Download CASK (LIN2) interacts with Cx43 in wounded skin and their

Research Article
695
CASK (LIN2) interacts with Cx43 in wounded skin and
their coexpression affects cell migration
Lucrecia Márquez-Rosado, Deepika Singh, Hector Rincón-Arano, Joell L. Solan and Paul D. Lampe*
Molecular Diagnostics Program, Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue
North, Seattle, WA, 98109, USA
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 22 September 2011
Journal of Cell Science 125, 695–702
ß 2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.084400
Summary
Vertebrate gap junctions are composed of proteins from the connexin family. Co-immunoprecipitation, in vitro binding and far western
experiments demonstrate that mammalian CASK (also known as LIN2) directly interacts with Cx43. Immunoprecipitation studies
indicate that the CASK mainly interacts with the hypophosphorylated form of Cx43. Functional co-regulation of these proteins was
found in MDCK cells migrating into a scratch wound, where expression of either protein individually inhibits migration but their
coexpression abrogates this inhibitory effect. Immunofluorescence shows colocalization of Cx43 and CASK in mouse brain astrocytes
and in response to wounding in human foreskin. During wounding, CASK is mobilized to the plasma membrane where it colocalizes
with Cx43 and CADM1 1 hour after skin explant wounding. Together, these studies indicate that CASK interaction with Cx43 occurs
relatively early in the connexin life cycle and imply a plasma membrane targeting role for the interaction that apparently affects cellular
processes including cellular migration and wound healing.
Key words: Gap junctions, Connexin, CASK (Lin-2)
Introduction
In vertebrates, connexin proteins assemble into tightly packed
gap junctional channels to connect the cytoplasms of adjacent
cells via an aqueous-filled channel. Gap junctions allow the
passive diffusion of molecules lower than 1000 Da in size such as
ions, amino acids, nutrients and secondary messengers (Laird,
2010; Scemes et al., 2007). Gap junctional intercellular
communication has been shown to play important roles in
tissue differentiation, transport, development, synchronous
muscle contraction, homeostasis and conduction in excitable
tissues (Goodenough and Paul, 2009). Studies utilizing transgenic
mice with altered connexin genes and linkage of connexin gene
alterations to human disease support their role in regulation of
cell growth control and embryonic development (Dobrowolski
and Willecke, 2009; Mese et al., 2007; Naus and Laird, 2010).
Cx43, the most ubiquitous connexin, oligomerizes into a hexamer
or ‘‘connexon’’ in the trans-Golgi network (TGN) (Musil and
Goodenough, 1993) and, after transport to the plasma membrane
and association with other connexons, can form a gap junction.
Zona Occudens-1 (ZO-1), a member of the membraneassociated guanylate kinase (MAGUK) family of proteins,
interacts with Cx43 through one of its PSD95–Dlg–ZO-1
(PDZ) domain proteins (Giepmans and Moolenaar, 1998;
Toyofuku et al., 1998). MAGUK proteins are highly conserved
and characterized by the presence of multiple protein–protein
interaction domains that have been shown to be important in
localizing target membrane proteins to specialized membrane
domains, maintaining cell polarity and acting as scaffolds.
Another MAGUK, calcium/calmodulin-dependent serine kinase,
CASK (also known as LIN2 and PALS) is a causative gene in Xlinked mental retardation (Najm et al., 2008), and its deletion in
mice is lethal (Atasoy et al., 2007). In renal epithelial cells,
CASK exists as a complex with LIN7 (also known as VELI and
MALS) (Straight et al., 2000), whereas in the brain CASK and
LIN7 are complexed with LIN10 (also known as X11a and
MINT1) (Borg et al., 1998; Butz et al., 1998). In Caenorhabditis
elegans, Lin-2 is responsible for the basolateral targeting of the
Let-23 growth factor receptor (homologous to the mammalian
EGF receptor) in vulval precursor cells (Hoskins et al., 1996;
Kaech et al., 1998). In mammalian epithelial and neuronal cells,
the CASK protein scaffolding complex is involved in the
assembly of junctional components (Borg et al., 1998; Butz
et al., 1998; Craven and Bredt, 1998; Dimitratos et al., 1999;
Kaech et al., 1998; Kamberov et al., 2000; Perego et al., 2000).
Here, we demonstrate that the C-terminus of Cx43 associates
directly with the mammalian CASK protein in a non-PDZdependent interaction. Furthermore, the CASK protein is
strongly immunoprecipitated by an antibody that preferentially
interacts with the hypophosphorylated form of Cx43
compared to other phosphospecific and general Cx43
antibodies. Immunofluorescence studies in skin tissues show
specific colocalization between Cx43, CASK and CADM1
[a protein that regulates epidermal adhesion and wound repair
(Giangreco et al., 2009)] that varies in a temporally and spatially
regulated manner during wound healing.
