1 Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA, and Department
of Pathobiology, University of Washington, WA 98195, USA
2 Cell Signaling Technology, Beverly, MA 01915, USA
3 Genetics, Cell Biology and Development, University of Minnesota, St Paul, MN
55108, USA
* Author for correspondence (e-mail: plampe{at}fhcrc.org)
Accepted 12 February 2003
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Summary |
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Key words: Gap junctions, Connexins, Tumor promoter, Phosphorylation, Carcinogenesis
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Introduction |
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Transient changes in gap junctional communication, probably regulated by
signaling cascades, have been observed and appear necessary for normal cell
cycling. For example, gap junctional communication was reported to be moderate
during G1/S, increased through S and decreased in G2/M
(Bittman and LoTurco, 1999;
Stein et al., 1992
). The
downregulation of junctional communication during G2/M has been
correlated with increased p34cdc2 kinase-dependent phosphorylation
of Cx43 (Kanemitsu et al.,
1998
; Lampe et al.,
1998a
) and redistribution of Cx43 from gap junctions to the
cytoplasm (Lampe et al.,
1998a
; Xie et al.,
1997
). Gap junctional structures reassemble and communication is
gradually restored as cells proceed through G1
(Stein et al., 1992
;
Xie et al., 1997
). Cx43 is
phosphorylated at multiple serine residues in vivo
(Berthoud et al., 1992
;
Brissette et al., 1991
;
Crow et al., 1990
;
Kadle et al., 1991
;
Laird et al., 1991
;
Musil et al., 1990
), and upon
phosphorylation, Cx43 migration in polyacrylamide gel electrophoresis
(SDS-PAGE) is reduced. Although apparently not required for the formation of
functional channels (Dunham et al.,
1992
; Fishman et al.,
1991
), phosphorylation of gap junction proteins appears to
regulate channel function (gating) and the rates of channel assembly and
turnover (Brissette et al.,
1991
; Kwak et al.,
1995a
; Kwak et al.,
1995b
; Kwak et al.,
1995c
; Lampe,
1994
; Lampe et al.,
2000
).
In the sustained absence of connexin expression, tumorigenesis is enhanced
(Laird et al., 1999;
Moennikes et al., 1999
). The
correlation between neoplastic transformation and reduced gap junctional
communication (Atkinson et al.,
1981
; Azarnia and Loewenstein,
1984
; de Feijter et al.,
1990
) has led to the hypothesis that reduced cell-cell
communication is a critical step in multistage carcinogenesis
(Fitzgerald and Yamasaki,
1990
; Trosko et al.,
1990
). PKC has received considerable attention because PKC
activators (e.g. TPA), which promote tumorigenesis, both increase Cx43
phosphorylation and decrease gap junction communication in several different
cell types (Berthoud et al.,
1992
; Berthoud et al.,
1993
; Brissette et al.,
1991
; Lampe, 1994
;
Reynhout et al., 1992
). PKC
has been shown to phosphorylate Cx43 at S368, and this site has been shown to
underlie a TPA-induced reduction in intercellular communication and alteration
of single channel behavior (Lampe et al.,
2000
). However, in some cell types, TPA treatment did not lead to
a shift in Cx43 mobility in SDS-PAGE (which is thought to indicate increased
Cx43 phosphorylation) but did change gap junctional communication (e.g.
Rivedal and Opsahl, 2001
),
leading to confusion as to the role of Cx43 phosphorylation in this
process.
Here, we report that Cx43 phosphorylation at S368 was indeed increased by TPA treatment in all cell types tested, but that Cx43 mobility was not significantly affected in some. Furthermore, S368 phosphorylation was increased during key stages of the cell cycle where gap junctional assembly is reduced. Thus, in addition to its role in the regulation of gap junction channel gating, phosphorylation at S368 was negatively correlated with gap junction assembly.
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Materials and Methods |
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Metabolic labeling and Cx43 immunoprecipitation
NRK cells were cultured, metabolically labeled with
[32P]orthophosphate (ICN, 64014L) or 35S-Trans label
(ICN, 5100607), and immunoprecipitated essentially as previously described
(TenBroek et al., 2001).
