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Wnt signaling in development and disease

Wnt signaling in development and disease. Vítězslav Bryja, PhD. Institute of Experimental Biology Faculty of Science, Masaryk University Brno, Czech Republic. Wnts can activate diverse pathways. family of ligands glycosylated and palmitoylated extracellular proteins

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Wnt signaling in development and disease

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  1. Wnt signaling in development and disease Vítězslav Bryja, PhD. Institute of Experimental Biology Faculty of Science, Masaryk University Brno, Czech Republic

  2. Wnts can activate diverse pathways • family of ligands • glycosylated and palmitoylated extracellular proteins • short range of action, bind to extracellular matrix • only in multicellular animals non-canonical (eg. Wnt-5a) canonical (eg. Wnt-1 or Wnt-3a)

  3. Wnt/-kateninová dráha (= kanonická dráha) • induce axis duplication in Xenopus • induce transformation of mammary cell line C57mg • signal via nuclear translocation of -catenin

  4. Wnt/-catenin pathway

  5. Wnt secretion

  6. no detergent detergent added Purification of Wnt ligands Wnt-3a

  7. Medium Blue Sepharose Superdex Heparin WB: Wnt-5a silver staining Wnt-5a purification Wnt-conditioned medium 4 liters of media conditioned by fibroblasts expressing HA tagged Wnt-5a Blue Sepharose column Superdex gel filtration Heparin column

  8. Frizzled – crucial receptor of most (all?) Wnt pathways

  9. Lrp5/6 – crucial co-receptor of the canonical Wnt pathway

  10. Dishevelled PDZ 267-339 DEP 433-507 C-term 697-736 DIX 11-93 • phosphorylated by numerous kinases, significance often unknown • bound by many proteins - significance often unknown • various cellular localization (membrane, cytoplasm, nucleus) • capable of polymerization (Dvl aggregate vs. monomere) • required for signal transduction of most Wnt signalling pathways

  11. Dishevelled 61.2% 79.5% 57.3%

  12. Destruction complex • A working model for the destruction complex. (1) Initially, the destruction complex contains Axin, GSK3, CK1 and APC (with the 15 aa and 20 aa repeat regions shown). The complex contains other components such as PP2A, which are not shown here. (2) -Catenin enters the complex by binding Axin and potentially the APC 15 aa repeats. This positions the N-terminus of -catenin near CK1 and GSK3. (3) CK1 phosphorylates -catenin at Ser45. (4) GSK3 phosphorylates -catenin at, successively, Thr41, Ser37 and Ser33. (5) The 20 aa repeats, particularly repeat 3, are phosphorylated by a CK1 (and possibly GSK3) which greatly increases their affinity for -catenin. The binding of a phosphorylated 20 aa repeat to -catenin displaces Axin from -catenin. (6) -TRCP1 binds the phosphorylated N-terminus of -catenin, causing the ubiquitination of -catenin by an E2 ligase. APC is then either desphosphorylated within the complex, allowing the ubiquitinated -catenin to leave the complex, or the ubiquitinated -catenin bound to APC leaves the complex and is separated from APC at the proteasome. The complex then returns to Step 1

  13. Tyr142 of -catenin: phosphorylated – binds Bcl9 dephosphorylated – binds -catenin

  14. Epithelio-mesenchymal transition (EMT)

  15. Transactivation of target genes

  16. Maternal Wnt/-catenin pathway determines the dorsal pole of the zygote and embryo in Xenopus

  17. axis duplication assay:

  18. Wnt/-catenin signaling:transcription, proliferation and cell fate

  19. Mouse embryo at E8.5: Wnt/-catenin target genes are expressed in the posterior part of the embryo. Uncx4.1/Mesogenin

  20. Loss of Wnt/-catenin pathway during gastrulation = loss of posterior body parts wild type Wnt-3a knockout

  21. Loss of Wnt/-catenin inhibitors = loss of anterior body parts wild type vs. Dkk1 knockout

  22. Wnt/-catenin pathway specifies neural crest

  23. Neural crest development: Wnt-3a - Source of peripheral nervous system,Melanocytes, facial muscles/bones and heart outflow tract

  24. Wnt1/3a DKO

  25. Aberrant activation of the Wnt/-catenin pathway leads to cancer

  26. Familial adenomatous polyposis (FAP) Mutations in the adenomatosis polyposis coli (APC)

  27. Wnt pathway activators are known oncogenes and Wnt pathway inhibitors are tumor supressors

  28. Stem cell niche Bone marrow Intestinal epithelium Hair folicle Reya & Clevers 2005, Nature

  29. The effects of Wnts on stem cells in their niche Wnt

  30. -catenin gain-of-function in the epidermis Lo Celso, C. L. et al. Development 2004;131:1787-1799

  31. Wnt pathway induces de novo formation of hair follicles Wnt signaling pathway related polymorphism?

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