From the Eleven isoforms of G protein Heterotrimeric G
proteins1 play a major role
in signal transduction from cell surface receptors to intracellular
effectors. The The To examine the role of Plasmids and Antibodies--
cDNAs of several
Transfection and Staining--
NIH 3T3 cells were grown in
Dulbecco's modified essential medium (DMEM) supplemented with 10%
calf serum. Transfection was performed using LipofectAMINE Plus reagent
(Life Technologies, Inc.) as described. After 48 h, cells were
fixed in 4% paraformaldehyde in phosphate-buffered saline and
immunostained using antibodies against Fractionation of Cells Using Triton X-100--
After
transfection, the cells (5 × 106 cells) washed with
phosphate-buffered saline were incubated for 5 min at 0 °C with
0.5% Triton X-100 in 20 mM Hepes, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, and 2 µg/ml trypsin inhibitor and
centrifuged at 12,000 × g for 3 min at 4 °C (11).
The pellet was washed once with the same buffer. The supernatant and
pellet were used as the Triton X-100-soluble and Triton X-100-insoluble fractions, respectively (11).
Phosphorylation of Cell Migration Assay--
Migration through a membrane in
response to serum was assayed with the use of a Chemotaxicell chamber
(Kurabo) with an 8-µm polycarbonate filter (24). Transfected cells
were trypsinized and counted, and 5 × 104 cells/well
were loaded into the top wells in DMEM. The bottom wells were similarly
filled with DMEM with 10% calf serum so that the cells were exposed to
a gradient of serum factors. The chambers were incubated for 2 h
at 37 °C, and the membranes were removed and stained. Under these
conditions, a negligible number of cells fell off the bottom of the
filter. The number of cells that had migrated through the membrane was
counted for each well. To determine cell migration by chemokinesis, top
and bottom wells were filled with media with 10% calf serum so that
the cells were exposed to no gradient of serum factors. Cell migration
in the absence of a gradient was about 40% of that in a gradient,
indicating that the observed migration consisted of both chemotaxis and chemokinesis.
Immunocytochemical double staining of normal NIH 3T3 cells with
phalloidin showed the complete overlap of In contrast with the cells co-transfected with The round shape of the cells suggested a decrease of cell adhesion,
which might influence cell migration (24-26). To examine this
possibility, Boyden chamber cell migration assays were performed for
cells transfected with various Department of Biochemistry,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit have
been found thus far, but the precise roles of individual
subunits
are not known. The
12 subunit has two unique
properties: phosphorylation by protein kinase C and association with
F-actin. To elucidate the role of
12, we overexpressed
12 and other
subunits in NIH 3T3 cells together with
the
1 subunit. The overexpressed
12 as
well as endogenous
12, but not
2,
5, and
7 subunits, associated with
cytoskeletal components. Expression of
12 induced
remarkable changes including cell rounding, disruption of stress
fibers, and enhancement of cell migration, but expression of other
subunits did not induce significant changes. Deletion of the N-terminal region of
12 decreased the abilities of
12 to associate with cytoskeletal fractions, to induce
cell rounding, and to increase cell motility. Replacement by alanine of
Ser2 of
12 (Ser1 of a mature
12 protein), a phosphorylation site for protein kinase
C, eliminated these effects of
12, whereas a mutant in which Ser2 was replaced with glutamic acid showed effects
equivalent to wild-type
12. These results indicate that
phosphorylation of
12 at Ser2 enhances the
motility of cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
complexes, as well as
subunits, directly
regulate various effectors, including adenylyl cyclase, phospholipase
C-
, phosphatidylinositol 3-kinase, K+ channels, and
Ca2+ channels (1, 2). At present, 11 isoforms of the
subunit have been found (2-6). Although the biological properties of the
complex containing
1 are noticeably different
from those of
complexes containing the other
subunits (7,
8), the precise roles of individual
subunits are not known.
