LymphedemaV1, Pmc, Checkpoint, bibRecord, 001C89

Casein Kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in the cytoplasm

Identifieur interne : 001C89 ( Pmc/Checkpoint ); précédent : 001C88; suivant : 001C90

Casein Kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in the cytoplasm

Auteurs : Diana Golden ; Lloyd G. Cantley

Source :

Oncogene [ 0950-9232 ] ; 2014.

RBID : PMC:4459945

Abstract

Nuclear Foxc2 is a transcriptional regulator of mesenchymal transformation during developmental EMT and has been associated with EMT in malignant epithelia. Our laboratory has shown that in normal epithelial cells Foxc2 is maintained in the cytoplasm where it promotes an epithelial phenotype. The Foxc2 amino terminus has a consensus casein kinase 2 phosphorylation site at serine 124, and we now show that CK2 associates with Foxc2 and phosphorylates this site in vitro. Knock-down or inhibition of the CK2α/α′ kinase subunit in epithelial cells causes de novo accumulation of Foxc2 in the nucleus. Mutation of serine 124 to leucine promotes constitutive nuclear localization of Foxc2 and expression of mesenchymal genes, whereas an S124D phosphomimetic leads to constitutive cytoplasmic localization and epithelial maintenance. In malignant breast cancer cells the CK2β regulatory subunit is downregulated and FOXC2 is found in the nucleus, correlating with an increase in α-SMA expression. Restoration of CK2β expression in these cells results in cytoplasmic localization of Foxc2, decreased α-SMA expression and reduced cell migration and invasion. In contrast, knockdown of CK2β in normal breast epithelial cells leads to FOXC2 nuclear localization, decreased E-cadherin expression, increased α-SMA and vimentin expression, and enhanced cell migration and invasion. Based on these findings we propose that Foxc2 is functionally maintained in the cytoplasm of normal epithelial cells by CK2α/α′-mediated phosphorylation at serine 124 that is dependent on proper targeting of the holoenzyme via the CK2β regulatory subunit.

Url:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459945

DOI: 10.1038/onc.2014.395
PubMed: 25486430
PubMed Central: 4459945

Affiliations:

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the cytoplasm</title>
<author><name sortKey="Golden, Diana" sort="Golden, Diana" uniqKey="Golden D" first="Diana" last="Golden">Diana Golden</name>
<affiliation><nlm:aff id="A1">Center for Vascular Biology, University of Connecticut Health Center</nlm:aff>
<wicri:noCountry code="subfield">University of Connecticut Health Center</wicri:noCountry>
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<author><name sortKey="Cantley, Lloyd G" sort="Cantley, Lloyd G" uniqKey="Cantley L" first="Lloyd G." last="Cantley">Lloyd G. Cantley</name>
<affiliation><nlm:aff id="A2">Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine</nlm:aff>
<wicri:noCountry code="subfield">Yale University School of Medicine</wicri:noCountry>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Casein Kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in
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<author><name sortKey="Golden, Diana" sort="Golden, Diana" uniqKey="Golden D" first="Diana" last="Golden">Diana Golden</name>
<affiliation><nlm:aff id="A1">Center for Vascular Biology, University of Connecticut Health Center</nlm:aff>
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<author><name sortKey="Cantley, Lloyd G" sort="Cantley, Lloyd G" uniqKey="Cantley L" first="Lloyd G." last="Cantley">Lloyd G. Cantley</name>
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<front><div type="abstract" xml:lang="en"><p id="P1">Nuclear Foxc2 is a transcriptional regulator of mesenchymal transformation during
developmental EMT and has been associated with EMT in malignant epithelia. Our laboratory
has shown that in normal epithelial cells Foxc2 is maintained in the cytoplasm where it
promotes an epithelial phenotype. The Foxc2 amino terminus has a consensus casein kinase 2
phosphorylation site at serine 124, and we now show that CK2 associates with Foxc2 and
phosphorylates this site <italic>in vitro</italic>. Knock-down or inhibition of the
CK2α/α′ kinase subunit in epithelial cells causes de novo
accumulation of Foxc2 in the nucleus. Mutation of serine 124 to leucine promotes
constitutive nuclear localization of Foxc2 and expression of mesenchymal genes, whereas an
S124D phosphomimetic leads to constitutive cytoplasmic localization and epithelial
maintenance. In malignant breast cancer cells the CK2β regulatory subunit is
downregulated and FOXC2 is found in the nucleus, correlating with an increase in
α-SMA expression. Restoration of CK2β expression in these cells results in
cytoplasmic localization of Foxc2, decreased α-SMA expression and reduced cell
migration and invasion. In contrast, knockdown of CK2β in normal breast epithelial
cells leads to FOXC2 nuclear localization, decreased E-cadherin expression, increased
α-SMA and vimentin expression, and enhanced cell migration and invasion. Based on
these findings we propose that Foxc2 is functionally maintained in the cytoplasm of normal
epithelial cells by CK2α/α′-mediated phosphorylation at serine 124
that is dependent on proper targeting of the holoenzyme via the CK2β regulatory
subunit.</p>
</div>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
  <pmc-dir>properties manuscript</pmc-dir>
  <front><journal-meta><journal-id journal-id-type="nlm-journal-id">8711562</journal-id>
<journal-id journal-id-type="pubmed-jr-id">6325</journal-id>
<journal-id journal-id-type="nlm-ta">Oncogene</journal-id>
<journal-id journal-id-type="iso-abbrev">Oncogene</journal-id>
<journal-title-group><journal-title>Oncogene</journal-title>
</journal-title-group>
<issn pub-type="ppub">0950-9232</issn>
<issn pub-type="epub">1476-5594</issn>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">25486430</article-id>
<article-id pub-id-type="pmc">4459945</article-id>
<article-id pub-id-type="doi">10.1038/onc.2014.395</article-id>
<article-id pub-id-type="manuscript">NIHMS627033</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Casein Kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in
the cytoplasm</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Golden</surname>
<given-names>Diana</given-names>
</name>
<degrees>PhD</degrees>
<xref ref-type="aff" rid="A1">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Cantley</surname>
<given-names>Lloyd G.</given-names>
</name>
<degrees>MD</degrees>
<xref ref-type="aff" rid="A2">2</xref>
