Morphological Changes and Detachment of Adherent Cells
Induced by p122, a GTPase-activating Protein for Rho*
Masayuki
Sekimata
,
Yukihito
Kabuyama
,
Yasufumi
Emori§, and
Yoshimi
Homma
¶
From the
Department of Biomolecular Sciences,
Institute of Biomedical Sciences, Fukushima Medical College, 1 Hikariga-oka, Fukushima 960-1295 and the § Department of
Biophysics and Biochemistry, Faculty of Science, University of
Tokyo, Bunkyo, Tokyo 113-0033, Japan
 |
ABSTRACT |
We recently cloned a novel signaling molecule,
p122, that shows a GTPase-activating activity specific for Rho and the
ability to enhance the phosphatidylinositol
4,5-bisphosphate-hydrolyzing activity of phospholipase C
1 in
vitro. Here we analyzed the in vivo function of p122.
Microinjection of the GTPase-activating domain of p122 suppressed the
formation of stress fibers and focal adhesions induced by
lysophosphatidic acid, suggesting a GTPase-activating activity for Rho
as in in vitro. Transfection of p122 also induced the
disassembly of stress fibers and the morphological rounding of various
adherent cells. Analyses using deletion and point mutants demonstrated
that the GTPase-activating domain of p122 is responsible for the
morphological changes and detachment and that arginine residues at
positions 668 and 710 and a lysine residue at position 706 in the
GTPase-activating domain are essential. Using Fluo-3-based Ca2+ microscopy, we found that p122 evoked a rapid
elevation of intracellular Ca2+ levels, suggesting that
p122 stimulates the phosphatidylinositol 4,5-bisphosphate-hydrolyzing
activity of phospholipase C
1. These results demonstrate that p122
synergistically functions as a GTPase-activating protein specific for
Rho and an activator of phospholipase C
1 in vivo and
induces morphological changes and detachment through cytoskeletal reorganization.
 |
INTRODUCTION |
Recent studies have indicated a close association between the
regulation of cytoskeletal assembly and phosphatidylinositol (PI)1 metabolism. That is, a
number of PI 4,5-bisphosphate (PIP2)-binding proteins
including gelsolin, cofilin, profilin, and
-actinin are known to
bind to actin and regulate cytoskeletal assembly (1-4). In addition,
Rho has been shown to enhance the activity of PI 4-phosphate 5-kinase,
the PIP2-synthesizing enzyme (5), and the overexpression of
PI 4-phosphate 5-kinase induces massive actin polymerization in COS-7
cells (6). Alternatively, microinjection of antibodies against
PIP2 inhibits the formation of stress fibers and focal
adhesions (7). These results strongly suggest that PIP2
newly synthesized by PI 4-phosphate 5-kinase, which exists downstream
of Rho, binds to PIP2-binding proteins (i.e.
actin-binding protein) to release free G-actins. These reactions
increase the intracellular concentrations of free G-actins, resulting
in their reorganization to form actin fibers (8).
On the other hand, Rho is inactivated by GTPase-activating proteins
(GAPs) (9-12). It is possible that Rho GAPs, as downstream components
of Rho, regulate cytoskeletal disassembly. We recently cloned a GAP as
a novel molecule that interacts with phospholipase C
1 (PLC
1)
(13). In addition to the GAP activity specific for Rho in
vitro, p122 possesses the ability to enhance the
PIP2-hydrolyzing activity of PLC
1 in vitro.
Therefore, it is possible that p122 is an ideal link between Rho and
cytoskeletal disassembly. To address this issue, we tried to isolate
cells stably overexpressing p122. However, we have never succeeded in
obtaining such cells, suggesting that p122 affects not only
cytoskeletal assembly but also other physiological properties such as
the ability to attach to the substrate. In this study, we analyze the
in vivo functions of p122 by microinjection and transient
expression into adherent cultures. The results demonstrate that p122
functions at least as a GAP for Rho and a stimulator of PLC
1
in vivo and phenotypically induces morphological changes
including the disappearance of stress fibers and focal adhesions and
detachment from the substrate.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Microinjection--
Madin-Darby canine kidney
(MDCK) cells, baby hamster kidney (BHK21) cells, and mouse Swiss 3T3
fibroblasts were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum and L-glutamine.
For microinjection of proteins, 1 × 104 Swiss 3T3
cells were seeded onto glass coverslips as described previously (14).