Results
Cx43 interacts directly with CASK and not with
LIN7 protein
Multiple protein interaction domains and phosphorylation sites in
the C terminal tail of Cx43 have been implicated in regulating
Cx43 trafficking, assembly, gating and turnover. Previously, we
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applied an approach combining liquid chromatography with
tandem mass spectrometry (LC-MS/MS) to identify Cx43
interacting proteins (Singh and Lampe, 2003). Briefly, the Cterminal tail of Cx43 (amino acid residues 246–382) fused to
GST was incubated with whole cell lysates from normal rat
kidney (NRK) cells. After washing, elution and trypsinization,
peptides were analyzed by LC-MS/MS. Based on the MS data,
we reported multiple possible Cx43 interacting partners including
LIN7 (Singh and Lampe, 2003). Given that LIN7 exists as a
hetero-trimeric complex with CASK, we performed a far western
overlay assay to investigate whether Cx43 interacted directly
with either protein. Cell lysates from 293T cells transfected with
either pRK5-Myc-CASK (amino acids 1–897) or pRK5-MycLIN7 (amino acids 1–197) were run on a SDS-PAGE and then
transferred to a nitrocellulose membrane. The membrane was
blocked and then incubated with a 32P-labeled GST–Cx43CT
(amino acids 236–382) probe. After washing, a band
corresponding to CASK (approximately 120 kDa) but no LIN7
band was observed (Fig. 1A), suggesting that the direct
interaction between GST–Cx43CT and CASK might have led
to the LIN7 pulldown by linkage via CASK.
Because PDZ domains recognize a three-residue peptide motif
in the C-termini of their binding partners (Songyang et al., 1997)
and the terminal Leu-Glu-Ile (LEI) residues of Cx43 are crucial
for interaction with the PDZ-domain-containing proteins ZO-1
and ZO-2, we suspected that the interaction with CASK involves
the C-terminal region of Cx43. Therefore, we utilized different
GST–Cx43 fusion constructs including the entire cytoplasmic tail
(CT) region (residues 236–382) of Cx43 and the same construct
lacking residues 260–280 (D270) or lacking the terminal three
amino acids (DLEI) in a GST pull-down assay. As shown in
Fig. 1B, both CT and DLEI could effectively pull down CASK.
Because these constructs eliminate the PDZ binding domain of
Cx43, the CASK binding to Cx43 was probably not via the PDZ
domain. However, the construct lacking residues 260–280 was
significantly (P,0.01) less effective at pulling down CASK and
was comparable to the GST-alone control level (Fig. 1C)
indicating that this region, known to be important for Cx43
regulation of turnover and gating (Thomas et al., 2003; WarnCramer et al., 1996), is crucial for CASK binding.
CASK interaction shows a preference for
hypophosphorylated Cx43
Further examination of the Cx43–CASK interaction involved
incubation of GST–CASK with cell lysates from NRK cells in a
pull-down assay. In SDS-PAGE, Cx43 separates into multiple
bands typically indicated as P0, P1 and P2. GST–CASK indeed
interacted with endogenous Cx43 in the cell lysates with a
preference for hypophosphorylated Cx43, i.e. P0 (Fig. 2A).