Briefly, cells were labeled with [32P]orthophosphate at 1.0 mCi/ml
for 3 hours in phosphate-deficient medium (Gibco-Invitrogen, Grand Island, NY)
and, where indicated, were treated with 50 ng/ml TPA during the final 30
minutes. Alternatively, cells were washed three times and labeled with
35S-Trans label at 0.1 mCi/ml for 3 hours in methionine-free media
(Gibco-Invitrogen) and, where indicated, were treated with 50 ng/ml TPA during
the final 30 minutes. The cells were rinsed in PBS, lysed in RIPA buffer [25
mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 50 mM NaF, 500 µM
Na3VO4, 0.25% Triton X-100, 2 mM phenylmethylsulphonyl
fluoride (PMSF) and 1x Roche Complete protease inhibitors], clarified
with protein A beads, and immunoprecipitated with p368 antibody [a rabbit
anti-Phospho-Cx43 (Ser368) antibody #3511; Cell Signaling Technology, Beverly,
MA], rabbit antibody C6219 from Sigma (St Louis, MO) and/or monoclonal Cx43CT1
antibody. Cx43CT1 antibody is an antibody prepared to a peptide representing
the last 23 amino acids of Cx43 (described in
Cooper and Lampe, 2002
).
Cx43CT1 behaves like antibody 13-8300 from Zymed, which was prepared to the
same region of Cx43, in that it immunoprecipitates primarily the `NP' form of
Connexin unless cells are treated with TPA, when slower migrating forms were
detected (Cruciani and Mikalsen,
1999
). After four washes in RIPA buffer, the immunoprecipitates
were treated with Laemmli sample buffer and run via SDS-PAGE (10%
polyacrylamide, Tris-glycine gels).
Immunoblotting
Cells were lysed in sample buffer containing 50 mM NaF, 500 µM
Na3VO4, 2 mM PMSF and 1x Complete protease
inhibitors (Roche Diagnostics, Indianapolis, IN) and cellular proteins were
separated by SDS-PAGE on 10% Tris-glycine gels. For alkaline phosphatase
treatment, cells were lysed in 0.2% SDS, 2 mM PMSF and 1x protease
inhibitors, and briefly sonicated followed by addition of one-tenth volume of
10x phosphatase buffer (M183A; Promega, Madison, WI) and incubation with
10 units of calf intestinal alkaline phosphatase (M182A; Promega) for 1 hour
at 37°C. After electrophoresis, protein was transferred to nitrocellulose,
the membrane was blocked, and antibodies were incubated as previously
indicated (Lampe et al.,
1998a). Primary and secondary antibodies utilized were p368
antibody, mouse anti-Cx43 (Cx43NT1 described in
Goldberg et al., 2002
), mouse
anti-vinculin (Sigma), peroxidase-conjugated donkey anti-mouse or mouse
anti-rabbit secondary antibodies (Jackson Immunoresearch Laboratories, West
Grove, PA). Where indicated, the blots were `stripped' for 30 minutes at
50°C in 62.5 mM Tris pH 6.8, 1% SDS and 5% ß-mercaptoethanol buffer
followed by washing for 2 hours with at least six changes of PBS. Signal was
visualized with SuperSignal West Pico or Femto Chemiluminescent Substrate
(Pierce Chemicals, Rockford, IL) followed by exposure to Kodak Biomax MR film.
Densitometry of autoradiographs was performed on a Macintosh G3 using a Sharp
JX-325 scanner to collect the image and the public domain NIH Image program
(developed at the US National Institutes of Health and available at
http://rsb.info.nih.gov/nih-image).
Immunofluorescence
NRK cells were untreated or treated with TPA for 30 minutes at 37°C,
washed twice in PBS, and fixed in cold methanol/acetone (50:50) for 1 minute
followed by blocking for 1 hour in 1% bovine serum albumin in PBS. Cells were
incubated with anti-Cx43 antibody p368 and/or Cx43IF1 (see
Cooper and Lampe, 2002;
TenBroek et al., 2001
) in
blocking solution for 1 hour. Following several PBS washes, the cultures were
incubated with Alexa594-conjugated goat anti-rabbit antibody (Molecular
Probes, Eugene, OR) and/or fluorescein isothiocyanate-conjugated donkey
anti-mouse antibody (Jackson Immunoresearch Laboratories) for 30-60 minutes
and counterstained with DAPI (Molecular Probes), followed by several washes in
PBS. The coverslips were mounted onto slides with DABCO antifade medium [25
mg/ml of 1,4-diazobicyclo-(2,2,2)octane (Sigma) diluted in Spectroglycerol
(Kodak) and 10% PBS, pH 8.6] and viewed with a Nikon Diaphot TE300
fluorescence microscope, equipped with a 40x (1.3 n.a.) oil objective
and a Princeton Instruments cooled digital camera driven by an attached PC and
Metamorph imaging software.