After 4-7 days, the cells were washed extensively and starved in
serum-free culture medium for 24-36 h. Recombinant proteins were
microinjected at a concentration of 1 mg/ml along with 10 mg/ml
FITC-dextran (average Mr 20,000 Sigma) as a
marker into the cytoplasm of the quiescent, serum-starved cells. For microinjection of plasmids, 1 × 104 MDCK cells were
seeded onto glass coverslips and cultured for 1 day. Various plasmids
were microinjected at a concentration of 0.1 mg/ml into the nuclei.
Microinjection was performed using Eppendorf 5170 micromanipulator and
5246 microinjector (Eppendorf, Germany).
Expression and Purification of Recombinant Proteins for
Microinjection--
For construction of the N- and C-terminal
recombinant p122 proteins, p122-N and p122-C, p122 cDNA was
mutagenized by the polymerase chain reaction (PCR) to create an
NdeI site at the starting ATG and codon 617 using the
following primer: 5'-TAtctagaCATATGATCCTAACACAAATTGAAGCC-3' and 5'-AGCATATGAAAAGGATCAAGGTTCCAG-3' (the XbaI
site is indicated by lowercase letters, and the NdeI site is
underlined), respectively. The PCR product encoding amino acid residues
1 to 533 was digested with XbaI and EcoRV,
subcloned into the corresponding sites of pBluescript SK (Stratagene,
CA) and then sequenced. The plasmid was digested with NdeI
and XhoI (which cuts in the multiple cloning sites of
pBluescript) and subcloned into the
NdeI-XhoI sites of a bacterial expression vector
pET-15b (Novagen, WI) to obtain the plasmid p122-N. For
construction of the plasmid p122-C, the PCR product encoding amino acid
residues 617 to 1083 was digested with NdeI and
BamHI (which cuts in the 3'-untranslated segment of p122
cDNA), subcloned into the NdeI-BamHI sites of
pET-15b, and then confirmed by sequence analysis. The plasmids were
routinely transformed into Escherichia coli BL21(DE3)pLysS
to allow for the expression of (His)6-tagged recombinant
p122-N and p122-C proteins (Fig. 1A). The recombinant
proteins were purified with a nickel-bound HiTrap chelating column
(Amersham Pharmacia Biotech) and dialyzed against phosphate-buffered
saline (PBS) for microinjection. The purified proteins were probed with
a 1:1000 dilution of nickel-nitrilotriacetic acid horseradish
peroxidase conjugate (Qiagen, CA) and visualized by enhanced
chemiluminescence detection using an ECL kit (Amersham Pharmacia
Biotech) (Fig. 1B).
Plasmid Construction and Mutagenesis--
The various p122
constructs are summarized in Fig. 4A. To express a green
fluorescent protein (GFP) fused to p122 in mammalian cells, a 5'
XhoI site was introduced into the 5'-flanking sequence of
the reading frame by PCR amplification using the primer
5'-AGACTCGAGACCATATGATCCTAACACAAATTGAAGCC-3' (the
XhoI site is underlined) and verified by sequencing. To
construct pGFP-WT, the full-length p122 cDNA was digested with
XhoI and KpnI (which cuts in the 3'-untranslated
segment of p122 cDNA) and then inserted into the corresponding
sites of the mammalian expression vector, pEGFP-C1
(CLONTECH, Palo Alto, CA), resulting in a fusion
protein with GFP at the N terminus. Deletion derivatives of p122 were
generated using the following restriction sites: XhoI and
BamHI at positions 1-117 (pGFP-117
C), XhoI
and EcoRV at positions 1-534 (pGFP-534
C),
EcoRI and KpnI at positions 798-1083 (pGFP-798
N), and SalI and KpnI at positions
949-1083 (pGFP-949
N). For construction of pGFP-863
C, p122
cDNA was mutagenized by PCR to create a KpnI site at
codon 864 using the primer
5'-CCGGTACCTCCAGTGTCAGGGGCTTCAG-3' (the KpnI
site is underlined). The PCR product encoding residues 1 to 863 was
digested with XhoI and KpnI and then subcloned
into the corresponding sites of pEGFP-C1. For construction of
pGFP-617
N, p122 cDNA was mutagenized by PCR to create a
SacI site at codon 617 using the primer
5'-AGGAGCTCATATGAAAAGGATCAAGGTTCCAG-3' (the SacI
site is underlined). The PCR product encoding residues 617 to 1083 was
digested with SacI and KpnI and then subcloned
into the corresponding sites of pEGFP-C1.