This result was confirmed in co-immunoprecipitation assays
using several Cx43 antibodies with affinities for different
phosphorylated isoforms of Cx43 (Fig. 2B). We observed
that the CT1 antibody, which shows a strong preference for
the hypophosphorylated isoform (Sosinsky et al., 2007),
immunoprecipitated the most CASK, whereas IF1, an antibody
that shows more preference for phosphorylated isoforms,
precipitated 84% less (n55, a representative example is
shown). Similar results (albeit with fewer repetitions) were
obtained with MDCK cells expressing Cx43 (see supplementary
material Fig. S1). A polyclonal pan-Cx43 antibody (PNRF) and
an antibody specific for Cx43 phosphorylated at Ser365 also
immunoprecipitated less CASK than the CT1 antibody. We then
performed the reverse immunoprecipitation experiments with
CASK antibodies and probed for the presence of Cx43 in an
immunoblot. The anti-CASK antibody and an antibody to ZO-1
(a known Cx43-interacting protein) could co-immunoprecipitate
Cx43, with both proteins showing some preference for the P0
isoform (Fig. 2C). A control precipitation of lysate performed
with protein A beads did not co-precipitate Cx43. To map the
portion of CASK that interacts with Cx43, we utilized N- and Cterminal constructs of CASK representing residues 1–612 (L2N,
containing intact kinase, L27 and PDZ domains), 578–897 (L2C,
containing SH3 and GUK domains) and 1–897 (full length). Both
the full length and L2C, C-terminal piece of CASK, could bind to
Fig. 1. Interaction of Cx43 with LIN7 and CASK. (A) Lysates from 293T cells transfected with pRK5-MycCASK (CASK; amino acids 1-897) or pRK5MycLin7 (LIN7; amino acids 1–197) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Ponceau S staining of the membrane is shown
in the left panel). The membrane was then probed with a 32P-labeled GST–Cx43CT probe and exposed to an X-ray film (autoradiographic image is shown in the
right panel). (B) GST–Cx43CT (CT), GST–Cx43CT missing residues 260–280 (D270) and GST–Cx43 missing residues 380–382 (DLEI) were incubated with
NRK cell lysates. Pulled down proteins were analyzed via immunoblot with anti-GST and CASK antibodies. (C) The mean ratio of CASK signal to recombinant
Cx43 for five experiments is shown in the bar graph (x + s.d.). **P,0.01, ***P,0.001 compared with CT values.
CASK and Cx43 functionally interact
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Journal of Cell Science
Fig. 2. Non-phosphorylated Cx43 binds to CASK.
(A) Cell lysates from NRK cells were incubated with either GST
alone or GST CASK in a pull-down assay. Bound Cx43 was
detected by immunoblotting with a Cx43 antibody. One tenth of the
lysate used in the pull-down experiment was run as a whole cell
control. (B) Cx43 antibodies including mouse anti-Cx43 antibodies
CT1 and IF1 and rabbit antibodies to the C-terminal region of Cx43
(PNRF) and specific for Cx43 phosphorylated at Ser365 (pS365)
were used for immunoprecipitation studies. The resulting
immunoblot was cut and incubated with either a CASK antibody
(upper panel) or a Cx43 antibody (lower panel). (C) Coimmunoprecipitation of Cx43 from NRK cells using antibodies to
CASK, ZO-1 (positive control), Cx43 (positive control) or beads
alone (negative control). (D) Different portions of CASK including
full-length (residues 1–897), N-terminal (L2N; residues 1–612) and
C-terminal (L2C; residues 578–897) were expressed in 293-T cell.
Lysates were pulled down with GST–Cx43 or GST alone (negative
control).
Cx43 (Fig. 2D), but not L2N, indicating that the interaction is not
through the PDZ domain but could involve the SH3 or GUK
domains.
Immunolocalization of Cx43 and CASK in tissues
To investigate whether CASK could interact with Cx43 prior to
cellular lysis in tissues, we examined co-immunolabeling in brain
and skin using confocal microscopy. In Fig. 3B, CASK (green)
and Cx43 (red) show specific colocalization in sections of mouse
brain. DAPI and glial fibrillary acidic protein (GFAP) staining of
the same field indicate higher Cx43 expression in areas of higher
astrocyte cell density (Fig. 3A). CASK and Cx43 were found to
colocalize in what appear to be cell bodies (arrowheads) and
along astrocytic processes (emphasized in Fig. 3C).
We next examined Cx43 and CASK localization during
wounding in human foreskin explant tissue. Samples were
examined in unwounded tissue and at 1 hour after epidermal
wounding. The dashed line in Fig. 4 marks the basement
membrane, indicating the boundary between dermis (below)
and epidermis (above). In Fig. 4B, the arrowhead indicates the
edge of the wound. The colored panels show overlay of Cx43
(red) and CASK (green) staining with nuclei indicated in blue.
Note that in some cases (especially in Fig. 4A) the CASK
antibody was bound nonspecifically to the upper, dead cornified
layer of cells, which is commonly observed with some antibodies
(Aho et al., 2004; Harper et al., 2005; Lampe et al., 1998).
Extensive colocalization was observed 1 hour after wounding
(Fig. 4B). Projections (positions indicated by white lines) of the
x–z and y–z panels confirmed colocalization in all three
dimensions. The grayscale images show immunostaining for
the individual antibodies. In unwounded skin (Fig. 4A),
both Cx43 and CASK are concentrated in the upper
differentiating layers of the epidermis, though CASK appears
to be localized to the cytoplasm whereas Cx43 is found around
the boundaries of cells, in apparent gap junction plaques. This
subcellular localization is most apparent in the grayscale images.
