Purified Wnt 5a protein activates or inhibits

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urified Wnt 5a protein activates or inhibits tenin-TCF signaling depending on receptor co Mikels et al., 2006, PLOS Biology

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Purified Wnt 5a protein activates or inhibits β -catenin-TCF signaling depending on receptor context. Mikels et al., 2006, PLOS Biology. Wnts antagonize Wnts. Mouse limb bud. Over-expression of Wnt 5 blocks the ability of Wnt 8 to promote dorsal cell fate in Xenopus. - PowerPoint PPT Presentation

Transcript of Purified Wnt 5a protein activates or inhibits

Page 1: Purified Wnt 5a  protein activates or inhibits

Purified Wnt 5a protein activates or inhibits β-catenin-TCF signaling depending on receptor context

Mikels et al., 2006, PLOS Biology

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Mouselimb bud

Over-expression of Wnt 5 blocks the ability of Wnt 8 to promote dorsal cell fate in Xenopus. - Torres et al., 2003, J. Cell Biol.

In the distal tip of mouse limb buds, Wnt 5a antagonizes b-catenin signaling, since in Wnt 5a -/-limbs higher levels of b-catenin can be detected in the distal tip. - Topol et al., 2003, J. Cell Biol.

Removal of maternal and zygotic functions of zebrafish Wnt 5 result in variable degrees of dorsalization, including formation of secondary axis. - Westfall et al., 2003, Dev. Biol.

Wnts antagonize Wnts

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Dominant negative Wnt 11 and CaMK II promotes dorsal cell fates. - Kuhl et al., 2000, J. Biol. Chem.

Over-expression of Wnt 5 can activate Nemo-like kinase, which in C. elegans and mammalian cellsInhibit Tcf transcription factors to which b-catenin binds to regulate gene expression. - Ishitani et al., 2003, Mol. Cell. Biol.

Siah 2 (b-catenin destruction complex) is transcriptionally activated by Wnt 5a. - Topol et al., 2003, J. Cell Biol.

How to inhibit the canonical signaling…?

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SuperTopflash in 293 cells

Purified Wnt 5a protein inhibits canonical Wnt signaling

wnt 3a wnt 5a (after 1, 3hr)wnt 5a 6hr after washing wnt 3a

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Wnt 5a does not affect b-catenin protein stabilization

Wnt 5a protein competes with Wnt 3a for Fz receptor context.

Siah 2, which targets b-catenin for b-TrCP independent proteasomal degradation, is transcriptionally activated by Wnt 5a. - Topol et al., 2003, J. Cell Biol.

293 cells

Cytosolic fraction of 293 cells

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Wnt 5a - mediated inhibition of canonical signaling is not associated with Wnt- stimulated Ca++ flux

* PTX (pertussis toxin) : inhibition of G protein binding with membrane receptor

* LPA (lysophosphatidic acid) : agonist of G protein signaling

wnt 5a

Ca ++

CaMKII

TAK/NLK mitogen activatedSignaling cascade

TCF/LEF - phosphorylation

DNA

b-cat

G-protein

* Ionomycin :raises the intracellular level of Ca2+

Wnt stimulatedcalcium flux

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Wnt 5a protein activates b-catenin signaling depending on receptor context

XWnt 5a RNA with Xfz4 RNA leads to the synergistic activation of Wnt/b-cat target genes in Xenopus animal caps. - Umbhauer et al., 2000, EMBO J

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An alternative Wnt receptor, mRor2, mediates Wnt 5a’s inhibitory activity

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Expression of mRor2 is required for Wnt 5a- mediated inhibition of canonical Wnt signaling

SuperTopflashLRPcotransfection

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Membrane tethered form(no transmembrane & cytoplasmic domain)

293

Transmembrane domain : OCytoplasmic domain : X

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A member of the Fz protein family (hFz5) mediating axis induction by Wnt 5a. - He et al., 1997, Scinece.