From the
Several laboratories have demonstrated a decrease in gap
junctional communication in cells transformed by the src oncogene of the Rous sarcoma virus. The decrease in gap junctional
communication was associated with tyrosine phosphorylation of the gap
junction protein, connexin 43 (Cx43). This study was initiated to
determine if the phosphorylation of Cx43 is the result of a direct
kinase-substrate interaction between the highly active tyrosine kinase,
pp60
Gap junctions, which are found in plasma membranes of almost all
animal cells, mediate cell-to-cell communication and maintain normal
organ function
(1) , embryonic development
(2) , and
perhaps growth
control
(3, 4, 5, 6, 7) . Gap
junctions are formed when a connexon (hemichannel) from one cell docks
with a symmetrically opposed connexon from an adjacent cell forming a
conduit which permits the passive diffusion of ions and small molecules
(<1000 daltons) between neighboring cells. Each connexon consists of
an oligomer of six connexin proteins that form the central aqueous
pore
(8) .
Connexins are a family of gap junction proteins
that are composed of four transmembrane domains, two extracellular
loops, an intracellular loop, and amino- and carboxyl-terminal domains
located in the cytoplasm
(9, 10, 11) . The
transmembrane domains and extracellular loops are highly conserved
between family members and are believed to be responsible for channel
structure, whereas the cytoplasmic domains are less conserved and may
account for the differential regulation of gap junctions
(12) .
Gap junctional communication (GJC)
The permeability of the gap junction channel is
affected by various factors such as changes in free intracellular
calcium levels
(25) , intracellular pH
(26) ,
voltage
(27) , growth factors
(28, 29) , and
transforming oncogene activity
(19, 30) . The mechanisms
that mediate changes in gap junctional permeability are at present
incompletely understood. However, the increased phosphorylation of
connexin induced by various stimuli is associated with modulation of
channel gating (opening and closing)
(31, 32) . For
example, cyclic AMP treatment of liver hepatocytes increased GJC and
connexin 32 (Cx32) phosphorylation, which may be the result of
activated cyclic AMP-dependent serine kinases
(33, 34) .
Expression of the ras oncogene or TPA treatment of mouse
primary keratinocytes decreased GJC and increased serine
phosphorylation on Cx43
(15) . Epidermal growth factor treatment
of T51B rat liver epithelial cells produced a rapid, transient decrease
in GJC, which was associated with increased serine phosphorylation on
Cx43
(28, 35) .
RSV possesses the oncogene
v-src, which encodes a plasma membrane-associated tyrosine
kinase, pp60
Further evidence for this
proposed mechanism came from experiments utilizing Xenopus oocytes expressing Cx43 and/or
pp60
pp60
To test the hypothesis that
Cx43 is a substrate of activated pp60
Recombinant
baculovirus was generated by cotransfecting a monolayer of Sf-9 cells
with recombinant transfer plasmid and BaculoGold baculovirus DNA
(Pharmingen). Recombinant baculoviruses were then subjected to one
round of plaque purification.
Samples for the biochemical subcellular
localization experiments were obtained by lysing recombinant
baculovirus-infected cells with cytosol extraction buffer (50
mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA (pH
7.5), 1 mM EGTA, 25 µg/ml leupeptin, and 25 µg/ml
aprotinin) in a Dounce homogenizer. The samples were centrifuged at
131,000
Protein concentrations were measured with the
Bio-Rad protein assay kit. Whole and fractionated cell samples were
boiled in an equal volume of 2
The immunoaffinity
matrix was prepared with the D-7 monoclonal antibodies covalently bound
to protein G-Sepharose 4B Fast Flow beads (Sigma). Briefly, mouse
ascites fluid was subjected to a 50% ammonium sulfate
precipitation
(52) . The ammonium sulfate precipitate was
resuspended in PBS and dialyzed overnight against PBS. The antibodies
were then incubated with the protein G-Sepharose beads overnight at 4
°C with gentle end-over-end mixing. The beads were washed with 200
mM triethanolamine (TEA) in PBS (pH 8.0) and incubated for 2 h
at room temperature with end-over-end mixing in 40 mM
dimethylpalmilidate, 200 mM TEA, PBS (pH 8.0), followed by a
200 mM TEA (pH 8.0) wash and overnight incubation at room
temperature in 40 mM dimethylpalmilidate, 200 mM TEA
(pH 8.0) with end-over-end mixing. The beads were finally washed with
200 mM TEA (pH 8.0), incubated with 40 mM
dimethylpalmilidate, 200 mM TEA (pH 8.0) for 2 h and incubated
in 40 mM ethanolamine at room temperature for 2 h with
end-over-end mixing. The beads were thoroughly washed with PBS and
stored at 4°C in PBS containing merthiolate to prevent bacterial
contamination
(52, 53) .
Activated
pp60
For depletion
experiments, the kinase reaction was performed in two incubations. The
first incubation included partially purified Cx43,
pp60
For in vitro kinase assays, 15 µl of GST or GST-Cx43CT bound to
glutathione-Sepharose 4B beads (washed once in kinase buffer) were
incubated with purified kinase-active
pp60
To determine if Cx43,
expressed in recombinant baculovirus-infected Sf-9 cells, was also
phosphorylated,
Our laboratory and others have previously demonstrated that
Cx43 is phosphorylated on tyrosine in Rous sarcoma virus-transformed
fibroblasts
(38, 40) . This phosphorylation event is
tightly associated with the kinase activity and membrane localization
of pp60
We chose the baculovirus eucaryotic expression system for the
production of Cx43 because it has been used successfully to express
high levels of native, post-translationally modified
protein
(55, 64) . Cx43 was expressed in the recombinant
baculovirus-infected Sf-9 cells at levels 33 times greater than those
observed with Rat-1 fibroblasts (Fig. 1). Biochemical subcellular
fractionation and immunofluorescence microscopy demonstrated that Cx43
was found in the plasma membranes of the Sf-9 cells (Fig. 2). We
also observed that the expressed Cx43 was postranslationally modified
by endogenous serine kinases in insect cells (Fig. 3).
Baculovirus-expressed Cx43, partially purified with a monoclonal
antibody immunoaffinity matrix, was used in in vitro kinase
assays with immunoaffinity-purified, activated
pp60
This conclusion was
reinforced by our demonstration of
pp60
To
examine whether the ability of activated
pp60
We also examined the
phosphorylation of Cx43 in Sf-9 cells coinfected with the Cx43 and
pp60
Taken together, these data demonstrated that Cx43 is
phosphorylated directly by activated pp60
The apparent ability of
pp60
An increased serine
phosphorylation on Cx43 has also been observed in
v-src-transformed cells
(40, 61) . v-src has been demonstrated to activate protein kinase C and thus may
induce serine phosphorylation of Cx43 by this mechanism
(65) .
pp60
Multiple pp60
We thank E. Beyer for Cx43 cDNA, J. Brugge for mAb
327, D. Morgan and R. Erikson for pp60
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, and Cx43. Previous
biochemical studies have been limited by the low levels of Cx43 protein
in fibroblast cell lines. To obtain larger quantities of Cx43, we
constructed a recombinant baculovirus expressing Cx43 in Spodoptera
frugiperda (Sf-9) cells and subsequently purified the expressed
Cx43 by immunoaffinity chromatography. We observed that this partially
purified Cx43 was phosphorylated on tyrosine in vitro in the
presence of kinase-active pp60
. Phosphotryptic
peptide mapping indicated that the in vitro phosphorylated
Cx43 contained phosphopeptides which comigrated with a subset of
tryptic peptides prepared from Cx43 phosphorylated in vivo.
