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J Biol Chem
2004 Mar 26;27913:13011-7. doi: 10.1074/jbc.M304682200.
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Regulation of casein kinase I epsilon activity by Wnt signaling.
Swiatek W
,
Tsai IC
,
Klimowski L
,
Pepler A
,
Barnette J
,
Yost HJ
,
Virshup DM
.
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The Wnt/beta-catenin signaling pathway is important in both development and cancer. Casein kinase Iepsilon (CKIepsilon) is a positive regulator of the canonical Wnt pathway. CKIepsilon itself can be regulated in vitro by inhibitory autophosphorylation, and recent data suggest that in vivo kinase activity can be regulated by extracellular stimuli. We show here that the phosphorylation state and kinase activity of CKIepsilon are directly regulated by Wnt signaling. Coexpression of XWnt-8 or addition of soluble Wnt-3a ligand led to a significant and rapid increase in the activity of endogenous CKIepsilon. The increase in CKIepsilon activity is the result of decreased inhibitory autophosphorylation because it is abolished by preincubation of immunoprecipitated kinase with ATP. Furthermore, mutation of CKIepsilon inhibitory autophosphorylation sites creates a kinase termed CKIepsilon(MM2) that is significantly more active than CKIepsilon and is not activated further upon Wnt stimulation. Autoinhibition of CKIepsilon is biologically relevant because CKIepsilon(MM2) is more effective than CKIepsilon at activating transcription from a Lef1-dependent promoter. Finally, CKIepsilon(MM2) expression in Xenopus embryos induces both axis duplication and additional developmental abnormalities. The data suggest that Wnt signaling activates CKIepsilon by causing transient dephosphorylation of critical inhibitory sites present in the carboxyl-terminal domain of the kinase. Activation of the Wnt pathway may therefore stimulate a cellular phosphatase to dephosphorylate and activate CKIepsilon
FIG. 1. CKI is activated by Wnt signaling. A, endogenous CKI is
activated by expression of XWnt-8. HEK 293 cells were transfected with
empty vector () or pCS2-XWnt-8, directing expression of XWnt-8. 24 h
after transfection, lysates were prepared, and endogenous CKI was
immunoprecipitated using UT31, an affinity-purified polyclonal antibody
that recognizes the amino terminus of CKI. The activity of the
immunoprecipitated kinase was assayed in an in vitro kinase assay
using MBP-axin as a substrate. Samples were then analyzed by SDSPAGE
and autoradiography as described under âExperimental Procedures.â
Left panel, average of data obtained from two experiments, each
performed in duplicate. Data for each sample were normalized to the
total level of CKI, and background kinase activity in the absence of
specific antibody was subtracted. Right top panel, representative autoradiogram
of 32P-axin. Right bottom panel, immunoprecipitates were
probed with CKI mAb to verify equal recovery of CKI. A.U., arbitrary
units. B, endogenous CKI is activated by Wnt-3a-conditioned medium.
HEK 293 cells were treated with Wnt-3a-conditioned or control medium
for 30 min prior to lysis, immunoprecipitation with UT31 antibody, and
kinase assay as above. Left panel, cumulative data obtained from two
experiments performed in duplicate. Data for each sample were normalized
to the total level of CKI. Right top panel, representative
autoradiogram of 32P-axin. Right bottom panel, immunoprecipitates
were probed with CKI mAb to verify equal recovery of CKI. C, Wnt-3a
rapidly induces -catenin stabilization. Mouse L-cells were incubated
with either Wnt-3a-conditioned control medium for 30 min. 75 g of
protein lysates was analyzed by Western blotting with mouse anti--
catenin antibodies.
CKI Is
FIG. 2. Wnt signaling regulates the activity of CKI through
changes in its phosphorylation state. A, activated CKI is reinhibited
by incubation with ATP. HEK 293 cells were treated with Wnt-3aconditioned
or control medium for 30 min. Cell lysates were then
subjected to immunoprecipitation with UT31 antibody. The isolated
immunocomplexes containing CKI were then incubated with buffer
(lanes 1 and 2), 500 M ATP (lanes 3 and 4), or with protein phosphatase
(lanes 5 and 6) for 20 min. After two washes with kinase buffer lacking
ATP, kinase activity was assayed as described. Top panel, data obtained
from two experiments each performed in duplicate. Data for each
sample were normalized to the total level of CKI. A.U., arbitrary units.
Middle panel, representative autoradiogram of 32P-axin. Bottom panel,
immunoprecipitates were probed with CKI mAb to verify equal recovery
of CKI. B, XWnt-8 coexpression alters the isoelectric point of CKI.