12 subunit, which is widely distributed and
especially rich in fibroblasts and smooth muscle cells (6), has unique properties. First,
12 is a selective substrate for
protein kinase C (PKC) (6, 9). In Swiss 3T3 cells,
12 is
phosphorylated upon exposure of cells to various reagents such as
phorbol 12-myristate 13-acetate, serum, lysophosphatidic acid,
endothelines, and growth factors (9). Phosphorylated
12
enhanced the association of
12 with Go
(6) and weakened the ability of
12 to stimulate type
II adenylyl cyclase (10), but the magnitudes of the changes induced by
phosphorylation were relatively small. The second unique property is
that
12 associates with F-actin in cells and in a cell-free system (11). In addition to
12, various
subunits, such as Gi2
, Gs
(12), and
Gq/11
(13), and the
subunit (14) as well as enzymes
involved in signal transduction, such as phospholipase C,
phosphoinositide 3-kinase, and PKC (15-17) are found associated with
the cytoskeleton in a variety of cells, but the physiological
significance of these associations is unclear.
12, we overexpressed
12, other
subunits and their mutants together with
1 in NIH 3T3 cells. The results indicate that only
12 subunit induces cell rounding and enhances cell
migration and that phosphorylation of
12 is involved in
these processes.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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12 and
2 mutants were prepared using
synthetic polymerase chain reaction primers (3, 6). cDNAs of bovine
1 (18) and
2 (3) were generously provided
by Dr. M. I. Simon (California Institute of Technology) and Dr. T. Nukada (Tokyo Institute of Psychiatry), respectively. All cDNAs of
G protein subunits (3-6) and a C-terminal fragment (amino acids 495-689) of the
-adrenergic receptor kinase (
ARKct) (19) were subcloned into pCMV5 vector as described previously (20). Antibodies against the
and
subunits of G protein have been described previously (6, 9, 21, 22).
or
, followed by
fluorescein isothiocyanate-labeled secondary antibody (MBL) as
described (11). The cells were also stained for F-actin with
tetramethylrhodamine isothiocyanate-phalloidin.
12 in Transfected
Cells--
24 h after transfection, the culture medium was replaced
with DMEM for 24 h prior to the 32P incorporation
experiment. The cells were washed twice with phosphate-free medium
(Eagle's minimum essential medium without sodium phosphate), preincubated for 1 h at 37 °C in the same medium containing
[32P]orthophosphate (0.2 mCi), and then incubated for 30 min with 10% calf serum in DMEM. After labeling, the cells were washed with the ice-cold phosphate-buffered saline and then suspended in 0.2 ml of a solution containing 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.2 mM
phenylmethylsulfonyl fluoride, 1 µg/ml trypsin inhibitor, 10 nM calyculin A, and 1% CHAPS. The suspension was mixed
with a vortex mixer and centrifuged at 100,000 × g for 15 min at 4 °C. The supernatant was incubated at 4 °C for 2 h with 5 µg of affinity-purified antibody against
7,
which had been preincubated with protein A-Sepharose beads. The
Sepharose beads were washed with the solution containing 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml trypsin inhibitor, 10 nM calyculin A, and 0.1%
CHAPS. The beads were suspended in 30 µl of sample buffer for
electrophoresis, and each resultant supernatant was subjected to
Tricine/SDS-polyacrylamide gel electrophoresis (23) with subsequent
autoradiography (6). For immunoblotting, cells were trypsinized 48 h after transfection, seeded into culture dishes, and incubated for
2 h at 37 °C in DMEM with 10% calf serum. Then cell lysates
were subjected to Tricine/SDS-polyacrylamide gel electrophoresis for
immunoblotting with the antibody against phosphorylated
12 (p-
12) (9).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12 staining
with staining of actin stress fibers, which were found crossing the entire cell (Fig. 1A) as
previously reported for Swiss 3T3 cells (11). To investigate the role
of
12, we co-transfected
12 or various
subunits with
1 in NIH 3T3 cells. Most cells
overexpressing
12 were rounded, with disruption of
F-actin architecture (Fig. 1B), whereas some cells
overexpressing
12 had decreased stress fibers with a
flattened shape (Fig. 1C). Because the fluorescence of
subunits in transfected cells was much stronger than that in normal
cells, photographs with short exposure were shown in Fig. 1
(B-G). Therefore, the staining of endogenous
12 in normal cells was faint or not observed in Fig. 1
(B and C). In contrast with
12,
overexpression of
2,
5, and
7 did not induce such dramatic changes in cell shape,
but some of cells overexpressing these
subunits showed decreased
stress fibers in the center and adopted a flattened, rounded shape
(Fig. 1, D-F). Immunoblotting analyses showed that similar
amounts of
1 and transfected
subunits were expressed
in these experiments, whereas basal levels of
5 and
12, major endogenous
subunits in NIH 3T3 cells, were
detected in all cells (Fig.