Furthermore, coinfection of Sf-9 cells with recombinant baculoviruses
encoding pp60
and Cx43 resulted in
the accumulation of phosphotyrosine in Cx43. Taken together, the
evidence presented in this paper demonstrates that kinase active
pp60
is capable of phosphorylating
Cx43 in a direct manner. Since the presence of phosphotyrosine on Cx43
is correlated with the down-regulation of gap-junctional communication,
these results suggest that pp60
regulates gap junctional gating activity via tyrosine
phosphorylation of Cx43.
(
)
has been
implicated in the regulation of growth control based on experiments
linking the permeability of the gap junction channel with cellular
transformation (reviewed in Ref. 4). Several groups have demonstrated
that the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate
(TPA), can inhibit GJC (13, 14). Transformation of cells by viral
oncogenes such as v-ras(15, 16) ,
v-src(17, 18) , v-mos (19), and
polyomavirus middle T antigen
(20) has been correlated with an
inhibition of GJC. Furthermore, Mehta et al.(21) demonstrated that treatment of chemically transformed CH3
10T1/2 cells with retinoids simultaneously increased GJC and inhibited
transformation. The most direct evidence supporting the role of GJC in
transformation involved the introduction of connexin 43 (Cx43) into
communication-deficient, carcinogen-transformed cells. Re-expression of
Cx43 resulted in restoration of GJC, an inhibition of growth, focus
formation
(5) , and tumorigenicity
(22) . Similar results
were observed in communication-deficient C6 glioma cells transfected
with Cx43
(23) and human tumor cells (SKHep) transfected with
Cx32
(24) .
(36, 37). Expression
of pp60
results in a loss of growth
control and cellular transformation. Tyrosine phosphorylation on Cx43
in v-src-transformed cells correlates with a decrease in
GJC
(38, 39, 40) . Cells infected with a
temperature-sensitive mutant of RSV demonstrated a rapid reduction in
GJC and a transformed morphology when shifted to the permissive
temperature, where pp60
is active.
At the nonpermissive temperature, where
pp60
is inactive, the cells
exhibited high levels of GJC and a normal
morphology
(18, 30) . Phosphorylated Cx43, purified from
cells shifted to the permissive temperature, rapidly accumulated
phosphotyrosine (Tyr(P)), concomitant with the disruption of
GJC
(39) . In contrast, cells grown at the nonpermissive
temperature, with normal levels of GJC contained Cx43 phosphorylated
only on phosphoserine (Ser(P)) residues. These results indicated that
pp60
may decrease GJC by inducing
the phosphorylation of Cx43 on tyrosine.
. When Cx43 was coexpressed
with pp60
, junctional conductance
was disrupted and Cx43 was phosphorylated on tyrosine. Oocytes
coexpressing a mutated form of Cx43 (tyrosine 265 mutated to
phenylalanine) and pp60
did not
demonstrate a decrease in GJC or tyrosine phosphorylation on
Cx43
(41) . Although these results suggested that
pp60
may directly regulate channel
gating via tyrosine phosphorylation of Cx43 on residue 265, it is
possible that these events in the oocytes do not completely reflect the
events in mammalian cells.
may activate a secondary tyrosine kinase, which then
phosphorylates Cx43. One such tyrosine kinase could be the focal
adhesion kinase, pp125
(42) , which is
phosphorylated on tyrosine, activated in v-src-transformed
cells
(43) , and associated with
pp60
(44) .
, we
examined the ability of purified pp60
to phosphorylate either whole Cx43 purified from
baculovirus-infected insect cells or the carboxyl tail region of Cx43
fused to glutathione S-transferase (GST). In addition, we
examined the phosphorylation of Cx43 coexpressed with
pp60
in insect cells. The results
from this study indicated that Cx43 can serve as a substrate of
activated pp60
in in vitro kinase
reactions and in intact mammalian and insect cells. Furthermore, these
data strongly suggest that the tyrosine residue(s) phosphorylated by
pp60
are located in the carboxyl tail region of
Cx43. Since tyrosine phosphorylation on Cx43 has been correlated with a
down-regulation in GJC, these results suggest that activated
pp60
has a direct role in modulating the gating
of gap junctions.
Cell Culture
Sf-9 insect cells (Invitrogen) were
grown as monolayers in Grace's insect medium (Life Technologies,
Inc.) supplemented with 3.3 g/liter yeastolate (Life Technologies,
Inc.), 3.3 g/liter lactalbumin hydrolysate (Life Technologies, Inc.)
and 10% fetal calf serum (HyClone) at 27 °C. Sf-9 cells were
infected with recombinant virus at a multiplicity of infection of
5-10 and harvested 48-72 h post-infection. For coinfection
experiments, cells were infected at a ratio of 1:2 for the recombinant
pp60 and Cx43 baculoviruses,
respectively. Rat-1 and v-src-transformed Rat-1 fibroblasts
were grown as described previously
(45) .
Construction of Recombinant Transfer Vector and
Baculovirus
pVL1393-Cx43 was constructed from the transfer
vector pVL1393 (Pharmingen)
(46) and the Cx43 cDNA (G2 clone)
(47) contained in Bluescript (Stratagene). The Bluescript-G2
construct was digested with BanI (New England Biolabs) and the
1.8-kilobase pair BanI fragment containing the Cx43 cDNA was
blunt-ended with Klenow (Promega), then digested with EcoRI
(U. S. Biochemical Corp.). This fragment was then inserted into a
SmaI-EcoRI site of pVL1393 transfer vector resulting
in the recombinant transfer plasmid, pVL1393-Cx43.