Cell synchronization
G0 cells were prepared by contact-inhibiting NRK cells at least
3 days past confluency without addition of fresh media. To obtain
G1 cells, confluent cells were trypsinized, then diluted to 60-80%
confluency and allowed to progress 8-10 hours for early G1 and
14-16 hours for late G1. Cell-cycle analysis showed that by 18
hours these cells begin to enter S phase. For preparation of G1/S,
S, G2 and G2/M cells, confluent cells were trypsinized
then diluted to 60-80% confluency in media containing 1 mM thymidine for 16
hours to induce a G1/S block. Cells were released from
G1/S by washing and replacement of 37°C complete media.
Cell-cycle analysis showed that S phase lasts 4-6 hours in these cells and
that cells cycle through G2/M to G1 by 9-11 hours after
washout. We typically observed 70-90% synchrony as cells progress through S to
G1. Cell-cycle analysis was performed by fluorescence activated
cell sorting. Specifically, cells were trypsinized, then pelleted in PBS with
2% fetal bovine serum and fixed in 70% EtOH. Cells were pelleted, washed and
incubated with 5 µg/ml RNase at 37°C for 30 minutes, and then stained
with 50 µg/ml propidium iodide on ice for 1 hour. DNA content was assessed
on a Becton Dickinson FACScalibur and data analyzed using CellQuest
software.
Gap junctional communication/assembly
Gap junctional communication was assayed via dye transfer according to
published methods using either an assembly-preloading assay with calcein-AM
(Lampe et al., 1998b) or by
microinjection of fluorescent dyes. Briefly, for the preloading assay, one 10
cm plate of NRK cells was labeled with 0.5 µM calcein-AM (Molecular
Probes), the cell-permeant ester of calcein that is cleaved to
membrane-impermeant calcein by cellular esterases. Three other culture plates
were labeled with 0.25 µM DiI (Molecular Probes). After washing twice with
PBS, the two populations of cells were each trypsin/EDTA suspended, treated
with trypsin inhibitor and pelleted. The cells were suspended in the
appropriate media, mixed, plated on culture dishes and placed in a 37°C
incubator. Cells were allowed to adhere for 2 hours then digital images of
calcein and DiI were captured. The assignment of a cell as an acceptor of dye
via transfer rather than a poorly loaded or leaking donor is checked by
digitally overlaying images of DiI and calcein fluorescence. If a cell
adjacent to a calcein-loaded, DiI-negative cell contains both punctate DiI and
more-diffuse calcein fluorescence, gap junction assembly and dye transfer
occurred. If a DiI-labeled cell adjacent to a calcein-loaded cell does not
contain calcein, then dye transfer did not occur at that interface. A
more-complete description of this assay is published elsewhere
(Lampe, 1994
;
Lampe et al., 1998b
). The
fraction of cells that transferred dye were determined by dividing the number
of DiI-labeled cells that contained calcein (i.e. transfers) by the number of
cell interfaces between calcein-loaded and DiI-labeled cells (i.e. total).
Dye transfer in established cultures was analyzed by microinjection of a 10 mM solution of each of the gap junction permeable dyes, Alexa hydrazide 488 and 594 (Mr=570.5 and 758.8, respectively; Molecular Probes) in 0.2 M KCl. The dyes were microinjected using a 5 millisecond pulse of air at 10 psi from a General Valve Picospritzer II, and the number of cells receiving dye was analyzed after 10 minutes using the imaging system described above.
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Results |
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To verify further that this antibody recognized a phosphorylated species of
Cx43, NRK cell lysates from control cells and TPA-treated cells were incubated
with alkaline phosphatase and analyzed by immunoblot. As above, immunoblots
were first processed with the p368 antibody, then stripped and reprobed with
the anti-Cx43 antibody, allowing precise alignment and determination of the
extent of migration of the bands. Immunoblots incubated with anti-Cx43 showed
little change in response to TPA treatment
(Fig. 2, -Cx43 panel;
compare CON and TPA lanes). When these cell lysates were incubated with
alkaline phosphatase, all of the Cx43 migrated as the NP form
(Fig. 2,
-Cx43 panel, AP
lanes), which is consistent both with effective alkaline phosphatase treatment
and with what has been shown by other investigators, as noted above.