Consistent with expression in these different compartments, we
see very little colocalization of these proteins in unwounded skin
but extensive Cx43–CASK colocalization at 1 hour (Fig. 4B).
This indicates that CASK is mobilized from the cytoplasm to the
plasma membrane at this time point. Previous reports have shown
that Cx43 (Goliger and Paul, 1995; Lampe et al., 1998; Saitoh
et al., 1997) and CASK (Ojeh et al., 2008) expression are lost
near the wound edge as wound healing proceeds.
Cx43 and CASK expression affect cell migration
Fig. 3. Confocal immunofluorescence staining of CASK and Cx43 in
mouse brain tissue. (A) Single tissue section was immunostained with a
GFAP (green) antibody and DAPI (blue). (B) Single tissue section was
stained with CASK (green) and Cx43 antibodies (red), with colocalization
indicated by arrowheads. (C) Higher magnification of the boxed region of B.
Colocalization of Cx43, GFAP and CASK is shown in white (indicated by
arrow). Scale bars: 50 mm (A,B) and 5 mm (C).
In order to further investigate the role that Cx43 and CASK
expression play in migration, we exogenously expressed these
proteins in MDCK cells that normally do not express Cx43 and
only express low levels of CASK (Fig. 5A). We then examined
their migration in a scratch wound assay (Fig. 5B). When CASK
or Cx43 were expressed alone, the migration distance travelled in
24 hours was 50–60% less than in parental cells (Fig. 5C).
However, coexpression of Cx43 and CASK resulted in a
significant rescue of this inhibition (coexpression of Cx43 and
CASK led to significantly more migration than either CASK or
Cx43 expression alone; P,0.001). These results indicate that
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Fig. 4. Human foreskin analyzed by immunostaining with antibodies to
Cx43 and CASK. The x–y, x–z and y–z plane projections are shown in color
and individual antibody staining is shown in grayscale. Cx43 staining is red
and CASK staining is green. Nuclei were counterstained with DAPI (blue).
Dashed line indicates basement membrane. Foreskins were wounded via
punch biopsy and wound margin is indicated by white arrowhead.
(A) Unwounded foreskin. (B) Skin at wound margin after 1 hour. Note that
the CASK antibody, like many other antibodies, sticks nonspecifically to dead
cells within the upper cornified layer, leading to the diffuse green staining.
Scale bars: 50 mm.
CASK and Cx43 functionally cooperate during migration,
whereas expression of either protein alone actually inhibits
migration.
Fig. 5. Migration of MDCK cells transfected with CASK and/or Cx43 in
a scratch wound assay. (A) Overexpression of Cx43 and CASK in MDCK
cells. (B) Examples of migrating cells (note that image is heavily contrasted and
manipulated to emphasize the cell front and deemphasize the marks used for
orientation and migration measurement). Scale bar: 100 mm. (C) The distance
migrated in 24 hours was determined, quantified and the means and standard
deviations determined. There was a significant difference between the migration
of cells expressing CASK or Cx43 alone and the migration of parental cells or
cells coexpressing Cx43 and CASK; ***P,0.001.
We next wanted to test whether CASK knockdown in cells that
endogenously express significant levels affected migration after
scratch wounding. NRK cells express readily detectable levels of
CASK, and expression of short hairpin RNA (shRNA) to CASK
reduced the level of the protein by 60–80% in different
transduction experiments. Because the cells showing 80%
knockdown did not retain this reduced level, we used the cells
that maintained stable 60% knockdown in the experiments shown
(Fig. 6A). Examples of NRK cells at 0 time and 24 hours after
scratch wounding are shown in Fig. 6B. Measurement of the
distance migrated in 24 hours showed approximately a 50%
reduction in the presence of shRNA against CASK compared
with the control (Fig. 6C). We also noticed that the migratory
front of the shRNA-expressing cells appeared to be more
irregular than in the control. We quantified this difference by
measuring the length of a line drawn immediately after scratching
and compared it with a similar line drawn after 24 hours of
migration and found almost a twofold difference (control,
5.662.5; shRNA, 11.866.3, x- 6 s.d., P50.015) potentially
indicating a loss of migrational control when CASK expression
was reduced.
Next, we were interested in determining how Cx43 and CASK
levels and their coordinated function might be regulated. Because
CADM1 has been shown to regulate CASK expression levels
(Giangreco et al., 2009), we decided to investigate its
colocalization in the Cx43- and CASK-expressing MDCK cells
and found extensive colocalization in specific regions (apparent
as white staining in the overlay in Fig. 7) at 6 hours after
scratching (Fig. 7A) and at 1 hour after foreskin wounding
(Fig. 7B) with less colocalization at the other time points studied
(data not shown). Specifically, the MDCK cells at 6 hours after
scratching showed colocalization of all three, or of two of the
three proteins, at different sites in the plasma membrane,
primarily 2–3 cell diameters from the edge of the scratch.