2A). To examine whether the
expressed
subunits associated with cytoskeletal components,
transfected cells were fractionated using Triton X-100. A large portion
of the expressed
12 was present in the Triton
X-100-insoluble fraction in transfected cells, and a similar
distribution of endogenous
12 was observed in control
cells, whereas most
2,
5, and
7 were present in Triton X-100-soluble fractions (Fig.
2B). These results indicate that expressed
12, but not other isoforms, associates with cytoskeletal components, which is consistent with the localization of endogenous
subunits in Swiss 3T3 cells (11).
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Fig. 1.
Immunocytochemistry of normal and transfected
NIH 3T3 cells. A, co-localization of 12
and F-actin in normal cells. NIH 3T3 cells were double stained for
12 (upper panels) and F-actin (lower
panels). B-F, effect of transfection of various
isoforms on morphology. NIH 3T3 cells were co-transfected with
1 and
12 (B and C),
2 (D),
5 (E), or
7 (F) and double stained for the respective
(upper panels) and F-actin (lower panels).
Most cells (60-70%) overexpressing
12 were rounding
(B), whereas a small number of cells overexpressing
12 had decreased stress fibers with a flattened shape
(C). G, prevention of
12-induced
cell rounding by co-transfection of
ARKct. NIH 3T3 cells were
co-transfected with
1,
12, and
ARKct
and double stained for
12 (upper panel) and
F-actin (lower panel). Scale bar, 50 µm.
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Fig. 2.
Expression of and
subunits in transfected cells and
association of expressed
subunits with
cytoskeletal components. A, expression of
and
subunits in transfected cells. NIH 3T3 cells were co-transfected with
1 and the various
subunits as indicated. Equal
amounts of cell lysates were subjected to Tricine/SDS-polyacrylamide
gel electrophoresis for
subunits or SDS-polyacrylamide gel
electrophoresis for
subunit and immunoblotted with antibodies
against
subunit and various
subunits. The standards
(Std, from top to bottom) were
purified bovine
2 (10 ng),
5 (5 ng),
7 (5 ng),
12 (5 ng), and
2 (5 ng). Because the antibody used to detect
7 cross-reacted with
2,
3
and
12 (22), staining for
7 artifactually
stained
2 and
12 as well as
7. The other antibodies used to stain the
subunits
were specific for their respective isoforms (6, 22). B,
association of transfected
subunits with cytoskeletal components.
Transfected cells were fractionated into Triton X-100-soluble
(Sol) and -insoluble (Insol) fractions. Fractions
were then subjected to immunoblotting with respective antibodies.
1 and
12, the cells transfected with
12 alone
were unchanged, suggesting that
1 and
12
were co-expressed in cells and the
1
12
complex induced cell rounding (data not shown). To test further whether the
1
12 complex was indeed involved in
induction of cell rounding, we expressed
ARKct (19) and
Gi2
, both of which are expected to bind and sequester
free
. Co-expression of
ARKct (Fig. 1G) or
Gi2
(data not shown) prevented
1
12-induced cell rounding, supporting the
involvement of the
complex in these changes.
subunits. Cell migration markedly
increased in cells transfected with
12 but did not
significantly change in cells transfected with
2,
5, or
7 (Fig.
3). Co-transfection of
ARKct again
suppressed the increase of cell motility induced by
12.
These results suggested that
12, but not other
subunits, was involved in enhancement of cell motility. Expression of
ARKct alone in normal cells decreased cell motility, suggesting that
12-induced cell migration occurred in normal cells
(data not shown).
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Fig. 3.
Effect of transfection of various
subunits on cell migration in response to calf
serum. NIH 3T3 cells were co-transfected with
1,
various
subunits, and
ARKct, and cell migration was examined.
Data represent the means ± S.E. of nine experiments.
Comparison of amino acid sequences of various isoforms of revealed
diverged residues concentrated at the N-terminal region (6). To test
whether the ability of
12 to associate with F-actin is
determined by its N-terminal sequence, the N-terminal truncated
12 (
12
N5, Fig.
4A) was transfected into NIH
3T3 cells. The deletion of the N terminus decreased the ability of
12 to associate with cytoskeletal fractions (Fig.