Western Blot Analysis
Expression of Cx43 in Sf-9
cells was examined by harvesting cells at specified time points. Cells
were then lysed in 200 µl of lysis buffer (100 mM NaCl, 10
mM Tris (pH 8.0), 2 mM EDTA, 1% Triton X-100), and
clarified in a Beckman TL-100 ultracentrifuge at 400,000 g for 20 min at 4 °C.
g for 20 min at 4 °C in a Beckman TL-100
ultracentrifuge. The supernatant was collected and stored as the
cytosolic fraction. The pellet was resuspended by passage through a
22-gauge, 3/8-inch needle five times in cytosol extraction buffer
supplemented with 1% Triton X-100, followed by centrifugation at
400,000
g for 20 min at 4 °C in a Beckman TL-100
ultracentrifuge. The supernatant was collected and stored as the
particulate fraction.
sample buffer (to achieve a
final concentration of 2% SDS, 5%
-mercaptoethanol, 5% glycerol,
0.004% bromphenol blue, and 0.03 M Tris (pH 6.8)), separated
on 12% SDS-polyacrylamide gels
(48) , and electrotransfered to
Immobilon-P membranes (Millipore Corp.). The membranes were blocked
overnight with blocking buffer (25 mM Tris-buffered saline (pH
7.4), 1% bovine serum albumin), washed 30 min with wash buffer (25
mM Tris-buffered saline (pH 7.4), 0.05% Tween 20, 1
mM EDTA), and incubated with rabbit antiserum directed against
peptide 368-382 of Cx43 (anti-CT 368; 1:500 dilution) for 2 h at
room temperature. The membranes were then washed for 45 min in wash
buffer, incubated with 1 µCi of
I-labeled goat
anti-rabbit IgG (ICN Pharmaceuticals, Inc.) for 1 h at room
temperature, and washed for 30 min. Membranes were then
autoradiographed using Kodak X-Omat XAR-5 film with the aid of an
intensifying screen at -70 °C. Immunoreactive bands were
excised and quantitated with a Micromedics four-channel
counter.
Immunofluorescence Microscopy
Sf-9 cells were
infected as described above and harvested 48 h post-infection. Cells
were fixed in 3% paraformaldehyde/PBS for 20 min at 4 °C, washed
two times in PBS, permeabilized in 0.2% Triton X-100/PBS for 2 min at 4
°C, and washed two times in PBS. Cells were incubated with Cx43
rabbit antiserum (anti-CT 368; 1:500 dilution) overnight at 4 °C,
washed two times, and blocked with normal goat serum (1:50 dilution)
for 30 min at 4 °C. After washing twice with PBS, the cells were
incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit
secondary antibody (1:80 dilution) (Sigma) for 1 h at 4 °C and
washed three times in PBS. Cells were wet-mounted in 50% PBS, 50%
glycerol containing 1 mg/ml p-phenylenediamine (Sigma) and
visualized and photographed with a Zeiss Axioplane Universal microscope
equipped with epifluorescence.
Radiolabeling, Immunoprecipitation, and Quantitation of
Cx43
Sf-9 cells were infected with recombinant baculovirus
encoding Cx43 as described above. At 24 h post-infection, the cells
were radiolabeled with either EXPRES
S protein
labeling mix (NEG-072; DuPont NEN) at 100 µCi/ml in Grace's
methionine-free medium (Life Technologies, Inc.) or
[
P
] (NEX-053; DuPont NEN)
at 0.5 mCi/ml in Grace's phosphate-deficient medium (Cell Culture
Facility, University of California, San Francisco) supplemented with
0.4% fetal calf serum, 5% complete TNM-FH medium, 20 µM
MES (pH 6.1) (Sigma). Cells were labeled overnight (16 h) at 27 °C
and harvested. Fibroblasts were grown to confluence and labeled with
EXPRE
S
S protein labeling mix at 100
µCi/ml or [
P
] at 0.5
mCi/ml in phosphate-deficient medium for 3 h at 37 °C. The cells
were lysed in RIPA buffer (150 mM NaCl, 1% sodium
deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 10
mM Tris (pH 7.2), 50 mM NaF, 160 µM
Na
VO
, 1 mM phenylmethylsulfonyl
fluoride), clarified, and immunoprecipitated with normal rabbit serum,
rabbit serum directed against a Cx43 (CT368), or monoclonal antibody
directed against pp60
(mAb 327, generously
provided by Joan Brugge; 54) as described previously
(38) .
Immunoprecipitated proteins were resolved on a 7.5-15%
SDS-polyacrylamide gradient gel. Gels were dried and autoradiographed
using Kodak X-Omat XAR-5 film at -70 °C. Gels containing
[
S]-labeled proteins were fluorographed prior to
drying. Gel pieces containing
S- or
P-labeled
Cx43 proteins were excised, rehydrated, and counted in 7.5% Scintigest
(New England Nuclear)-Scintiverse fluid (Fisher Scientific) using a
Beckman LS5000 liquid scintillation counter.
Phosphoamino Acid Analysis
P-Labeled
Cx43 was immunoprecipitated and gel-purified as described. The proteins
were electrotransfered to an Immobilon-P membrane (Millipore) and
acid-hydrolyzed. The phosphopeptides were resolved on thin-layer
cellulose plates in two dimensions at pH 1.9 and 3.5, as described
previously in Ref. 49. Autoradiography was performed with Kodak X-Omat
XAR-5 film with the aid of an intensifying screen at -70 °C.
Preparation of Cx43 Monoclonal Antibodies and
Immunoaffinity Matrix
Monoclonal antibodies were made according
to established techniques
(50) against a synthetic peptide
corresponding to amino acids 241-254 of the COOH-terminal
cytoplasmic domain of Cx43. The peptide was conjugated to rabbit serum
albumin via an amino-terminal cysteine
(51) and BALB/c mice were
immunized with the conjugate in Freund's adjuvant. 5
10
immune spleen cells were fused with 5
10
X63-Ag8.653 myeloma cells. Hybridoma clones were selected in HAT
medium, and supernatants were screened against the Cx43 peptide by
enzyme-linked immunosorbent assay. Two clones, F-3 (IgG
)
and D-7 (IgG
), out of the 208 hybridomas screened were
found to be reactive with the Cx43 peptide.
Immunoaffinity Purification of Cx43 and
pp60
Cx43 was purified from recombinant
baculovirus-infected Sf-9 cells. At 72 h post-infection, 4
10
cells were harvested from a suspension culture and
washed in PBS at 4 °C. The following steps were completed at 4
°C. Cells were disrupted with 75 strokes (pestle A) in a Dounce
homogenizer in 5 ml of homogenization buffer (50 mM Tris-HCl
(pH 7.2), 150 mM NaCl, 2 mM EDTA, 25 µg/ml
leupeptin, 25 µg/ml aprotinin, 1 mM phenylmethylsulfonyl
fluoride). This homogenate was clarified in a Beckman TL100
ultracentrifuge at 100,000
g for 30 min at 4 °C.