Processing of the blot with anti-p368 antibody showed a sixfold increase in
signal upon TPA treatment (Fig.
2,
-p368 panel; compare CON and TPA) and this signal was
completely lost upon alkaline phosphatase treatment
(Fig. 2,
-p368 panel, AP
lanes). These data show that the p368 antibody reactivity appears to be
specific for S368 only when it is phosphorylated (i.e. it is
phosphorylation-state specific) and that a dramatic increase in
phosphorylation at S368 is generated in response to TPA.
|
The `NP' form of Cx43 can be phosphorylated on S368
Figs 1 and
2 indicate that an isoform of
Cx43 that migrated similarly to NP Cx43 reacted with the p368 antibody. To
determine more directly whether a phosphorylated species migrated to the same
extent as the NP form of Cx43, we performed metabolic labeling on NRK cells
with [32P]orthophosphate or [35S]methionine. Cx43 from
[32P]orthophosphate-labeled cells was immunoprecipitated, run on
SDS-PAGE and blotted to nitrocellulose. These samples were analyzed first by
autoradiography (Fig. 3,
32P panel) and then immunoblot analysis using p368 (-p368
panel) and Cx43NT1 (
-Cx43) monoclonal antibodies. In the
autoradiograph, Cx43 immunoprecipitated from untreated cells showed two band,
indicated as P1 and P2, whereas cells treated with TPA showed a more-broad
phosphorylation pattern some of which appeared to migrate at the same position
as the NP form. The
-p368 panel, which represents the chemiluminescent
signal obtained from the same blot probed with p368 antibody, shows a dramatic
TPA-dependent increase in signal co-migrating with the NP form, whereas
probing the same blot with the
-Cx43 antibody showed minor differences
in the typical pattern for Cx43 with or without TPA treatment. Thus, the
TPA-dependent increase in Cx43 phosphorylation levels found by autoradiography
was not nearly as extensive as that observed with the p368 antibody
immunoreactivity. This result confirms that S368 phosphorylation, in
particular, is increased dramatically via TPA treatment, whereas
phosphorylation at many other residues was not as TPA responsive
(Lampe et al., 2000
),
essentially diluting the p368 signal. Furthermore, the total Cx43 signal and
the ratio of `phosphorylated' (i.e. P1 + P2) to nonphosphorylated
(Fig. 2, a-368 panel) were
quite similar regardless of TPA treatment, in spite of the fact that dramatic
changes in S368 phosphorylation occurred.
|
NRK cells were also labeled with [35S]methionine, and
immunoprecipitations were carried out using either the p368 antibody or an
anti-Cx43 (Cx43CT1) antibody that shows a strong preference for the NP
migratory isoform. These samples were run on SDS-PAGE and analyzed by
autoradiography. In NRK cells, the -Cx43 antibody immunoprecipitated a
single band that did not change significantly in intensity upon TPA treatment
(Fig. 4, NRK panel,
-Cx43). As expected, this band migrates at the same position as NP. The
p368 antibody also immunoprecipitated a single band that migrated exactly with
the band immunoprecipitated with the
-Cx43 antibody and showed
increased signal intensity upon TPA treatment
(Fig. 4, NRK panel,
-p368). Thus, the p368 antibody was able to immunoprecipitate a Cx43
isoform that migrates the same as the NP form in our typical Laemmli gel
system, and TPA-treated cells contained more of this isoform than control
cells.
|
Taken together, these metabolic labeling data show that a phosphorylated
species of Cx43 essentially co-migrates with the NP form and that this
phosphoform can be clearly detected using the p368 antibody. Nonphosphorylated
Cx43 and Cx43 phosphorylated at S368 probably could be separated given the
appropriate separation technique since these species vary in net charge. It is
noteworthy that standard isoelectric focusing and two-dimensional analysis of
Cx43 has been shown to be difficult
(Stockert et al., 1999).