</contrib>
</contrib-group>
<aff id="A1"><label>1</label>
Center for Vascular Biology, University of Connecticut Health Center</aff>
<aff id="A2"><label>2</label>
Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine</aff>
<author-notes><corresp id="FN1">Correspondence: Diana Golden, PhD, University of Connecticut Health
Center, Center for Vascular Biology, 263 Farmington Ave, Farmington, CT 06030-3501,
(860)-604-6144, <email>dgolden@uchc.edu</email>
</corresp>
</author-notes>
<pub-date pub-type="nihms-submitted"><day>11</day>
<month>9</month>
<year>2014</year>
</pub-date>
<pub-date pub-type="epub"><day>08</day>
<month>12</month>
<year>2014</year>
</pub-date>
<pub-date pub-type="ppub"><day>3</day>
<month>9</month>
<year>2015</year>
</pub-date>
<pub-date pub-type="pmc-release"><day>03</day>
<month>3</month>
<year>2016</year>
</pub-date>
<volume>34</volume>
<issue>36</issue>
<fpage>4702</fpage>
<lpage>4712</lpage>
<pmc-comment>elocation-id from pubmed: 10.1038/onc.2014.395</pmc-comment>
      <permissions><license xlink:href="http://www.nature.com/authors/editorial_policies/license.html#terms"><license-p>Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:<ext-link ext-link-type="uri" xlink:href="http://www.nature.com/authors/editorial_policies/license.html#terms">http://www.nature.com/authors/editorial_policies/license.html#terms</ext-link>
</license-p>
</license>
</permissions>
<abstract><p id="P1">Nuclear Foxc2 is a transcriptional regulator of mesenchymal transformation during
developmental EMT and has been associated with EMT in malignant epithelia. Our laboratory
has shown that in normal epithelial cells Foxc2 is maintained in the cytoplasm where it
promotes an epithelial phenotype. The Foxc2 amino terminus has a consensus casein kinase 2
phosphorylation site at serine 124, and we now show that CK2 associates with Foxc2 and
phosphorylates this site <italic>in vitro</italic>. Knock-down or inhibition of the
CK2α/α′ kinase subunit in epithelial cells causes de novo
accumulation of Foxc2 in the nucleus. Mutation of serine 124 to leucine promotes
constitutive nuclear localization of Foxc2 and expression of mesenchymal genes, whereas an
S124D phosphomimetic leads to constitutive cytoplasmic localization and epithelial
maintenance. In malignant breast cancer cells the CK2β regulatory subunit is
downregulated and FOXC2 is found in the nucleus, correlating with an increase in
α-SMA expression. Restoration of CK2β expression in these cells results in
cytoplasmic localization of Foxc2, decreased α-SMA expression and reduced cell
migration and invasion. In contrast, knockdown of CK2β in normal breast epithelial
cells leads to FOXC2 nuclear localization, decreased E-cadherin expression, increased
α-SMA and vimentin expression, and enhanced cell migration and invasion. Based on
these findings we propose that Foxc2 is functionally maintained in the cytoplasm of normal
epithelial cells by CK2α/α′-mediated phosphorylation at serine 124
that is dependent on proper targeting of the holoenzyme via the CK2β regulatory
subunit.</p>
</abstract>
<kwd-group><kwd>Foxc2</kwd>
<kwd>E-cadherin</kwd>
<kwd>Epithelial Cell</kwd>
<kwd>Epithelial to Mesenchymal Transition</kwd>
<kwd>CK2 Signaling</kwd>
</kwd-group>
</article-meta>
</front>
<floats-group><fig id="F1" orientation="portrait" position="float"><label>Figure 1</label>
<caption><title>Foxc2 localization</title>
<p>(A) MPT cells, NIH3T3 cells and MPT cells transiently transfected with FLAG-Foxc2 were
subjected to cell fractionation followed by SDS-PAGE and immunoblotting for Foxc2,
LaminA/C (nuclear marker) and GAPDH (cytoplasmic compartment marker). Representative
images from an <italic>n</italic> of 3 replicates per condition. (B) MPT cells transiently
transfected with either GFP-Foxc2, GFP-Foxc2-S124D or GFP-Foxc2-S124L were fixed and
imaged for GFP (green) and DAPI (blue). Images obtained at 40x magnification. (C) MPT
cells transiently transfected as above were subjected to cell fractionation followed by
immunoblotting with anti-GFP, anti-LaminA/C and anti-GAPDH. Representative blots from an n
of 4–6 replicates per condition.</p>
</caption>
<graphic xlink:href="nihms627033f1"></graphic>
</fig>
<fig id="F2" orientation="portrait" position="float"><label>Figure 2</label>
<caption><title>CK2 is required to maintain cytoplasmic localization of Foxc2</title>