The supernatant was discarded and the pellet resuspended in 5 ml cell
extraction buffer (10 mM Tris-HCl (pH 7.2), 150 mM
NaCl, 0.1% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA, 25
µg/ml leupeptin, 25 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride) by gentle passage through a 22-gauge,
1.5-inch needle. The resuspended sample was Dounce homogenized with 20
strokes (pestle A) and incubated on ice for 15 min. The sample was
clarified in a Beckman TL100 ultracentrifuge at 400,000
g for 20 min at 4 °C. The supernatant was incubated with 1 ml of
Cx43 monoclonal antibody-protein G-Sepharose for 3 h with end-over-end
mixing at 4 °C. The slurry was then packed into a column and washed
with cell extraction buffer, high salt wash buffer (10 mM
Tris-HCl (pH 7.2), 1 M NaCl, 1% Triton X-100), high salt
glycine wash buffer (100 mM glycine (pH 4.0), 1 M
NaCl, 1% Triton X-100), and glycine wash buffer (100 mM
glycine (pH 5.5), 1% Triton X-100). Cx43 was eluted from the
immunoaffinity matrix with a low pH glycine buffer (100 mM
glycine (pH 2.5), 1% Triton X-100), and the eluate was neutralized
immediately with 1 M Tris-HCl (pH 8.0).
was immunoaffinity-purified
from Sf-9 cells infected with the recombinant baculovirus containing
the c-src gene (Ref. 55; kindly provided by David Morgan). The
immunoaffinity column was generated by covalently coupling the
monoclonal antibody directed against pp60
, mAb
327
(54) to protein A-agarose beads (Bio-Rad). The purification
steps were performed as described previously
(55) .
In Vitro Protein Kinase Assay
In vitro protein kinase assays were performed using
immunoaffinity-purified, kinase-active
pp60 and/or partially purified Cx43
in a reaction volume of 20 µl containing 0.10 µg of Cx43, 0.10
µg of pp60
, kinase buffer (20
mM HEPES (pH 7.4), 12 mM MnCl
, 20
mM MgCl
, 1 mM dithiothreitol, 100
µM Na
VO
) and 10 µCi of
[
-
P]ATP (DuPont NEN, NEG-002Z, 6000
Ci/mmol) for 15 min at room temperature. The reactions were stopped by
the addition of 2
sample buffer and boiling.
, kinase buffer, and 5
nM unlabeled ATP. Following incubation for 15 min at room
temperature, pp60
was
immunodepleted from the reaction mixture by the addition of
pp60
monoclonal antibodies (mAb 327) chemically
coupled to protein A-agarose. The
pp60
-depleted sample was then
divided into two aliquots. To one portion, 10 µCi of
[
-
P]ATP alone was added, and to the other
portion, 10 µCi of [
-
P]ATP plus
purified pp60
were added. These
reactions were then incubated an additional 15 min at room temperature
to permit radiolabeling to occur. The reactions were stopped with the
addition of 2
sample buffer and boiling. The proteins were
resolved on a 12% SDS-polyacrylamide gel. The gels were dried and
autoradiographed using Kodak X-Omat XAR-5 film.
Generation and in Vitro Phosphorylation of a GST-Cx43CT
Fusion Protein
A GST fusion protein containing the COOH tail
region of Cx43 was generated by polymerase chain reaction (PCR)
amplification (Perkin Elmer 480) of nucleotide sequences between
907-1356 of the Cx43 cDNA corresponding to amino acids 236-382
(GST-Cx43CT). The primer sequences for PCR amplification were
5`-GTATGGATCCGTTAAGGATCGCGTCAAGGG-3` and
5`-CTATGAATTCGCCGGTTTAAATCTCCAGGT-3`. The amplified DNA product was
sequenced to confirm its authenticity. The PCR products were cloned
into BamHI and EcoRI sites of the pGEX-KG expression
vector (Ref. 56; generous gift from Jack Dixon). GST fusion proteins
were expressed in Escherichia coli upon induction with 2
mM isopropyl--D-thiogalactopyranoside for 2 h at
37 °C. The cells were lysed by brief sonication in PBS and then
incubated in 1% Triton X-100 for 15 min on ice. Insoluble material was
removed by a 10 min spin on a microcentrifuge at 4 °C. The lysate
was then incubated with glutathione-Sepharose 4B beads (Pharmacia) for
30 min followed by extensive washes with PBS.
, kinase buffer (20 mM
HEPES (pH 7.4), 12 mM MnCl
, 1 mM
dithothreitol, 100 µM Na
VO
) and 10
µCi of [
-
P]ATP (DuPont NEN, NEG-002Z,
6000 Ci/mmol) in a total volume of 40 µl for 15 min at room
temperature. Reactions were stopped with the addition of an equal
volume of 2
sample buffer, boiled, and resolved on a 12%
SDS-polyacrylamide gel.
Two-dimensional Tryptic Phosphopeptide
Mapping
Tryptic phosphopeptide maps of phosphorylated Cx43 were
prepared as described in Ref. 57. Briefly, v-src-transformed
Rat-1 fibroblasts were metabolically labeled with 3 mCi of
[P
] for 3 h at 37 °C
and then solubilized in RIPA buffer. Cx43 was then immunoprecipitated
from clarified lysates with CT368 peptide antiserum. For in vitro labeling experiments, partially purified Cx43 from infected Sf-9
cells was phosphorylated by activated
pp60
in in vitro kinase
reactions as described above. The radiolabeled proteins were resolved
on 12% SDS-polyacrylamide gels and autoradiographed. Phosphorylated
Cx43 was extracted from the wet, unfixed gel pieces and precipitated on
ice with trichloroacetic acid. The precipitated Cx43 was then digested
overnight with trypsin (Worthington, Freehold, NJ). The phosphotryptic
peptides were resolved on cellulose thin-layer chromatography (TLC)
plates (Curtin Matheson Scientific, Houston, TX) by electrophoresis in
the first dimension using pH 1.9 buffer (2.5% (v/v) formic acid (88%),
7.8% (v/v) glacial acetic acid) for 1.25 h at 1000 V, followed by
ascending chromatography in the second dimension using isobutyric acid
buffer (62.5% (v/v) isobutyric acid, 1.9% (v/v) n-butanol,
4.8% (v/v) pyridine, 2.9% (v/v) glacial acetic acid). The positions of
the phosphopeptides were determined by autoradiography using Kodak
X-Omat XAR-5 film with the aid of an intensifying screen at -70
°C.
Expression of Cx43 in Sf-9 Cells
The time course
of Cx43 production in recombinant baculovirus-infected Sf-9 cells was
examined by harvesting cells at the specified times after infection.
Cell lysates prepared from an equal number of cells were examined by
Western blotting with antibody against the COOH-terminal peptide of
Cx43. We observed that Cx43 was produced in the infected cells as early
as 24 h post-infection at approximately 2.5-fold greater levels than
detected in the Rat-1 fibroblast control (Fig. 1, A,
lane3, and B). Expression of Cx43 was even
more dramatic at 48 and 72 h post-infection with increases of 18- and
33-fold, respectively over Rat-1 fibroblast levels (Fig. 1,
A, lanes5 and 7, and B).
At these later times, multiple slower migrating forms of Cx43 were
observed which are reminiscent of the phosphorylated Cx43 isoforms
observed previously in mammalian cells
(28, 38) .
Uninfected Sf-9 cells did not express immunoreactive Cx43
(Fig. 1A, lanes2, 4, and
6).