4B), indicating that the N-terminal region of
12 is important for the association with F-actin. The weak association of
12
N5 with cytoskeletal fractions
was not due to an inability to form the
complex, because
12
N5 was coimmunoprecipitated with an antibody
against Gi2
when co-transfected with
and
Gi2
but not when co-transfected with only
Gi2
(data not shown). The
12
N5 mutant
neither caused cell rounding nor enhanced cell migration (Fig. 4,
C and F), suggesting that the association of
12 with F-actin is important to induce these
changes.
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On the other hand, the N-terminal region contains a site phosphorylated
by PKC (6, 9), and therefore it is possible that phosphorylation of
12 is involved in induction of these changes. When
phosphorylation of
12 was analyzed by immunoblotting with the antibody against p-
12, the expressed
12 was strongly phosphorylated, whereas weak
phosphorylation of endogenous
12 was observed (Fig.
4D). The phosphorylation of the expressed
12 determined with 32P incorporation was also greater than
that of the endogenous
12 observed in control cells, but
the increase appeared to be smaller than that observed in immunoblots
(Fig. 4E). The conditions of these experiments were not
identical, but the main reason for the small increase of
phosphorylation was that some
12 subunits in transfected
cells had already been phosphorylated before the 32P
incorporation experiment (data not shown). We then transfected NIH 3T3
cells with the mutant
12S2A, in which Ser2,
a phosphorylation site,2 is
replaced with alanine (Fig. 4A). The
12S2A,
which was not phosphorylated, could associate with the cytoskeleton but
had no effect on morphology and motility of cells (Fig. 4). Because glutamic acid can substitute for phosphoserine in some proteins activated by phosphorylation, we next transfected cells with mutant
12S2E, in which Ser2 is replaced with
glutamate (Fig. 4A). This mutant caused similar effects in
transfected cells to wild-type
12, except of the
increase of phosphorylation (Fig. 4). These results supported the idea that phosphorylation of
12 induced cell rounding and
enhanced cell motility. In Fig. 4 (D and E),
smaller increases of phosphorylation were observed in the cells
transfected with
12
N5,
12S2A, and
12S2E, in comparison with control cells. The reason for
these increases will be discussed later (see "Discussion").
To further evaluate the effect of phosphorylation on the activity of
subunits, we mutated the N-terminal region of a different
,
2, to introduce SSK, the phosphorylation motif for PKC
(
2SSK, Fig. 4A) (9). The
2SSK
was phosphorylated as well as
12 in cells when judged by
32P incorporation, whereas the antibody against
p-
12 weakly recognized phosphorylated
2SSK, probably due to a low reactivity with the mutant.
However, the
2SSK, which hardly associated with
cytoskeletal fractions, neither induced cell rounding nor increased
cell migration (Fig. 4), suggesting that phosphorylation is not
sufficient for the changes induced by phosphorylated
12.
The weak association of this mutant with cytoskeletal fractions (Fig.
4B) suggested that the association with the cytoskeleton
might be essential for enhancing cell migration.
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DISCUSSION |
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The present study showed that transfection of 12
markedly induced cell rounding and increased cell migration, but
transfection of
2,
5, and
7 induced only minor changes, clearly indicating a
functional difference among
subunits. This specific function of
12 is derived from its unique property of selective
phosphorylation by PKC. However, the phosphorylation is not sufficient
to account for the activity of
12, because the
2SSK mutant, which could be phosphorylated, did not
induce these changes in cells. This is in contrast to the result that
the activity of the
complex containing phosphorylated
10G4K (
10SSK) mutant was similar to that
of the phosphorylated
12 in stimulating type II
adenylyl cyclase (10). Some part(s) of the structure of
12 other than the N terminus might also be important for
enhancing cell migration or/and some other characteristic of
12 important for associating with F-actin might be
essential for inducing these changes. The present results demonstrated
that
subunits unable to associate with cytoskeletal factions did
not increase cell migration, but further experiments are necessary to
show whether association with F-actin is essential.