Nevertheless, when analyzed by a standard Tris/glycine SDS-PAGE system that
has been used by most investigators, the migration of Cx43 phosphorylated on
S368 often coincided with nonphosphorylated Cx43. For this reason, we believe
that it is probably most accurate to refer to the fastest-migrating form as P0
rather than NP when discussing standard SDS-PAGE separation of Cx43 from cells
that have been treated with kinase effectors or growth factors, and we do so
below. This is an interim solution since different cell types and slightly
modified gel systems appear to produce Cx43 with varying migratory properties.
A better definition of terms will probably require a thorough understanding of
the molecular events that underlie the shift in migration.
The TPA-dependent shift in Cx43 migration, but not phosphorylation of
S368, is cell-type specific
Although the migration of Cx43 derived from NRK cells does not shift
significantly in the presence of TPA, many other cell types can show a
dramatic shift, essentially leaving little faster-migrating species as shown
for HeLa cells in Fig. 1. We
found that CHO cells also show a dramatic shift in response to TPA, as is
shown via immunoprecipitation in Fig.
4 and western immunoblot in
Fig. 5 (CHO panels).
Fig. 4 shows
[35S]methionine-labeled CHO cell lysates immunoprecipitated with
-Cx43 or
-p368 antibodies. In TPA-treated CHO cells,
-Cx43 (Cx43CT1) immunoprecipitated both the NP/P0 migratory isoform and
the slower-migrating isoforms (Fig.
4, CHO panel,
-Cx43; see Materials and Methods for antibody
description). Immunoprecipitation of TPA-treated CHO cell lysates with
-p368 antibody shows primarily the slower-migrating isoforms
(Fig. 4, CHO panel,
-p368) indicating that, in this cell line, phosphorylation on S368 was
coincident with a shift in migration.
|
Similarly, Fig. 5 shows an
immunoblot of NRK and CHO whole cell lysates that was probed with the antibody
for p368 (-p368) and stripped/reprobed for Cx43 (
-Cx43).
Consistent with the immunoprecipitation results, Cx43 from NRK cells did not
shift its migration in response to TPA while the protein extensively shifts to
slower-migrating phosphoforms in CHO cells. Both cell types show large
TPA-dependent increases in reactivity to the p368 antibody, but the p368
signal in NRK cells primarily migrated at the P0 and P1 positions whereas the
p368 signal was highly shifted in the CHO cells. HeLa cells containing wt Cx43
(Fig. 1) were intermediate
between the two, as p368 is found in the P1 and P2 forms. Notably, in all cell
lines examined, a low level of p368 was present in untreated cells and often
co-migrated with the NP/P0 isoform, which indicates that phosphorylation of
S368 is part of the normal lifecycle of Cx43 in these cells. To examine
whether phosphorylation at S368 might be consistent with the early
phosphorylation event found in the presence of Brefeldin A (BFA)
(Laird et al., 1995
), we
treated NRK and CHO cell lysates with BFA and found decreased p368 antibody
labeling. However,
-p368 binding was not eliminated, so no firm
conclusions can be drawn with respect to this event. Thus, we have used the
p368 antibody to examine TPA-induced phosphorylation of Cx43 in NRK, CHO and
Cx43-transfected HeLa cells and found that all cell types examined show
increased phosphorylation on S368, but the degree to which this resulted in a
shift in the migration of Cx43 varied between cell types.
TPA-induced phosphorylation of S368 occurs on both intracellular and
plasma membrane Cx43
To determine whether a specific pool of Cx43 is phosphorylated in response
to TPA, immunofluorescence was performed on NRK cells with an antibody
specific for Cx43 (Cx43IF1) and the p368 antibody. NRK cells show extensive
immunofluorescence for Cx43 at cell-cell interfaces
(Fig. 6, upper left). Upon TPA
treatment, Cx43 immunofluorescence showed no apparent change although the
cells adopted a slightly more fibroblastic appearance
(Fig. 6, lower left). The p368
antibody also showed some cell-cell interface labeling and a light reticulate
pattern throughout the cytoplasm (upper center panel). The apparent
cytoplasmic pool of p368 staining does appear to be at least partly associated
with the endoplasmic reticulum as there was co-localization of p368 with an
endoplasmic reticulum-specific dye, R6 (data not shown). After TPA treatment,
the p368 signal was greatly increased in both cytoplasmic and interface
membranes (lower center panel). The plasma membrane pool of p368 shows
co-localization with the Cx43IF1 antibody, whereas less-distinct
co-localization of this antibody with the intracellular pool was observed. The
increase in intracellular fluorescence does appear to be specific to the p368
epitope as co-incubation of p368 antibody with the peptide antigen used to
generate the antibody blocked antibody binding, while co-incubation of the
antibody with a nonphosphorylated peptide representing 360-382 of Cx43 did not
block binding (data not shown). We have observed that there is competition
between Cx43IF1 and p368 antibody binding at cell-cell contacts. This was
manifest by a decrease in p368 signal when p368 and Cx43IF1 antibodies were
added together, but was reversed by inclusion of the nonphosphorylated
peptide, which removed the Cx43IF1 signal.