Figure 1:
Production and accumulation of Cx43 in
recombinant baculovirus-infected Sf-9 insect cells. A, Western
blotting of cell extracts from infected and uninfected Sf-9 cells
harvested at the specified time points. Cell lysates from equal numbers
of Rat-1 fibroblasts (lane1), uninfected Sf-9 cells
(lanes2, 4, and 6), and infected
Sf-9 cells (lanes 3, 5, and 7) were resolved
on a 12% SDS-polyacrylamide gel and transferred to an Immobilon-P
membrane. The samples were immunoblotted with Cx43 antibody and
[I]-goat anti-rabbit IgG, as described under
``Materials and Methods.'' B, quantitation of Cx43
production in Sf-9 Cells. Immunoreactive protein bands were excised and
quantitated with a Micromedics four-channel
counter. The values
were plotted as an increase of Cx43 in Sf-9 cells relative to the Cx43
level in Rat-1 fibroblasts.
Subcellular Localization of Cx43
Endogenously
expressed Cx43, like other members of the gap junction protein family,
localize in the plasma membranes of mammalian cells or Xenopus oocytes microinjected with connexin mRNA
(11, 41) .
To determine if Cx43 was also localized to the plasma membrane in
recombinant baculovirus-infected Sf-9 cells, biochemical subcellular
fractionation of infected and uninfected Sf-9 cells was performed.
Cytosolic and particulate proteins were separated by SDS-polyacrylamide
gel electrophoresis and Cx43 was detected by Western blot analysis. The
particulate fraction of infected Sf-9 cells contained Cx43 with
apparent molecular weights ranging from approximately 43,000 to 47,000
(Fig. 2A, lane4). Cx43 was nearly
undetectable in the soluble, cytosolic fraction
(Fig. 2A, lane3), and it was not
detected in either the particulate or cytosolic fractions of uninfected
Sf-9 cells (Fig. 2A, lanes1 and
2).
Figure 2:
Localization of Cx43 in Sf-9 cells by
biochemical subcellular fractionation and immunofluorescence
microscopy. A, cytosolic (lanes1 and
3) and particulate (lanes2 and 4)
proteins of uninfected (lanes1 and 2) and
infected (lanes3 and 4) Sf-9 cells 72 h
post infection were resolved on a 12% SDS-polyacrylamide gel,
transferred to an Immobilon-P membrane, and reacted with Cx43 antibody,
as described under ``Materials and Methods.'' B,
uninfected (panels1 and 2) and infected
(panels3 and 4) Sf-9 cells 48 h
post-infection were fixed, permeabilized, and then examined by indirect
immunofluorescent staining with Cx43 antibody and fluorescein
isothiocyanate-conjugated goat anti-rabbit antibody. Phase (panels
1 and 3) and fluorescent (panels2 and
4) images were photographed with a Zeiss Axioplane Universal
microscope with epifluorescence.
To confirm these biochemical results and to identify
the specific cellular membranes containing Cx43, immunofluorescence
microscopy studies were conducted on Cx43 baculovirus-infected Sf-9
cells. Triton X-100-permeabilized Sf-9 cells were incubated with Cx43
COOH-terminal peptide antibody, and the immune complexes were detected
with fluorescein-conjugated secondary antibody. Infected cells
demonstrated a strong Cx43 signal in the cell periphery with only a
faint signal in the cytoplasm (Fig. 2B, panel4). These results are consistent with the notion that
Cx43 is localized predominantly to the plasma membranes of infected
Sf-9 cells. Specificity of the Cx43 antibody reaction was demonstrated
by the lack of a specific response in uninfected Sf-9 cells
(Fig. 2B, panel2).
Phosphorylation of Cx43 in Sf-9 Cells
The
postranslational modification of Cx43 by serine phosphorylation in
various cells is associated with the modulation of channel gating
activity and its membrane
localization
(35, 38, 58, 59) . Cx43
immunoprecipitated from fibroblasts migrates on SDS-polyacrylamide gels
predominantly as the nonphosphorylated 43-kDa form and two slower
migrating bands of 45 and 47 kDa, each of which represents serine
phosphorylated isoforms of Cx43 (28, 38, 39).
S- and
P-labeled Cx43 were
immunoprecipitated from uninfected and infected Sf-9 cells and Rat-1
fibroblasts and resolved by SDS-polyacrylamide gel electrophoresis.
Five major forms of Cx43 with apparent molecular mass values of 43, 44,
45, 47, and >47 kDa were isolated from
S-labeled
infected, but not uninfected, Sf-9 cells (Fig. 3A,
lanes4 and 6). Immunoprecipitation of
P-labeled Cx43 from infected Sf-9 cells demonstrated that
the slower migrating forms of Cx43 were phosphorylated
(Fig. 3B, lane 4). Four phosphoproteins with
corresponding molecular mass values of 44, 45, 47, and >47 kDa were
detected, indicating that the Cx43 produced in the baculovirus
expression system is phosphorylated in a manner similar to that
observed in Rat-1 fibroblasts. However, there were differences in the
extent of phosphorylation of the Cx43 isoforms isolated from the two
cell types (Fig. 3B, compare lanes2 and 4). The predominant phosphorylated Cx43 in Rat-1
fibroblasts was the 45-kDa protein, whereas the 47-kDa form of Cx43
predominated in infected Sf-9 cells. Also, the >47-kDa band was
readily detected in infected Sf-9 cells, but to a lesser extent in the
Rat-1 fibroblasts. Phosphoamino acid analysis showed that Cx43
expressed in infected Sf-9 cells and Rat-1 fibroblasts contained only
Ser(P) (Fig. 3C).
Figure 3:
Phosphorylation of Cx43 expressed in
recombinant baculovirus-infected Sf-9 insect cells. Rat-1 fibroblasts,
uninfected (U), and infected (I) Sf-9 cells were
labeled with either EXPRES
S (panelA) or [
P] (panelB). Cx43 was immunoprecipitated from cell lysates with
either nonimmune rabbit serum (odd numbered lanes) or
antiserum against Cx43 (even numbered lanes) and resolved on
7.5-15% SDS-polyacrylamide gel as described under
``Materials and Methods.'' C, phosphoamino acid
analysis of [
P]Cx43 from the Rat-1 fibroblasts
and infected Sf-9 cells. The positions of ninhydrin-stained unlabeled
Ser(P) (P-S), Thr(P) (P-T), and Tyr(P)
(P-Y) are outlined. The directions of
migration for the first dimension (pH 1.9) and second dimension (pH
3.5) on thin-layer cellulose plates are indicated by the
arrows. All autoradiograms were prepared on Kodak X-Omat XAR-5
film at -70 °C.
Immunoaffinity Purification of Cx43
For in
vitro phosphorylation studies, rapid partial purification of Cx43
was achieved by immunoaffinity chromatography using a monoclonal
antibody against a Cx43 peptide (residues 241-254, clone D-7)
covalently coupled to protein G-Sepharose. The solubilized, particulate
fraction prepared from 72 h post-infected Sf-9 cells was incubated with
the immunoaffinity matrix (Fig. 4, lane1).
Following extensive washing to remove nonspecific proteins, Cx43 was
eluted from the matrix with a low pH buffer. Coomassie Blue staining
showed that the predominant protein isolated was the nonphosphorylated
43-kDa form of Cx43 (Fig. 4, lanes 4 and 5).