Similarly, suprabasal cells relatively near the wound margin in
foreskin showed extensive CADM1 and Cx43 overlap and
frequent colocalization of all three proteins (Fig. 7). Increased
magnification and line scan analysis of regions of apparent
colocalization showed frequent co-incident increases and
decreases in these proteins indicating ‘real’ overlay (Fig. 7C).
Immunoprecipitation of Cx43 from MDCK cells expressing
Cx43 and CASK showed both CASK and a band in a region
reported for CADM1 when probed with their respective
antibodies (see supplementary material Fig. S1).
Discussion
We have described five findings: (1) Cx43 and CASK interact
directly in vitro and the colocalization can be detected in
vivo (skin and brain). (2) CASK interacts with the C-terminal
region of Cx43 independently of its PDZ domain. (3) Cx43
phosphorylation events that reduce the migration of Cx43 in
SDS-PAGE appear to negatively regulate the interaction,
i.e. the most extensive interaction occurs between the
hypophosphorylated isoform of Cx43 (P0) and CASK. (4)
Cx43 and CASK colocalize in brain and skin and the interaction
is dynamic and coordinated during epidermal wounding. (5)
Cx43, CADM1 and CASK appear to mechanistically cooperate
in regulating migration in MDCK cells.
Both Cx43 and CASK have independently been shown to be
important in brain and skin function. We decided to focus on skin
because we could examine the effect of well-known Cx43
CASK and Cx43 functionally interact
699
Fig. 6. Knockdown of CASK reduces the extent and
regularity of migration of scratch-wounded NRK cells.
(A) shRNA knockdown of CASK was performed and the
levels of CASK were reduced as shown in the western
immunoblot. Cells treated with scrambled (scr) shRNA
were used as control. (B) Examples of scratch-wounded
cells immediately after scratch (0 time) and after 24 hours.
The front of cells is marked with a dotted line. Scale bar:
100 mm. (C) Quantification of the difference in distance
migrated. ***P,0.001.
Journal of Cell Science
expression pattern changes in response to wounding (Di et al.,
2001; Goliger and Paul, 1995; Lampe et al., 1998; Richard, 2000;
Richards et al., 2004; Saitoh et al., 1997; Salomon et al., 1994).
Mice with reduced levels of Cx43 heal wounds more rapidly
(Kretz et al., 2003; Mori et al., 2006). Cx43 antisense application
Fig. 7. Cx43, CASK and CADM1 colocalize at specific times after
wounding. (A) MDCK cells stained 6 hours after scratch wounding show
different amounts of interaction between each of the three proteins, with the
most interaction between the three approximately 50 mm from the wound
edge. (B) Foreskin after 1 hour of wounding shows extensive co-labeling in
the suprabasal region within 50–150 mm of the wound edge (indicated by
arrowhead). (C) Regions of apparent colocalization (taken from A and B in
the regions between the two arrows) and line scan analysis of these regions
(2 pixel width, peaks indicating colocalization are shown by dashed lines) are
presented with size in mm indicated on the x-axis. Scale bars: 50 mm.
to wounds accelerated the rate of keratinocyte migration, reduced
inflammation and increased the rate of wound repair, resulting in
less scarring (Qiu et al., 2003). Ojeh and colleagues have also
recently shown that CASK expression changes during wounding
(Ojeh et al., 2008) and that the ‘migratory tongue’ of epithelial
cells shows reduced CASK expression. Consistent with
this observation, Giangreco and colleagues reported that
overexpression of CADM1 (Necl2) leads to increased CASK
expression and eliminates the reduction of CASK at the
migratory tongue, causing a reduction in keratinocyte migration
and delayed wound healing (Giangreco et al., 2009). Together
these data indicate that CADM1 regulation of CASK levels could
regulate cellular migration during wound healing, potentially
through their action on Cx43.
Previous data from cultured primary keratinocytes (Richards
et al., 2004) indicated that gap junctional communication
dropped near the wound edge at 1 hour after wounding but that
blockage of intercellular communication by carbenoxolone at the
time of wounding inhibited subsequent cell migration. This
indicates that gap junction communication is involved in
migration initiation. Thus, the combination of these data and
the data reported here indicate an important functional interaction
between Cx43 and CASK in initiating migration in response to
wounding. Given that it appears that reductions in both CASK
and Cx43 are necessary for proper wound healing, we speculate
that their removal is also coordinated and might involve CADM1.