Although the effects of 2,
5, and
7 were not very remarkable, decreases of stress fibers
(Fig. 1, E-G) and small increases of cell motility (Fig. 3)
were observed in cells transfected with these
subunits. One
previous report showed that transfection of
1
2 did not induce morphological changes
in Swiss 3T3 cells (27), but another report indicated that
microinjection of
1
2 reduced stress
fibers in CV-1 cells (28), which is basically consistent with our
present observations. These isoforms may have weak activities, but it
is also possible that the apparent effects of
2,
5, and
7 are due to
12
released from endogenous G protein due to displacement by the
overexpressed
. This speculation might be supported by the
evidence that phosphorylation of
12 slightly increased
in the cells transfected with
subunits other than wild-type
12 in comparison with control cells (Fig. 4,
D and E). Because similar amounts of endogenous
12 exist in all these cells, the increase of
phosphorylated
12 suggests the replacement of the
12 in endogenous G protein and an increase of the
free
12.
We have shown that phosphorylation of 12 enhances
fibroblast migration in response to serum. A variety of growth factors, including platelet-derived growth factor (26, 29), basic fibroblast growth factor (30), lysophosphatidic acid (26), and serum (24), were
shown to stimulate migration of fibroblasts. Down-regulation or
inhibition of PKC abolished the effect of platelet-derived growth
factor and basic fibroblast growth factor on cell migration, suggesting
the role of PKC in these processes (29, 30). Our previous observation
that platelet-derived growth factor, basic fibroblast growth factor,
lysophosphatidic acid, and serum stimulated phosphorylation of
12 in Swiss 3T3 cells (9) strongly suggests the
involvement of phosphorylation of
12 in cell migration
stimulated by these growth factors. For phosphorylation of
12, activation of G proteins is important as well as PKC
activation, because free
12 was a better substrate
for PKC than the trimer form (6). Lysophosphatidic acid could stimulate
Gi and Gq, so that both G protein and PKC could
be activated by this agonist. By contrast, receptor tyrosine kinases
are well known to stimulate PKC via activation of phospholipase C
,
and the evidence that basic fibroblast growth factor-stimulated
migration of endothelial cells was reduced by pertussis toxin suggests
the involvement of G protein activation in this process (31). Neptune
and Bourne (32) and Arai et al. (33) reported that expressed
Gi-coupled receptors, such as D2 dopamine and opioid
receptors, induced cell migration, which was prevented by
ARKct and
Gt
, suggesting involvement of
subunits. However,
they also suggest that Gi activation is necessary but
probably not sufficient for chemotaxis. Activation of PKC to
phosphorylate
12 might be necessary for maximal
stimulation of chemotaxis.
Cell migration requires dynamic and coordinated disassembly and
reassembly of stress fibers and focal adhesions, but the precise mechanisms regulating these processes are not clear (25). The present
observations that overexpression of 12 decreases stress fibers and induces cell rounding suggest that
12 may be
involved in disassembly of stress fibers. Because PKC and
phosphoinositide 3-kinase, of which activation increases cell
migration, have also been found to associate with the cytoskeleton (16,
17), the
12-associated cytoskeleton may be easily
accessed by these enzymes, facilitating their interaction. Recent
studies showed the role of Rho family GTPases such as Rho, Rac, and
Cdc42 in regulation of assembly and organization of the actin
cytoskeleton (34). In the budding yeast Saccharomyces
cerevisiae,
complex has been shown to associate with Cdc24,
a guanine nucleotide exchange factor for Cdc42, suggesting a cascade
from
to actin organization via Cdc42 (35). At present, this
cascade has not been shown in mammalian cells, but future experiments
will elucidate downstream signaling molecules linking
12 to cell migration, possibly including this cascade.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. I. Simon and T. Nukada for supplying the plasmids.
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FOOTNOTES |
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* This work was partly supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Inst. for Developmental Research, Aichi Human Service Center, Kamiya-cho, Kasugai, Aichi 480-0392, Japan. Fax: 81-568-88-0829; E-mail: toasano{at}inst-hsc.pref.aichi.jp.
2
A phosphorylation site of 12 for
PKC is the first serine residue from the N terminus of
12, which was previously designated Ser1
from the sequence of a mature
12 protein (6, 9, 10) but
is designated Ser2 in this paper.
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ABBREVIATIONS |
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The abbreviations used are:
G protein, guanine
nucleotide-binding protein;
PKC, protein kinase C;
DMEM, Dulbecco's
modified essential medium;
ARKct, C-terminal fragment of the
-adrenergic receptor kinase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine.
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