The phosphorylated Cx43 isoforms were also present to a lesser extent
in the purified preparations. The identity of this immunoaffinity
product as Cx43 was confirmed by Western blotting utilizing a
polyclonal antibody directed against COOH-terminal peptide of Cx43
(data not shown). Trace levels of non-Cx43 proteins were detected at 70
and 31 kDa by Coomassie staining. The identities of these proteins are
unclear at the present time.
Figure 4:
Immunoaffinity purification of Cx43 from
recombinant baculovirus-infected Sf-9 cells. Cx43 was purified from
recombinant baculovirus-infected Sf-9 cells using a Cx43 monoclonal
antibody chemically coupled to protein G-Sepharose, as described under
``Materials and Methods.'' After extensive washing, Cx43 was
eluted from the column with a low pH buffer. Samples were resolved on a
12% SDS-polyacrylamide gel and stained with Coomassie Blue. Lanes1 and 2 represent the loading sample
(LS) and flow-through (FT), respectively. Lanes
4-11 represent elution fractions 1-8
(E). Cx43 was eluted predominantly in
fractions 2 and 3 (lanes4 and 5). The sizes
of the molecular size markers are as indicated at the leftmargin.
pp60
Previous studies have demonstrated that Cx43 is
phosphorylated on tyrosine in mammalian cells transformed by
pp60 -mediated Phosphorylation of
Cx43 in Vitro
(38, 39, 40) and in Xenopus oocytes microinjected with both
Cx43 and pp60
mRNAs
(41) .
These studies suggested that the tyrosine kinase responsible for
phosphorylating Cx43 was pp60
. The
availability of these purified proteins permitted us to examine the
ability of pp60
to directly phosphorylate Cx43
in in vitro kinase assays. Several phosphorylated proteins
were observed in these kinase reactions. One major phosphorylated
protein of approximately 44-47 kDa comigrated with the
Coomassie-stained, purified Cx43 (Fig. 5, compare panelA, lane3 to panelB,
lane4). The second major phosphorylated protein
migrated around 60 kDa and represents autophosphorylated
pp60
, since it comigrated with the
major phosphorylated product present in reactions consisting only of
pp60
(Fig. 5A,
lanes1 and 3). Phosphoamino acid analyses
of these in vitro phosphorylated products revealed only Tyr(P)
(Fig. 5C). In similar experiments, a
baculovirus-expressed pp60
,
purified independently under different conditions (Ref. 60; generously
provided by Martin Broome, Salk Institute), also phosphorylated Cx43 on
tyrosine (data not shown). To determine if our Cx43 preparation
contained endogenous kinases capable of phosphorylating Cx43, a kinase
reaction was performed in the absence of
pp60
. No phosphorylated proteins
were observed in this control reaction (Fig. 5A,
lane2). These data are consistent with the idea that
activated pp60
phosphorylates Cx43
directly in vitro.
Figure 5:
Phosphorylation of Cx43 in
pp60 kinase reactions. pp60
and Cx43
were immunoaffinity-purified as specified under ``Materials and
Methods.'' A, results from the in vitro kinase
assay with pp60
alone (lane1), Cx43
alone (lane2), and pp60
together with
Cx43 (lane3). B, Coomassie-stained
SDS-polyacrylamide gel of the in vitro kinase reactions shown
in panelB. Cx43 is indicated by the
brackets. The samples were resolved on a 7.5-15%
SDS-polyacrylamide gel. The gel was dried and autoradiographed at
-70 °C. The sizes of the molecular size standards are
indicated between panelsA and B. Positions
of phosphorylated pp60 and Cx43 are shown at the left.
C, phosphoamino acid analysis of
[
P]pp60
and Cx43 from the in
vitro kinase reaction. The positions of ninhydrin-stained,
unlabeled Ser(P) (P-S), Thr(P) (P-T), and Tyr(P)
(P-Y) are outlined. The directions of migration for
the first dimension (pH 1.9) and second dimension (pH 3.5) on
thin-layer cellulose plates are indicated by the
arrows.
To rule out the possibility that Cx43
was phosphorylated by an activated endogenous tyrosine kinase that was
present in the partially purified Cx43 preparation and activated by
pp60 phosphorylation, we conducted a two-stage
kinase reaction in which pp60
was
initially present, but then immunodepleted to reveal the activity of
any activated endogenous tyrosine kinase(s). In this experiment, a
complete kinase reaction, containing activated
pp60
and Cx43, was first performed
with unlabeled ATP (5 nM) to permit
pp60
to phosphorylate and possibly
activate any putative tyrosine kinase(s) present in the reaction mix.
pp60
was then immunodepleted from
this reaction mix by incubation with a pp60
monoclonal antibody conjugated to protein A-agarose beads, and
the kinase reaction was continued in the presence of 10 µCi of
[
-
P]ATP to detect the activity of any
putative, activated kinases. Under these experimental conditions, it
was expected that any putative pp60
-activated
tyrosine kinase might reveal itself in the second kinase reaction by
phosphorylating Cx43 in the absence of
pp60
. However, our results
demonstrated that phosphorylation of Cx43 did not occur in
pp60
immunodepleted reactions
(Fig. 6, lane2). The absence of
autophosphorylated pp60
in this
reaction indicated that the immunodepletion of
pp60
was effective (Fig. 6,
lane2). Furthermore, when
pp60
was added back to the depleted
reaction, Cx43 phosphorylation and pp60
autophosphorylation were observed, which indicated that Cx43 was
not altered or removed during the immunodepletion step and that the
ability of the depleted reaction mix to support protein phosphorylation
was not compromised (Fig. 6, lane3). Thus,
these results demonstrated that the partially purified Cx43 preparation
did not contain any detectable endogenous kinase(s) capable of
phosphorylating Cx43. Control kinase reactions, performed in the
presence of 5 nM unlabeled ATP with 10 µCi of
[
-
P]ATP, supported phosphorylation of Cx43
and pp60
(Fig. 6, lane1), which suggested that
pp60
should have been capable of
phosphorylating a putative tyrosine kinase present in the kinase
reaction mix. The lower levels of radiolabeled Cx43 and
pp60
observed in this reaction,
compared to reactions containing 10 µCi of
[
-
P]ATP alone (see Fig. 5A,
lane3), was probably because of dilution of the
radiolabel by the unlabeled ATP.
Figure 6:
Phosphorylation of Cx43 in kinase
reactions depleted of pp60. Control kinase reaction
performed with pp60
and Cx43 in the presence of 10 µCi
of [
-
P]ATP and 5 nM ATP (lane1). Kinase reaction performed first with
pp60
and Cx43 in the presence of 5 nM ATP;
pp60
was then immunodepleted, and the reaction was
continued with 10 µCi of [
-
P]ATP
(lane2). Kinase reaction performed as described for
lane2 but pp60
was added back to the
depleted reaction (lane3). Samples were resolved on
a 10% SDS-polyacrylamide gel. The gel was dried and autoradiographed at
-70 °C. The positions of pp60src and Cx43 are indicated at
the leftmargin.