Although it is not yet clear exactly how these interactions
mediate their effects, the known functions of these proteins can
give us clues. CASK is a MAGUK protein which, like other
family members, exists as a complex with other proteins. It is part
of an evolutionarily conserved tripartite complex on the
membrane that is composed of modular adaptor proteins
(CASK, LIN7, LIN10). Many MAGUK proteins are known to
play key roles in the apical–basolateral targeting of proteins in
polarized cells, including sorting in the trans-Golgi network and
endocytic compartments. For instance, mutations in C. elegans
Lin proteins result in the mislocalization of the Let-23 receptor
(Budnik et al., 1996; Kaech et al., 1998), whereas mutations in
the Drosophila Dlg (Discs large protein), another MAGUK
protein, cause overgrowth of Drosophila imaginal discs and
abnormal larval neuromuscular junctions (Budnik et al., 1996).
We show here that CASK interacts preferentially with the P0 form
of Cx43. Notably, the P0 form of Cx43 is found predominantly in
cytoplasmic and nonjunctional plasma membranes, presumably
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en route to gap junction formation. Cx43 becomes increasingly
phosphorylated and exhibits retarded migration in SDS-PAGE as
it is incorporated into gap junction plaques. Our laboratory has
shown that phosphorylation at serine residues 365, 325, 328 and
330 occur as Cx43 assembles into a gap junction, and that these
events are responsible for most of the shift in the SDS-PAGE
migration of Cx43 (Lampe et al., 2006; Solan et al., 2007). CASK
localization is similarly dynamic, shifting from cytoplasmic to
membrane localization during differentiation and to the nucleus
upon entry into S phase of the cell cycle (Ojeh et al., 2008). The
fact that CASK preferentially interacts with P0 Cx43 suggests
that CASK is involved in the transport of non-phosphorylated
Cx43 to the plasma membrane where phosphorylation and
interaction with proteins involved in forming the gap junction
plaque could supplant the interaction, resulting in a ‘hand-off’ of
Cx43 to interacting proteins involved in gap junction assembly.
Recently, CASK has been shown to have Mg2+-independent
neurexin kinase activity that could conceivably be involved in
Cx43 phosphorylation (Mukherjee et al., 2008). However,
our very preliminary experiments did not show Cx43
phosphorylation in immunoprecipitated complexes of the two
proteins (data not shown), but the phosphorylation could also be
indirect. A model consistent with these results could involve an
initial wound and CADM1-induced recruitment of CASK to the
plasma membrane promoting Cx43 phosphorylation, loss of
interaction with CASK and gap junction assembly. At later time
points, a reduction in CADM1 expression or localization might
cause a reduction in CASK and Cx43 phosphorylation, thereby
reducing gap junction stability and resulting in cells that can
more efficiently migrate and close wounds. Because gap
junctional communication is necessary soon after wounding to
initiate migration (Richards et al., 2004), it could play a role in
reducing CADM1 levels and thus in triggering the loss of CASK
and Cx43 observed at 24 hours. Alternatively, dissociation of
interactions between CASK, CADM1 and/or Cx43 could trigger
degradation and/or migration.
Cx43 and CASK also play important roles in mammalian cell
development. Disruption of the CASK locus by transgenic
insertion in mice results in cleft palate and death within 24 hours
of birth (Laverty and Wilson, 1998). Similarly, Cx43 knockout is
neonatally lethal due to conotruncal defects in the heart (Reaume
et al., 1995). The crucial roles of these apparently co-regulated
proteins during development and wound repair, their temporally
and spatially regulated colocalization and their roles in the
control of migration indicate both a physical and functional
interaction between Cx43, CASK and CADM1 that is of
significant biological importance.
Materials and Methods
Reagents, antibodies and cDNA constructs
All general chemicals, unless otherwise noted, were purchased from Fisher Scientific.
Anti-Myc mouse monoclonal 9E10 (Cell Signaling Technology, Beverly, MA),
rabbit anti-Cx43 (C6219; Sigma, St Louis, MO), rabbit anti-CADM1 (SynCAM,
S4945, Sigma), rabbit anti-Cx43 [termed PNRF and obtained from A. Boynton
(Hossain et al., 1998)], rabbit antibody against Ser365-phosphorylated Cx43 (Solan
et al., 2007), mouse anti-Cx43 antibodies Cx43CT1 and Cx43IF1 raised against
amino acids 360–382 (Cooper and Lampe, 2002; Sosinsky et al., 2007), Cx43NT1
raised against residues 1–20 of Cx43 (Solan et al., 2007), rabbit polyclonal antiCASK (UM196) (Straight et al., 2000), anti-GFAP (Zymed/Invitrogen, Carlsbad,
CA), anti-CASK (BD Biosciences, San Jose, CA), and rabbit anti-CASK (Invitrogen)
antibodies were purchased or prepared as described in the associated reference.