Top panel, coexpression of 4HA-CKI and XWnt-8 causes accumulation
of endogenous -catenin in HEK 293 cells. Bottom panel, XWnt-8 expression
shifts the isoelectric point of the predominant 4HA-CKI immunoreactive
species almost 1 pH unit toward the basic side as analyzed
by two-dimensional gel electrophoresis. Lysates from HEK 293
cells expressing HA-CKI and XWnt-8 as indicated were subjected to
isoelectric focusing (pH range 3â10), SDS-PAGE, and immunoblotting
with anti-HA mAb. Similar results were obtained in multiple
replicate experiments.
FIG. 3.
FIG. 3. CKI mutant CKI(MM2), unable to autophosphorylate
and autoinhibit, is constitutively active and is not activated
further by Wnt signaling. A, Myc-tagged wild type CKI and
CKI(MM2) were transiently expressed in HEK 293 cells without or
with coexpressed XWnt-8. Cell lysates were then subjected to immunoprecipitation
with anti-HA (control) or anti-Myc antibodies, and the
immunoprecipitates were tested for kinase activity as described. Top
panel, cumulative data obtained from three experiments, each performed
in duplicate (mean S.E.). Data for each sample were normalized
to the total level of CKI, and kinase activity obtained from the
control immunoprecipitate was subtracted. A.U., arbitrary units. Middle
panel, representative autoradiogram of 32P-axin. Bottom panel,
immunoprecipitates were probed with UT31 to verify equal recovery of
CKI. B, similar to A, except that XWnt-8 expression vectors were
omitted, and cells were treated with Wnt-3a-conditioned or control
medium for 30 min prior to lysis and kinase assay.
130
FIG. 4. Time course of CKI activation after exposure to Wnt
ligand. A, activation of endogenous CKI by Wnt signaling is rapid.
HEK 293 cells were treated with the Wnt-3a-conditioned or control
medium for the indicated periods prior to lysis, and CKI was immunoprecipitated
with UT31 antibody. Top panel, cumulative data obtained
from two experiments, each in duplicate and normalized to the
total level of CKI. A.U., arbitrary units. Middle panel, autoradiogram
of 32P-axin. Bottom panel, immunoprecipitates were probed with CKI
mAb to verify equal recovery of CKI. B, the Wnt-3a-induced activation
of CKI is terminated by the elimination of signaling. HEK 293 cells
were transiently transfected with either wild type Myc-tagged CKI or
Myc-tagged CKI(MM2). Cells were all pretreated with Wnt-3a-conditioned
medium for 30 min and then either incubated further with
Wnt-3a (closed bars) or Dulbeccoâs modified Eagleâs medium (open bars)
for the indicated length of time prior to cell lysis. CKI was immunoprecipitated
with anti-Myc antibody and kinase activity assayed as
described. The average of data obtained from two experiments each
performed in duplicate is shown. Results were normalized to the quantity
of immunoprecipitated CKI.
CKI Is A
FIG. 5. Constitutively active CKI -
(MM2) is biologically more active than
wild type CKI . A, CKI(MM2) activates
a -catenin responsive promoter. Activation
of the TOPflash reporter plasmid by
CKI and CKI(MM2) was measured as described
under “Experimental Procedures.â€
B–D, CKI(MM2) expression in Xenopus
embryogenesis causes axis duplication and
an extreme phenotype. Xenopus embryos
at the four-cell stage were injected ventrally
with 0.5–1.0 ng of CKI(WT) (C) or
CKI(MM2) (D) RNA. Embryos were
scored after 24 and 72 h, and photos of
representative tadpoles after 72 h of development
are shown. Similar results
were obtained in replicate experiments
and at doses of CKI and CKI(MM2)
ranging from 0.5 to 1 ng of RNA.
FIG.
FIG. 6. Inhibition of serine/threonine phosphatases blocks CKI activation by Wnt signaling. A and B, calyculin A and cyclosporine A
inhibit CKI activation. Myc-tagged wild type CKI and CKI(MM2) were transiently expressed in HEK 293 cells. 22 h post-transfection cells were
treated with Wnt-3a conditioned (closed bars) or control medium (open bars) for 60 min followed by the addition of 20 nM calyculin A (CalA) (A)
or 1 M cyclosporine A (CyA) (B) for 30 min. Cell lysates were then subjected to immunoprecipitation with anti-Myc antibodies, and the
immunoprecipitates were tested for kinase activity as described. Data for each sample were normalized to the total level of CKI, and kinase
activity obtained from the control immunoprecipitate was subtracted. These studies were performed in duplicate twice with similar results. C,
PP2A can activate CKI in vivo. Myc-tagged wild type CKI was transiently expressed in HEK 293 cells without or with coexpressed 0.5 g of PP2C.
Cell lysates were then subjected to immunoprecipitation with anti-Myc antibodies, and the immunoprecipitates were tested for kinase activity.
Data for each sample were normalized to the total level of CKI, and kinase activity obtained from the control immunoprecipitate was subtracted.
These results were performed in duplicate on three separate occasions with identical results.
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