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Fig. 5. Axin is required for Lrp6 phosphorylation via its ability to bind Gsk3,
and inhibition of Gsk3 at the plasma membrane blocks Wnt/β-catenin
signaling. (A) Axin-/- and the wild-type ES
cells were treated with Wnt3a or control CM. Wnt3a-induced Lrp6 (endogenous)
phosphorylation was significantly reduced in Axin-/- ES
cells. (B,C) Reducing Axin2 expression in
Axin-/- ES cells further inhibited Lrp6 (endogenous)
phosphorylation (B). Axin-/- ES cells were infected with
lentiviral shRNAs against mouse Axin2. TfR, transferin receptor, loading
control. The efficiency of Axin2 mRNA knockdown was assessed by RT-PCR
analysis. GAPDH, a loading control (C). (D) The wild-type axin, but not
the GSK3 binding mutant axin (L396Q) promoted LRP6 phosphorylation by GSK3.
Axin and axin (L396Q) were cotransfected with VSVG-tagged LRP6 in the presence
or absence of GSK3 in HEK293T cells. Axin and GSK3 were detected by the Flag
and HA tags, respectively. (E) Gsk3
and Gsk3β share
redundant function in Wnt-induced LRP6 phosphorylation. ES cells null for both
Gsk3 and Gsk3β
(Gsk3-/-;β-/-),
null for either Gsk3 (Gsk3-/-) or
Gsk3β (Gsk3β-/-) and the control
wild-type ES cells were treated with Wnt3a or control CM for 1 hour. The
endogenous Lrp6 was examined. β-actin, loading control. (F) A
plasma membrane-targeted CAAX-GID blocked wnt8 (Xwnt8) signaling in
Xenopus embryo explants. GID induced nr3 (Xnr3) expression; CAAX-GID,
but not CAAX-GID-LP, blocked wnt8 (Xwnt8)-induced nr3 expression (each was
injected at 10-50 pg mRNA/embryo). wnt8 was injected at 10 pg mRNA/embryo. -,
without reverse transcriptase; Un, uninjected control embryo; EF-1;
loading control.
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