A Western blot of the purified
pp60 and Cx43 samples, performed
with polyclonal antibodies (BC3) against
pp125
(42) , was negative, which excluded the
possibility that the tyrosine kinase pp125
was associated
with either purified pp60
or Cx43
preparations (data not shown).
Phosphotryptic Peptide Mapping of Cx43 Phosphorylated in
Vivo and in in Vitro Kinase Reactions
Two-dimensional tryptic
phosphopeptide analysis was performed to determine if the sites of Cx43
phosphorylation mediated by pp60in vitro corresponded with sites of tyrosine
phosphorylation on Cx43 from in vivo labeled
v-src-transformed fibroblasts. Four phosphopeptides (labeled
1-4) of full-length Cx43, purified from Sf-9 cells and
phosphorylated in vitro by
pp60
, migrated similarly to
phosphopeptides from Cx43 radiolabeled with
[
P
] in
v-src-transformed cells (Fig. 7, panelsA and B). Analysis of a mixture of these two samples
confirmed that the four phosphopeptides labeled in vitro comigrated with phosphopeptides from the in vivo labeled
sample (Fig. 7, panelC). Peptides 1 and 4 of
Cx43 from v-src-transformed cells correspond to
phosphotyrosine-containing peptides a and c previously reported in Ref.
61. Phosphoamino acid analyses of peptides 2 and 3 revealed that they
also contained Tyr(P).(
)
Figure 7:
Two-dimensional tryptic phosphopeptide
maps of P-labeled Cx43. Cx43 from radiolabeled
v-src-transformed fibroblasts was immunoprecipitated with Cx43
CT368 peptide antiserum and resolved on a SDS-polyacrylamide gel as
described under ``Materials and Methods.'' Partially
purified, full-length Cx43 from infected Sf-9 cells and the GST
carboxyl tail Cx43 fusion protein were phosphorylated in vitro in the presence of pp60
as described under
``Materials and Methods.'' Figure shows phosphotryptic
peptides of: A, full-length Cx43 phosphorylated in
vitro; B, Cx43 from v-src-transformed
fibroblasts; C, mixture of in vivo labeled Cx43 and
full-length Cx43 phosphorylated in vitro; D,
GST-Cx43CT phosphorylated in vitro; E, mixture of
GST-Cx43CT phosphorylated in vitro and full-length Cx43
phosphorylated in vitro. Origins are indicated by the
arrowheads. The directions of migration are indicated by the
arrows. The inset in panelD shows
the phosphoamino acid analysis results of the phosphorylated GST-Cx43CT
fusion protein. The positions of the unlabeled Ser(P) (P-S),
Thr(P) (P-T), and Tyr(P) (P-Y) standards are
outlined.
A bacterially
expressed GST-fusion protein containing the carboxyl tail region (amino
acids 236-382) of Cx43 (GST-Cx43CT) was generated to determine if
the putative pp60 -mediated phosphorylation of
Cx43 occurred in this domain. The bacterially expressed GST-Cx43CT
protein was purified by absorption on glutathione-Sepharose. The
GST-Cx43CT fusion protein bound to glutathione-Sepharose was
phosphorylated only on tyrosine by activated
pp60
(Fig. 7, panelD, inset). Control GST protein bound to
Sepharose beads was not phosphorylated under identical conditions (data
not shown). The tryptic peptide map of phosphorylated GST-Cx43CT had a
similar pattern consisting of the same four phosphopeptides observed in
the in vitro phosphorylated whole Cx43 isolated from Sf-9
cells (Fig. 7, panelD). A mixture of the
tryptic digests of the two samples demonstrated the comigration of the
four phosphopeptides (Fig. 7, panelE).
However, the intensities of the phosphorylated peptides differed
between the two samples. Peptide 4 represented the major phosphotryptic
spot in the GST-Cx43CT protein, whereas peptides 1 and 2 were the major
phosphopeptides observed in in vitro phosphorylated
full-length Cx43 isolated from the Sf-9 cells (Fig. 7, compare
panelsA and D). Taken together, these
results suggested that
pp60
-mediated phosphorylation of
Cx43 in vitro mimics similar phosphorylation events occurring
in v-src-transformed fibroblasts and that the tyrosine
phosphorylation of Cx43 may be occurring primarily in the carboxyl tail
region of the molecule.
Phosphorylation of Cx43 in Sf-9 Cells Coinfected with
pp60
Several
studies have utilized Sf-9 cells coinfected with recombinant
baculoviruses to demonstrate protein kinase-substrate
interactions
(62, 63) . We also examined the ability of
pp60 and Cx43 Recombinant Baculoviruses
to phosphorylate Cx43 in
coinfected Sf-9 cells. Phosphorylated forms of both
pp60
and Cx43 were
immunoprecipitated from these cells (Fig. 8A, lanes8 and 9). Cx43 from the coinfected cells was
phosphorylated primarily on Ser(P), but also contained levels of Tyr(P)
(Fig. 8B) that were comparable to the phosphorylated
Cx43 expressed in v-src-transformed Rat-1 fibroblasts
(Fig. 8, panelA, lane3 and
panelB). In contrast, Cx43 expressed in Sf-9 cells
infected with the Cx43 baculovirus alone was phosphorylated only on
serine residues as described above (Fig. 8, panelA, lane6 and panelB). Thus, these coinfection experiments further support
the conclusion that a direct kinase-substrate interaction between
pp60
and Cx43 exists.
Figure 8:
Phosphorylation of Cx43 in insect Sf-9
cells co-expressing pp60. A, Rat-1
v-src-transformed fibroblasts, monoinfected (Cx43 recombinant
baculovirus), and coinfected (Cx43 and pp60
recombinant
baculoviruses) Sf-9 cells were labeled with [
P].
Cell lysates were immunoprecipitated with either nonimmune rabbit serum
(lanes1, 4, and 7), mAb 327
monoclonal antibody specific for pp60 (lanes2,
5, and 8), or antiserum directed against Cx43
(lanes3, 6, and 9).
Immunoprecipitates were resolved on a 7.5-15% SDS-polyacrylamide
gel and autoradiographed. The positions of pp60
and Cx43
are shown at the left. Molecular size standards are shown at
the right. B, phosphoamino acid analysis of
[
P]Cx43. The positions of ninhydrin-stained,
unlabeled Ser(P) (P-S), Thr(P) (P-T), and
Tyr(P) (P-Y) are outlined. The directions of
migration of the first dimension (pH 1.9) and second dimension (pH 3.5)
are indicated by the arrows.