Cx43CT (C-terminus of Cx43) encoding amino acids 236–382 of Cx43 was
cloned in pGEX-2T or pGEX-2TK vector to generate GST–Cx43CT, D270 (i.e.
missing amino acids 260–280), and LEI (missing amino acids 380–382).
Essentially, all of the CASK constructs were created in the Margolis laboratory
and have been previously described (Borg et al., 1999; Borg et al., 1998; Straight
et al., 2001; Straight et al., 2000). All GST fusion proteins were expressed and
purified as described earlier (Singh et al., 2005). Protein concentrations were
determined by running GST fusion proteins on SDS-PAGE and comparing to
known concentrations of bovine serum albumin (BSA). Approximately 5 mg of
protein was used in each binding study.
Cell culture and transfections
NRK epithelial cells (NRK-E51; American Tissue Culture Collection, Manassas,
VA), MDCK and 293-T cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (Atlanta Biologicals,
Atlanta, GA), 100 units/ml penicillin G and 100 mg/ml streptomycin in a
humidified chamber with 5% CO2 at 37 ˚C. Cell lysates were harvested in cold
RIPA lysis buffer [25 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 50 mM NaF,
500 mM Na3VO4, 0.25% Triton X-100, 0.25% deoxycholate, 0.02% NaN3, 2 mM
phenylmethylsulfonyl fluoride and 16 Complete Protease Inhibitors (Roche
Applied Science, Indianapolis, IN) pH 7.2]. The lysate was centrifuged at 15,870 g
for 10 minutes and pre-cleared with GST bound to glutathione beads or protein A
for use in pull-down or immunoprecipitation experiments, respectively. 293-T and
MDCK cells were grown to 80% confluence and transiently transfected using
Fugene reagent (Roche Applied Science) according to the manufacturer’s
instructions. Transfected cells were harvested in RIPA precipitation assay buffer
after 48 hours.
shRNA-based CASK knockdown
For CASK depletion, NRK cells were transduced with shRNA-expressing
lentiviruses. The following shRNA-encoding pGIPZ vectors were obtained from
OpenBiosystems: V2LMM_193314 (59-TGCTGTTGACAGTGAGCGCGGGAGTATTACCTTCAAGATTTAGTGAAGCCACAGATGTAAATCTTGAAGGTAATACTCCCTTGCCTACTGCCTCGGA-39), V2LHS_91774 (59-TGCTGTTGACAGTGAGCGCGGTGACATCATCCAGATTATTTAGTGAAGCCACAGATGTAAATAATCTGGATGATGTCACCATGCCTACTGCCTCGGA-39), and a scramble
shRNA. Lentiviral particles were generated by standard procedures (Kappes et al.,
2003). Briefly, 60% confluent 293FT cells were transfected with shRNA-encoding
lentiviral pGIPZ vector, psPAX2 and MD2.G (4:2:1 ratio, respectively) using
Lipofectamine 2000 accordingly to the manufacturer’s specifications (Invitrogen).
At 72 hours after transfection, supernatants were harvested and cleared by
centrifugation at 1,200 g for 10 minutes. Viral preparations were concentrated by
using PEG-it Virus Precipitation Solution according to the manufacturer’s
specifications (SBI, System Biosciences, Mountain View. CA). NRK cells were
transduced with the concentrated viral supernatant (MOI ,1) in the growth media
supplemented with 0.5 mg/ml polybrene for 16 hours at 31 ˚C. At 48 hours posttransduction, puromycin (4 mg/ml) was added to the medium and cells incubated for
5 days. shRNA-expressing cells were maintained in growth medium with 1 mg/ml
puromycin. Knockdown efficiency was estimated by western blot.
