receptors) • Positive regulator of the Shh pathway • Does not bind to Shh but is signaling component of the receptor complex • Smo is constitutively active in the absence of Ptc

  6. Cubitus interruptus (Ci)/Gli transcription factor family • Zn++ finger transcription factors • Recognize a 9bp consensus sequence in the promoters of several Hh target genes • In the absense of Hh the full length protein (Ci-155) is in the cytoplasm and gets processed into a 75 kD N-terminal repressor form (Ci-75)

  7. Modificaiton of Ci in DrosophilaFrom Munroe, et. al. (1999) Exp. Cell Res. 253:25-33

  8. Ci-75 repressor generation is: • Triggered in part by protein kinase A (Pka) phosphorylation of the C-terminus • Possible participation of Slimb, a ubiquitin targeting protein and the proteosome • Cleavage of Ci-155 is blocked by the presence of Hedgehog (Smo signaling)

  9. Hedgehog activation of “positively regulating” Ci-155 • Facilitated by a serine/threonine kinase called fused • Antagonized by “Supressor of fused” Su(fu) • Mutant embryos for Fu or Ci have defects resembling Hh deficiency • Pka or Ptc mutants display ectopic expression of Hh target genes • CBP/p300 is a co-activator for Ci-155

  10. Role of microtuble association • Cos-2 tethers Ci and Fu to the microtubles • Cos-2 is a negative regulator of the pathway • Hh signaling leads to Cos-2 dissociation and phosphorylation of both Fu and Cos-2 • Unanswered questions: • How does Smo signal to the microtuble associated complex? • What exactly is in the complex?

  11. Hedgehog signaling pathwayFrom Munroe, et. al. (1999) Exp. Cell Res. 253:25-33

  12. Vertebrate versions of Ci (the Gli proteins) • Behave differently in this pathway than Ci does in Drosophila • Gli-1, Gli-2 and Gli-3 appear to be transcriptionally regulated in response to Shh • See Munroe et. al. (1999) for several assorted speculations

  13. Evidence for Sonic Hedgehog involvement in development • Mouse knockout results in abnormal limb development and cyclopia • Gli-3/Ci mutations lead to Grieg's cephalopolysyndactyly and other inherited diseases • Activating mutations can cause cancers (basal cell carcinomas-epidermal)

  14. Wnt/wingless pathway • Ligands, Wnt-secreted glycoproteins • At least 16 members of the Wnt ligand family • Receptors are relatives of Frizzled (Frz) • 11 Frizzled homologs in vertebrates • Soluble Frz related proteins (Frps) exist • Antagonists of wnt signaling • Proposed to play a role in neural development

  15. Wnt signal transductionFig 6.23 of Gilbert

  16. Negative regulators of wnt signals • Glycogen synthase kinase (GSK)-3b • Binds and phosphorylates several proteins in the wnt pathway to downregulate b-catenin • Adenomatous polyposis coli (APC) • Tumor suppressor in which mutations eliminate binding sites for Axin and b-catenin • Axin: Binds APC, b-catenin, GSK-3b and dishevelled (Dvl) • Facilitates phosphorylation of APC and b-catenin by GSK-3b

  17. A closer look at b-catenin regulationFrom Polakis (2000) Genes Dev. 14:1837-1851

  18. b-catenin mutations in tumors Table 1 From Polakis (2000) Genes Dev. 14:1837-1851

  19. Clustered mutations in wnt signaling components affect negative interactions • b-catenin mutations in the N-terminal region affect amino acids necessary for its phosphorylation dependent interaction with protein degradation machinery • APC mutations affect axin and b-catenin binding • Axin mutations truncate the protein thus eliminating b-catenin binding sites

  20. The wnt pathway is loaded with proto-oncogenes and tumor suppressors Figure 1 From Polakis (2000) Genes Dev. 14:1837-1851

  21. Fibroblast Growth Factors (FGF) • 19-22 FGF family members (FGF-1 called acidic FGF, FGF-2 called basic FGF) • Some lack signal sequences for secretion • Contain heparin/heparan sulfate binding domains (critical for the function of some FGFs) • Four FGF receptors. Several FGFs bind to more than one FGF receptor with high affinity • FGFRs are transmembrane tyrosine kinases

  22. Evidence for FGF involvement in development: KO mice • FGF2: defects in vascular systems • FGF3: inner ear and tail development • FGF4: early post-implantation lethality • FGF5 and FGF7: Abnormal hair phenotype • FGF8: Early embryonic lethality. Conditional KO showed effect on brain development • FGF10: lung and limb development

  23. FGF Receptor KO phenotypes • FGFR-1: Postimplantation embryonic lethal with vertebral malformations • FGFR-2: Postimplantation embryonic lethal with abnormal limb development • FGFR-3: Defective chondrocyte generation • FGFR-4: Perfectly normal • Websites 6.2 and 6.6 deal with FGF & FGFR

  24. Facts on Retinoic Acid • RA is a teratogen, causes birth defects • Interfering with RA biosynthesis causes developmental abnormalities linked to Hox gene expression • Exerts it effect through the retinoic acid receptors (RAR) which dimerizes with other steroid receptors to activate transcription • Important in development of limbs, neural tube and in anterior-posterior patterning

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