|
Phosphorylation on S368 is regulated as cells progress through the
cell cycle
Given that phosphorylation on S368 appeared to be part of the normal
lifecycle of Cx43, we wanted to determine circumstances under which this event
was regulated. As it has previously been shown that Cx43 phosphorylation
increases as cells progress through the cell cycle
(Kanemitsu et al., 1998), we
looked at Cx43 and phosphorylation of S368 in cells synchronized at different
stages of the cell cycle in NRK cells. Fig.
7 shows an immunoblot probed first for p368 (
-p368) and
then stripped/reprobed for Cx43 (
-Cx43). Vinculin was also detected for
a loading control. Densitometry was performed for Cx43 and p368 antibody
binding, and the ratio of p368/Cx43 densitometry is shown at the bottom of the
figure. Cx43 phosphorylated at S368 was most abundant relative to total Cx43
during S and G2/M. This result is consistent with previous reports
where gap junctional communication was shut-down during mitosis
(Stein et al., 1992
;
Xie et al., 1997
) and
phosphorylation at S368 had been shown to reduce communication
(Lampe et al., 2000
). Here, we
found that G0 cells contain very little p368 and that S368 is
increasingly phosphorylated as cells approach and progress through S
phase.
|
Given the 7x increase in phosphorylation at S368 when G0 and S phase cells were compared (Fig. 7), we wanted to examine Cx43 distribution and intercellular communication in these two cell populations. G0 cells showed strong plasma membrane staining for Cx43 at cell-cell interfaces consistent with gap junctions (Fig. 8A), while S-phase cells showed both typical gap junctional labeling and also extensive perinuclear staining (Fig. 8B). Immunofluorescent labeling with the p368 antibody showed both cytoplasmic and gap junctional staining for both G0- and S-phase cells (data not shown).
|
Since TPA treatment of cells has been reported to decrease intercellular
communication via changes in channel gating (e.g.,
Kwak et al., 1995c;
Lampe et al., 2000
;
Moreno et al., 1994
) and gap
junction assembly (Lampe,
1994
), we assessed both the ability to transfer dye and the
ability to assemble junctions in G0- and S-phase cells. When we
microinjected G0- and S-phase cells with two fluorescent dyes of
the Alexa series (A488, Mr=570.5; A594,
Mr=758.8), we found that S-phase cells transferred both
dyes approximately twice as well as G0 cells
(Fig. 8C). However,
G0-phase cells were approximately twice as likely to transfer dye
to their neighbors than S-phase cells when the calcein/DiI assay, which
requires nascent gap junction assembly, was performed
(Fig. 8D).
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Discussion |
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Cx43 phosphorylation at S368 appears to occur normally in dividing cells.
The seven- to eightfold increase in the level of phosphorylation on S368 at S
and G2/M, respectively, correlates well with increased cytoplasmic
localization of Cx43 during S (Fig.
7) and G2/M (Lampe
et al., 1998a; Xie et al.,
1997
), consistent with a role for S368 phosphorylation in
regulating Cx43 trafficking/assembly into gap junctional structures.
Interestingly, we also occasionally observed a unique and apparently nuclear
envelope/endoplasmic reticulum localization of the p368 antibody at the early
stages of G2/M (data not shown). This immunolocalization was highly
transitory because it was lost as the nuclear envelope broke down as the cells
entered mitosis. Although this localization appeared specific for the antibody
based on antigen competition studies, Cx43IF1 antibody immunolabeling of the
nuclear envelope region of G2/M cells was not nearly as striking as
the p368 antibody. Thus, we cannot rule out the possibility that an
alternative non-connexin epitope that specifically reacts with the p368
antibody is expressed in early mitosis.
