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Glycogen synthase kinase-3 beta (GSK-3 beta/zeste-white-3/shaggy) is a negative regulator of the wnt signaling pathway which plays a central role in the development of invertebrates and vertebrates; loss of function and dominant negative mutations in GSK-3 beta lead to activation of the wnt pathway in Drosophila and Xenopus. We now provide evidence that lithium activates downstream components of the wnt signaling pathway in vivo, leading to accumulation of beta-catenin protein. Our data indicate that this activation of the wnt pathway is a consequence of inhibition of GSK-3 beta by lithium. Using a novel assay for GSK-3 beta in oocytes, we show that lithium inhibits GSK-3 beta from species as diverse as Dictyostelium discoideum and Xenopus laevis, providing a biochemical mechanism for the action of lithium on the development of these organisms. Lithium treatment also leads to activation of an AP-1-luciferase reporter in Xenopus embryos, consistent with previous observations that GSK-3 beta inhibits c-jun activity. Activation of the wnt pathway with a dominant negative form of GSK-3 beta is inhibited by myo-inositol, similar to the previously described effect of coinjecting myo-inositol with lithium. The mechanism by which myo-inositol inhibits both dominant negative GSK-3 beta and lithium remains uncertain.
FIG. 1. In vitro and in vivo phosphorylation of tau protein by
GSK-3b. (A) Recombinant, purified GSK-3b was incubated with [g32P]ATP in the presence of tau protein. [g-32P]ATP incorporation
can be seen at 5 min (lane 2) and continues to increase over 30
min. Phosphorylated tau protein was also detected by immunostaining with PHF1, an antibody specific for tau phosphorylated at
serine residues 396 and 404. GSK-3 phosphorylated tau can also be
seen in this assay at 5 min (lane 2) and continues to accumulate
over 30 min. (B) Stage 6 Xenopus oocytes expressing GSK-3b were
injected with tau protein and samples were harvested at 5 min, 30
min, 1.5 hr, 3 hr, and 6 hr for Western blot analysis with PHF-1
antibody. In control oocytes (0GSK-3b RNA) (lanes 1â 5) tau is
not phosphorylated at Ser-396 or 404 during the 6-hr incubation.
However, in oocytes injected with GSK-3b RNA(lanes 6â 10) PHF1
staining can be identified at 5 min and increases in intensity over
time. Equal loading of tau protein is shown in lower panels with
an antibody that recognizes all forms of tau protein.
FIG. 2. In vivo inhibition of GSK-3b by lithium. (A) Xenopus
oocytes expressing GSK-3b were transferred to OR2/ containing
0 mM LiCl (lanes 3 and 5) or 20 mM LiCl (lanes 4 and 6) and were
injected with the tau protein. After 2 hr oocytes were harvested
for Western blot. Lane 1, no tau protein; lanes 2â 6, tau injected.
GSK-3b RNA injection and lithium treatment are indicated in the
figure. PHF1 and T14/T46 are as described in the legend to Fig. 1.
Lanes 1, 3, and 4, injection of Xenopus GSK-3b RNA; lanes 5 and
6, injection of Dictyostelium GSK-3b RNA. (B) Selected samples
from (A) were analyzed by Western blot using an anti-GSK-3 anti
body, which recognizes Xenopus but not Dictyostelium GSK-3b.
(C) Dose response for lithium inhibition. Oocytes were treated as
in (A), but the concentration of LiCl was varied from 0 to 50 mM,
as indicated in the figure. After injection of tau protein, oocyte
extracts were analyzed by Western blot using PHF-1 or T14/46
antibodies as indicated.
FIG. 3. b-Catenin stabilization by lithium. (A) Xenopus oocytes
were injected with b-cateninRNA (lanes 1â4) and DN-GSK-3 RNA
(lane 3), wild-type GSK-3 RNA (lane 4), or incubated in 20 mM
LiCl (lane 2). Oocytes were incubated overnight in OR2/ and then
harvested for Western analysis. (B) b-Catenin protein was injected
into Stage 6 Xenopus oocytes and samples were taken for Western
blotting at 0, 1, 2, 4, or 6 hr after incubation in OR2/ with 0 (0)
or 20 mM (/) LiCl. un represents uninjected oocytes. (C) Two-cell
embryos
were injected with b-catenin protein and treated with
lithium
or control buffer as described in the text. Embryos were
harvested immediately after lithium treatment or at 4 hr (blastula
stage). 0 and / indicate control or lithiumtreated and un indicates
uninjected embryos. inj, injected b-catenin; end, endogenous b-
catenin protein. For all panels, b-catenin protein was visualized by
Western blot using anti-b-catenin antibody.
FIG. 4. Lithium activates AP-1 transcriptional activity in Xenopus embryos. (A) One-cell embryos were injected with an AP-1â luciferase
reporter plasmid and cultured in the presence of 0â20 mM LiCl until stage 12 when embryos were harvested for luciferase assays. (B)
Luciferase activity with AP-1âluciferase and SV40â luciferase constructs in the presence (20 mM) or absence of LiCl. The values are
normalized to maximum activity for each reporter. The experiment was repeated five times and in each case AP-1 activity was stimulated
in the presence of lithium (up to 145-fold), although the magnitude of the enhancement varied considerably in different batches of eggs.
FIG. 5. Myo-inositol blocks ectopic axis induced by DN-GSK-3. Single ventralâvegetal blastomeres of 16-cell Xenopus embryos were
injected with DN-GSK-3 / H2O or DN-GSK-3 / myo-inositol and were scored for formation of ectopic dorsal axis through stage 35. The
panel on the left (DN-GSK-3) shows induction of a complete second axis with cement gland, head, and eyes. The panel on the right (DNGSK-
3 / myo-inositol) shows an incomplete secondary axis without clear head structures. See Table 1.