<p>(A) MPT cells were transfected with GFP-Foxc2 and whole cell lysates immunoprecipitated
with anti-GFP or IgG isotype control and blotted for anti-GFP and anti-CK2. WCL is the
input material for the IP. (B) MPT cells were transfected with scrambled siRNA or siRNA
directed against CK2 followed 24 hours later by lysis and immunoblotting to determine
efficiency of CK2 protein reduction. Percentage knock-down determined by normalizing to
β-actin. Graph shows quantification for <italic>n</italic>=3;
**p<0.005 relative to siRNA scrambled control. (C) MPT cells
transfected as in (B) followed by cell fractionation and immunoblotting for Foxc2,
LaminA/C and GAPDH. Graph shows quantification for <italic>n</italic>=3;
***p<0.001, **p<0.005 relative to siRNA
scrambled control. (D) MPT cells were treated with vehicle control or
100<italic>μ</italic>M TBCA for 1h, followed by cell fractionation and
immunoblotting for endogenous Foxc2, LaminA/C and GAPDH. Relative Foxc2 expression in each
compartment was determined as described in Methods.
<italic>n</italic>=4;***p<0.001,
**p<0.005 relative to vehicle control. (E–F) MPT cells
transiently transfected with the indicated constructs were treated with vehicle control
(E) or 100<italic>μ</italic>M TBCA (F) for 1h, and subjected to cell fractionation
followed by immunoblotting with anti-GFP, anti-LaminA/C and anti-GAPDH. Relative
expression was determined as described in Methods. <italic>n</italic>=3;
**p<0.005.</p>
</caption>
<graphic xlink:href="nihms627033f2"></graphic>
</fig>
<fig id="F3" orientation="portrait" position="float"><label>Figure 3</label>
<caption><title>Serine 124 is phosphorylated <italic>in vitro</italic>
 by CK2</title>
<p>MPT cells were transfected with GFP-Foxc2 or GFP-Foxc2-S124L followed by
immunoprecipitation with anti-GFP antibody and <italic>in vitro</italic> phosphorylation
± CK2. Paired samples were subjected to SDS-PAGE and immunoblotting with anti-GFP
antibody to confirm immunoprecipitation of GFP-Foxc2 and GFP-Foxc2-S124L.</p>
</caption>
<graphic xlink:href="nihms627033f3"></graphic>
</fig>
<fig id="F4" orientation="portrait" position="float"><label>Figure 4</label>
<caption><title>Cytoplasmic localization maintains an epithelial phenotype</title>
<p>(A) MPT cells were transfected with GFP, GFP-Foxc2-S124D or GFP-Foxc2-S124L constructs x3
to achieve ~70% transfection efficiency and grown to 90% confluency. Whole
cell lysates were then blotted with the indicated antibodies. (B) Quantification of 3
experiments performed as in (A) with protein expression normalized to β-actin.
<italic>n</italic>=3; ***p<0.001,
**p<0.005 (compared to GFP control cells). (C) Real-time PCR was
performed using RNA from cells transfected as in (A) with RNA expression normalized to
Hprt1 and MPT control. WCL=whole cell lysate. <italic>n</italic>=3;
***p<0.001, **p<0.005 (compared to control
cells).</p>
</caption>
<graphic xlink:href="nihms627033f4"></graphic>
</fig>
<fig id="F5" orientation="portrait" position="float"><label>Figure 5</label>
<caption><title>FOXC2 nuclear expression correlates with metastatic phenotype in breast cancer cell
lines</title>
<p>(A) MCF10A, MDA-MB-436 and MDA-MB-231 whole cell lysates were subjected to immunoblotting
with anti-Foxc2 or anti-β-actin and quantified relative to β-actin.
<italic>n</italic>=3; *p<0.05 relative to MCF10A cells. (B) MCF10A,
MDA-MB-436 and MDA-MB-231 cells were subjected to cell fractionation followed by SDS-PAGE
and immunoblotting for FOXC2, Lamin A/C and GAPDH and quantified below. Relative FOXC2
expression was determined as described in Methods. WCL=whole cell lysate,
C=Cytosolic, N=Nuclear. <italic>n</italic>=3; *p<0.05
relative to MCF10A cells.</p>
</caption>
<graphic xlink:href="nihms627033f5"></graphic>
</fig>
<fig id="F6" orientation="portrait" position="float"><label>Figure 6</label>
<caption><title>Mesenchymal cell lines expressing nuclear FOXC2 have higher expression levels of EMT
markers and altered CK2α/α′:CK2β expression</title>
<p>(A) MCF10A, MDA-MB-436 and MDA-MB-231 whole cell lysates were subjected to immunoblotting
with the indicated antibodies. (B) Quantification of 3 experiments performed as in (A)
with protein expression normalized to β-actin loading control.
<italic>n</italic>=3; *p<0.05 relative to MCF10A cells. (C) MPT and
MDA-MB-231 whole cell lysates (left panel) along with MPT, MCF10A and NIH3T3 cell lysates