Much of the work examining TPA-mediated downregulation of Cx43 has been
motivated by the role of PKC activators as tumor promoters and the potential
role of gap junctional communication as a tumor suppressor. Although details
are still poorly understood, there is a wealth of data showing that Cx43
phosphorylation is increased and gap junctional communication is reduced upon
activation of PKC (reviewed by Lampe and
Lau, 2000). However, the use of different assays for
communication, several methods for assaying Cx43 phosphorylation and various
cellular systems expressing different isoforms of PKC
(Cruciani et al., 2001
;
Munster and Weingart, 1993
)
have confused the interpretation of the role PKC plays as a modulator of gap
junctional communication. For example, several reports have fueled the
controversy as to whether mitogen-activated protein kinase (MAPK) or PKC is
the actual kinase that phosphorylates Cx43 and reduces gap junctional
communication after growth factor or phorbol ester treatment, or whether Cx43
phosphorylation even plays a direct role (e.g.,
Hossain et al., 1999
;
Kanemitsu and Lau, 1993
;
Rivedal and Opsahl, 2001
;
Vikhamar et al., 1998
). One
presumption found in many of these reports that might cloud interpretation of
the data is that a shift in Cx43 migration has been equated with increased
phosphorylation. We know that Cx43 can be phosphorylated at many (>5) sites
in untreated cells and at many more sites in growth factor-treated cells
(Lampe and Lau, 2000
). At this
time, we have no understanding of the molecular events responsible for the
shift in migration, or of any of the serines involved. By comparing two cell
types where TPA led to a shift in Cx43 migration in one but no change in
another, the logical but potentially erroneous conclusion could be that Cx43
phosphorylation levels only changed in one of the cell types. For example,
from the
-Cx43 panel of Fig.
5, one could conclude that there was a large change in Cx43
phosphorylation in CHO cells upon TPA treatment, whereas NRK cells showed
little change and thus appeared unresponsive to TPA treatment; by contrast,
the
-p368 panel shows that phosphorylation was dramatically increased
at this site in NRK cells. In fact, there probably is some correlation with
the extent of shift and the overall level of Cx43 phosphorylation. However,
specific phosphorylation events and not the overall level of phosphorylation
probably elicit a specific regulatory event such as assembly, disassembly or
gating changes. We believe re-evaluation of many of these seemingly
conflicting results might be resolved by assaying for TPA and growth factor
effects with the p368 and other phosphorylation-site-specific antibodies.
Phosphorylation of Cx43 appears to regulate the trafficking of Cx43 to the
plasma membrane, assembly of Cx43 into gap junctional structures, single
channel behavior and Cx43 degradation. The latter three events have been
reported to be sensitive to TPA and, therefore, could be regulated by PKC
(Kanemitsu and Lau, 1993;
Kwak et al., 1995a
;
Kwak et al., 1995c
;
Lampe, 1994
). Our
immunofluorescence data with the p368 antibody and comparison of the kinetics
of the mobility shift in SDS-PAGE with decreases in gap junctional
communication (Kanemitsu and Lau,
1993
) indicate that S368 phosphorylation and potentially other
PKC-mediated events can occur prior to export to the plasma membrane
(Lampe, 1994
). Therefore, at
least some TPA-dependent phosphorylation at S368 occurs prior to gap junction
assembly.
Intercellular communication was reduced by TPA in quiescent but not
proliferating NRK cells (Paulson et al.,
1994). Data presented here indicates that, in addition to S368
being a TPA-responsive site, there is regulation of S368 phosphorylation
during the normal lifespan of Cx43 in untreated, cycling cells. Although
S-phase cells transferred dye more rapidly than G0 cells in
established cultures, S-phase cultures were less able to form new functional
gap junctions in an assembly assay (Fig.
8D). Clearly, cell-cycle regulation plays a key role during
tumorigenesis. The cell-cycle-mediated regulation shown here might indicate a
more-subtle and physiological role for gap junctional communication through
S368-mediated effects on assembly. Cell-cycle-mediated regulation of Cx43 has
been shown during mitosis, when there is a dramatic change in phosphorylation
and Cx43 is localized predominately to cytoplasmic membranes. A model in which
assembly is most efficient during G0/G1, and then
decreases as cells progress towards mitosis, this being partially due to
phosphorylation at S368, fits our data. During tumorigenesis or faulty
regulation of the cell cycle, this decrease in assembly could have dramatic
effects on gap junctional communication.
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Acknowledgments |
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References |
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