. In cells expressing
temperature-sensitive pp60
,
phosphorylation of Cx43 on tyrosine correlated closely with activation
of the pp60
kinase activity and the
reduction of GJC
(39) . Moreover, tyrosine phosphorylation of
Cx43 and disruption of its function were not observed in cells
containing kinase-active but nonmyristylated
pp60
, which does not localize to plasma
membranes
(39, 40) . In this study, we present data which
strongly suggest that pp60
is one of the
tyrosine kinases responsible for directly phosphorylating Cx43 in
src-transformed fibroblasts. In support of this conclusion, we
have demonstrated that purified, activated
pp60
phosphorylates Cx43 in in
vitro kinase reactions and that the tryptic phosphopeptides of
in vitro phosphorylated Cx43 represent a subset of those
obtained from Cx43 metabolically labeled in v-src-transformed
cells.
to demonstrate that Cx43 is
phosphorylated on tyrosine in the presence of activated
pp60
(Fig. 5). Several control reactions
assured us that the tyrosine kinase responsible for phosphorylating
Cx43 in vitro was indeed
pp60
. First, kinase reactions
performed with the partially purified preparation of Cx43 alone did not
result in the phosphorylation of Cx43 (Fig. 5B). Second,
because it was possible that another tyrosine kinase, which required
activation by pp60
, was present in either the
pp60
or Cx43 preparations, we
performed a two-stage kinase reaction in which the putative tyrosine
kinase was first activated by pp60
.
Its activity was then measured in the second stage in the absence of
pp60
. This experimental protocol
failed to produce in vitro phosphorylated Cx43. However, Cx43
from the pp60
-depleted reaction
mixture could be phosphorylated when pp60
was added back (Fig. 6). Third, p125
, which
has been reported to be associated with
pp60
(44) , was not detected in the
pp60
or Cx43 preparations by
immunoblotting. Therefore, these results suggest that Cx43 is
phosphorylated directly by activated pp60
in these in vitro assays.
's ability to directly
phosphorylate a GST-Cx43CT fusion protein expressed in bacteria. This
substrate, purified from bacterial cell lysates on
glutathione-Sepharose, is unlikely to contain endogenous tyrosine
kinases capable of phosphorylating Cx43 in vitro.
to phosphorylate Cx43 in
vitro reflected its ability to do so in intact
v-src-transformed cells, we compared the phosphotryptic
peptides of in vitro and in vivo phosphorylated Cx43
by two-dimensional peptide mapping. These experiments demonstrated that
four of the Cx43 peptides phosphorylated in vitro represented
a subset of those obtained from Cx43 phosphorylated in
v-src-transformed fibroblasts (peptides 1-4,
Fig. 7
). The corresponding four phosphopeptides of in vivo labeled Cx43 contained phosphotyrosine (Ref. 61, and unpublished
observations). Peptides 1 and 4 (Fig. 7) were identified in a
previous study characterizing the phosphorylation of Cx43 in
v-src-transformed cells (labeled as peptides a and c in Ref.
61). Peptides 2 and 3 (Fig. 7) were also detected in this
previous study, but at a reduced level of phosphorylation
(61) .
The reasons for the differences in phosphorylation levels of peptides 2
and 3 are not clear at this time.
baculoviruses. Phosphoamino
acid analysis demonstrated that the Cx43 expressed in these coinfected
cells contained both Tyr(P) and Ser(P). In contrast, Cx43 expressed in
Sf-9 cells infected with only the Cx43 recombinant baculovirus
contained only Ser(P). Our attempts to measure an alteration of the
gating activity of Cx43 in Sf-9 cells, expressed in the presence or
absence of pp60
, were unsuccessful
due to the decreased adhesiveness of the cells following baculovirus
infection.
in in vitro kinase reactions and support the hypothesis
that Cx43 is phosphorylated by pp60
in intact v-src-transformed fibroblasts. However,
because of the existence of enzymes like pp125
, which is
associated with pp60
, we cannot
exclude the possibility that Cx43 may also be phosphorylated in
vivo by other tyrosine kinases besides
pp60
.
to phosphorylate four phosphotryptic
peptides of full-length Cx43 suggested the possibility of multiple
Tyr(P) sites in Cx43. These results differ from those published
previously, which indicated that phosphorylation of tyrosine 265 in
Cx43 expressed in Xenopus oocytes was sufficient to disrupt
junctional conductance
(41) . Because these multiple
phosphopeptides may possibly result from events such as incomplete
tryptic cleavage or differential serine phosphorylation, we are
identifying the Tyr(P) sites present in these four Cx43 tryptic
peptides. The same phosphopeptides also resulted from
pp60
's phosphorylation of the GST-Cx43CT
fusion protein (Fig. 7), which contains the COOH-terminal
cytoplasmic region of Cx43. Therefore, the potential phosphorylation
sites of interest must be located between amino acids 236-382 of
Cx43. Thus, one of the four phosphopeptides from Cx43, phosphorylated
in v-src-transformed fibroblasts, may also contain
phosphorylated tyrosine 265. Site-directed mutagenesis studies should
help to clarify the relative importance of these potentially different
Cx43 phosphorylation sites in pp60
's
ability to interrupt GJC in mammalian cells.
also associates with
Shc
(66, 67) and activates the ras-raf signaling pathway which may result in mitogen-activated protein
kinase-mediated phosphorylation of Cx43
(68) . Additional
v-src-dependent serine kinases may also be involved but are
yet to be identified. Serine phosphorylation of Cx43 induced by the
ras oncogene or epidermal growth factor is correlated with
down-regulation of GJC
(15, 28, 35) . Epidermal
growth factor's effects on Cx43 phosphorylation are independent
of PKC, but may involve activated mitogen-activated protein
kinase
(35) . The function of the observed increase in serine
phosphorylation in v-src-transformed cells is at present
unknown. It is possible that a combination of serine and tyrosine
phosphorylation of Cx43 contribute to the regulation of the gap
junction channel.
substrates
have been identified in src-transformed cells, including
cytoskeletal proteins (69-71), the epidermal growth factor
receptor
(72) , focal adhesion kinase
(p125
)
(42, 43) , and an RNA-binding
protein
(73, 74) . The identification of
pp60
substrates is critical to the understanding
of the mechanisms involved in pp60
's
effects on growth and cellular transformation. It is possible that the
phosphorylation of only one or two substrates may be sufficient for
cellular transformation. Alternatively, multiple pp60
substrates may work in concert to initiate and maintain the
different properties associated with altered cell growth and behavior.
Based on previous studies and data presented here, we propose that Cx43
also serves as a direct substrate of pp60
.
Several studies have suggested that the maintenance of intercellular
GJC is one parameter critically involved in normal growth
control
(6, 75) .
pp60
-mediated tyrosine
phosphorylation of Cx43 and the associated disruption of GJC could be
one mechanism by which pp60
mediates some aspects of cellular transformation and loss of
growth control.
recombinant baculoviruses, M. Broome for purified, kinase-active
pp60
, J. T. Parsons for antibodies
against pp125
, and J. Dixon for pGEX clones. We also
thank K. Martyn for suggesting the depletion experiments, W. Kurata for
technical support, K. Martyn and B. Warn-Cramer for critical review of
manuscript, and L. Kuriyama for assistance in the preparation of the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.