(right panel) were subjected to immunoblotting with the indicated antibodies.
WCL=whole cell lysate.</p>
</caption>
<graphic xlink:href="nihms627033f6"></graphic>
</fig>
<fig id="F7" orientation="portrait" position="float"><label>Figure 7</label>
<caption><title>Restoration of CK2β expression in metastatic breast cancer cells promotes
FOXC2 cytoplasmic localization and reduced migration</title>
<p>(A) MDA-MB-231 cells were transfected with either an empty vector or a CK2β
expression construct x2 and grown to 90% confluency. Whole cell lysates were then
blotted with the indicated antibodies. (B) MDA-MB-231 cells transfected as in (A) were
subjected to cell fractionation and fractions immunoblotted for FOXC2, LaminA/C and GAPDH.
(C) MDA-MB-231 cells transfected as in (A) were subjected to cellular migration assays
± 10% FBS. <italic>n</italic>=3;
***p<0.001, *p<0.05 (compared to control cells). (D)
MDA-MB-231 cells transfected as in (A) were subjected to a basement membrane extract
cellular invasion assay ± 10% FBS. <italic>n</italic>=3;
***p<0.001, *p<0.05 (compared to control cells).
WCL=whole cell lysate, C=Cytosolic, N=Nuclear, FBS=Fetal
Bovine Serum.</p>
</caption>
<graphic xlink:href="nihms627033f7"></graphic>
</fig>
<fig id="F8" orientation="portrait" position="float"><label>Figure 8</label>
<caption><title>CK2β knockdown in normal breast epithelial cells promotes FOXC2 nuclear
localization and increased migration/invasion</title>
<p>(A) MCF10A cells were transfected with either a scrambled siRNA or siRNA directed against
CK2β and grown to 90% confluency. Whole cell lysates were then blotted
with the indicated antibodies. (B) MCF10A cells transfected as in (A) were subjected to
cell fractionation and fractions immunoblotted for FOXC2, LaminA/C and GAPDH. (C) MCF10A
cells transfected as in (A) were subjected to cellular migration assays ±
10% FBS. <italic>n</italic>=3; ***p<0.001,
*p<0.05 (compared to control scrambled siRNA cells). (D) MCF10A cells
transfected as in (A) were subjected to a basement membrane extract cellular invasion
assay ± 10% FBS. <italic>n</italic>=3;
***p<0.001 (compared to control cells). WCL=whole cell
lysate, C=Cytosolic, N=Nuclear, FBS=Fetal Bovine Serum.</p>
</caption>
<graphic xlink:href="nihms627033f8"></graphic>
</fig>
<fig id="F9" orientation="portrait" position="float"><label>Figure 9</label>
<caption><title>Foxc2-S124L can overcome the CK2β-mediated normalization of migration</title>
<p>(A) MCF10A cells were transfected with either empty vector, CK2β, GFP-Foxc2 or
GFP-Foxc2-S124L in the indicated combinations, grown to 90% confluency and
subjected to cell migration assays without fetal bovine serum (FBS).
<italic>n</italic>=3; ***p<0.001 (compared to control
cells). (B) MDA-MB-231 cells transfected as in (A) were subjected to cell migration assays
without FBS. <italic>n</italic>=3; ***p<0.001 (compared
to control cells). (C) MCF10A cells transfected as in (A) were subjected to a basement
membrane extract cellular invasion assay without FBS. <italic>n</italic>=3;
**p<0.01, *p<0.05 (compared to control cells). (D)
MDA-MB-231 cells transfected as in (A) were subjected to a basement membrane extract
cellular invasion assay without FBS. <italic>n</italic>=3;
**p<0.01 (compared to control cells).</p>
</caption>
<graphic xlink:href="nihms627033f9"></graphic>
</fig>
<table-wrap id="T1" position="float" orientation="portrait"><label>Table 1</label>
<caption><p>Potential CK2 site is conserved amongst Fox family members.</p>
</caption>
<table frame="void" rules="none"><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1"><graphic xlink:href="nihms627033f10"></graphic>
</td>
</tr>
</tbody>
</table>
</table-wrap>
</floats-group>
</pmc>
<affiliations><list></list>
<tree><noCountry><name sortKey="Cantley, Lloyd G" sort="Cantley, Lloyd G" uniqKey="Cantley L" first="Lloyd G." last="Cantley">Lloyd G. Cantley</name>
<name sortKey="Golden, Diana" sort="Golden, Diana" uniqKey="Golden D" first="Diana" last="Golden">Diana Golden</name>
</noCountry>
</tree>
</affiliations>
</record>

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Casein Kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in the cytoplasm

Casein Kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in the cytoplasm

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