Pull-down, immunoprecipitation and in vitro binding assays
For GST pull-down assays, 5 mg of the individual GST fusion proteins bound to
glutathione-agarose beads (Thermo Scientific, Rockford, IL) were incubated at 4 ˚C
for 1.5 hours with 400 ml of cell lysate (prepared as described above). For
immunoprecipitation experiments, the antibodies were incubated with cell lysate
for 1.5 hours followed by immunoprecipitation with protein A-Sepharose beads for
1 hour. The beads were washed extensively in PBS, and bound protein was eluted
in Laemmli sample buffer followed by separation on SDS-PAGE (10%) and
immunoblotting as previously described (Solan et al., 2007). For immunoblot
detection, primary antibodies were visualized with fluorescent dye-labeled
secondary antibodies. Alexa-Fluor-680-conjugated goat anti-rabbit (Invitrogen)
and IRDye800-conjugated donkey anti-mouse IgG (Rockland Immunochemicals,
Gilbertsville, PA) both extensively cross-reacted against other species. The
immunoblots were analyzed using the Li-Cor Biosciences Odyssey infrared
imaging system and associated software (inverted images are presented).
The far western assay has been described previously (Singh et al., 2005).
Briefly, 10–20 mg of GST–Cx43CT fusion protein cloned in the pGEX-2TK vector
was suspended in kinase buffer (50 mM potassium phosphate, pH 7.2, 10 mM
MgCl2, 5 mM NaF, and 5 mM dithiothreitol) to which the catalytic subunit of
protein kinase A (Sigma) and 50 mCi of [c-32P]ATP (Blu002; PerkinElmer Life
Sciences, Waltham, MA) were added for 30 minutes. After washing and elution of
the labeled Cx43, it was incubated with a blot containing lysates from cells
expressing CASK or LIN7. Ponceau S (Sigma) staining of the membrane was
performed according to the manufacturer’s directions.
Immunofluorescence and immunostaining
Mouse brain and neonatal human foreskin were frozen in OCT (VWR, West
Chester, PA) and cut as 8-mm sections on a cryostat microtome. The sections were
fixed in ice-cold acetone and methanol (1:1), washed twice in PBS and then
incubated with 5% normal serum and 1% BSA in PBS for 1 hour at room
CASK and Cx43 functionally interact
temperature. Slices were then incubated with rabbit anti-Cx43 (1:200) and mouse
anti-CASK monoclonal (1:200) in blocking solution for 1 hour. After four washes
in PBS, the cells were incubated with Alexa-Fluor-488-conjugated goat anti-mouse
and Alexa-Fluor-546-conjugated goat anti-rabbit secondary antibodies (Invitrogen)
for 1 hour. For the triple labeling experiments, Cx43 was labeled with anti-mouse
IgG-specific Alexa Fluor 546 and CADM1 with anti-rabbit IgG Alexa Fluor 633
(Invitrogen). The slides were washed in PBS and then labeled with anti-mouse
IgG1-specific Alexa Fluor 488 (Invitrogen) to detect CASK. After washing, the
slides were mounted in ProGold anti-fade medium (Invitrogen). Images were
acquired using a Zeiss LSM 510 Confocal Laser Scanning microscope with a 406
oil objective. Z-planes analysis of images including linescan analysis utilized
Metamorph 7.0 (Universal Imaging, Westchester, PA).
Wounding of human neonatal foreskin
Neonatal foreskin tissue was obtained from a local hospital under an approved
Institutional Review Board protocol. Human foreskins were placed in serum-free
keratinocyte growth media (KGM, Lonza, Walkersville, MD) for up to 24 hours.
Wounds were created via punch biopsy then fixed immediately in 10% neutral
buffered formalin or maintained in culture for 1 and 24 hours prior to fixation. The
tissue was analyzed via immunofluorescence with antibodies to Cx43 and CASK
as indicated and counterstained with DAPI.
Journal of Cell Science
In vitro scratch-wounding assay
Cells were cultured to confluence on 10-cm plates, and the medium was replaced
with Opti-MEM I (Invitrogen) reduced serum medium 24 hours before scratching
with a 200 ml pipette tip. Detached cells were removed, and the remaining cells
were incubated for 0 and 24 hours. At least eight scratched areas for each sample
were marked and photographed with a Nikon TE-400 inverted microscope
immediately and after 24 hours of incubation. Migration was evaluated as
previously described (Richards et al., 2004) by measuring the distance migrated at
24 hours compared to the position at 0 time. For measurements of migrating front
regularity, the length of a line drawn along the front edge of the leading cells was
compared to the length of a line drawn immediately after the scratch was
performed, as previously described (Richards et al., 2004).
Acknowledgements
We wish to thank Ben Margolis and Samuel Straight, previously in
the Margolis laboratory, for their advice and sharing of essentially all
of the CASK reagents used in this study. This research was supported
by grant GM55632 from the National Institutes of Health.
Funding
This research was supported by the National Institutes of Health
[grant number GM55632]. Deposited in PMC for release after 12
months.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.084400/-/DC1
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