Nucleotide changes in the p122 cDNA were engineered by
site-directed mutagenesis using a QuikChang mutagenesis kit
(Stratagene). The mutant primers used to generate pGFP-R668E,
pGFP-K706E, pGFP-R710E, and pGFP-N779V were
5'-GGTTGGGCTCTTCGAGAAGTCAGGTGTC-3',
5'-GCAGACATGTTAGAGCAATATTTCCG-3', 5'-GCAATATTTCGAGGATCTCCCCGAG-3', and
5'-CCAGATGACTCCCACCGTCCTGGCTGTGTGC-3', respectively (base
changes are underlined). All mutant cDNA sequences were confirmed
by DNA sequencing.
Immunofluorescence Microscopy--
Cells growing on glass
coverslips or glass-bottomed dishes were rinsed in PBS, fixed with
3.7% formaldehyde in PBS for 15 min at room temperature, and
permeabilized with 0.2% Triton X-100 in PBS for 5 min. After washing
three times with PBS, actin filaments were stained with
tetramethylrhodamine B isothiocyanate (TRITC)-labeled phalloidin
(Sigma) for 30 min at room temperature. To visualize focal adhesions,
cells were incubated with a 1:500 dilution of antiphosphotyrosine
antibody, 4G10 (Upstate Biotechnology), or a 1:100 dilution of
anti-vinculin antibody (VIN-11-5, Sigma) for 1 h, washed three
times with PBS, and then incubated for 30 min with a 1:100 dilution of
rhodamine B-conjugated goat anti-mouse immunoglobulins
(BIOSOURCE, Camalliro, CA). For visualization of
myc-tagged Val14-RhoA, monoclonal antibody 9E10 was used as a primary antibody.
Ca2+ Imaging--
MDCK cells were loaded with 5 µM fluo-3/AM (Dojindo, Japan) and 0.02% Pluronic F-127
(Molecular Probes, Eugene, OR) in a physiological salt solution (20 mM Hepes (pH 7.4), 115 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, and 13.8 mM glucose) for
20 min at 37 °C, incubated with the solution for 1 h at
37 °C, and then washed with PBS. The loaded cells were mounted on
the stage of an inverted microscope and microinjected with recombinant
p122-N or p122-C protein at a concentration of 1 mg/ml into the
cytoplasm. Fluorescence images were obtained by excitation at 480 nm
using xenon lamps. The emission signal at 520 nm was collected with a
cooled charge-coupled device camera, and the digitized signals were
stored and analyzed with Merlin digital imaging system (Olympus, Japan).
 |
RESULTS |
p122-C Inhibits Formation of Stress Fibers and Focal
Adhesions--
To address the physiological activities of the GAP
domain of p122 in signaling pathways specific for Rho in
vivo, purified recombinant p122-C proteins encompassing the
complete GAP domain (Fig. 1) were
microinjected into Swiss 3T3 cells. In agreement with the previous
results (14), removal of serum from the culture medium of Swiss 3T3
cells resulted in a reduction of both actin stress fibers and focal
adhesions containing phosphotyrosine and vinculin, whereas
lysophosphatidic acid (LPA) stimulation restored stress fibers and
focal adhesions in the serum-starved cells. Using this system, we
examined whether p122-C could inhibit LPA-stimulated changes in actin
fiber organization and focal adhesions in vivo. When cells
were microinjected with p122-C and then treated with LPA for 10 min,
the LPA-induced formation of stress fibers and focal adhesions was
completely abolished as compared with uninjected cells (Fig.
2, A-F). In contrast, p122-C
had no effect on platelet-derived growth factor-induced membrane
ruffling, which is mediated by Rac proteins (Fig. 2, G and
H). These results indicate that the GAP domain of p122
specifically inhibits LPA-induced Rho activation in vivo,
probably by stimulating the intrinsic GTPase activity of Rho.

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Fig. 1.
Structure and purification of p122-N and
p122-C. A, schematic representation of p122, p122-N,
and p122-C proteins. The hatched box denotes the
(His)6 tag at the N terminus; the solid box
denotes the GAP domain. The numbers refer to the positions
of amino acids in p122. B, the purified recombinant
proteins, p122-N (lanes a and c) and
p122-C (lanes b and d) were analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie
Brilliant Blue (CBB) staining (a and
b) or Western blotting (c and d). The
molecular masses of protein markers are indicated.
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Fig. 2.
p122-C prevents the formation of LPA-induced
stress fibers and focal adhesions but not platelet-derived growth
factor-induced membrane ruffling in Swiss 3T3 cells. Cells were
serum-starved to induce the disassembly of stress fibers and focal
adhesions and microinjected with recombinant p122-C protein. After
incubation for 30 min, the microinjected cells were stimulated with
culture medium containing 200 ng/ml LPA for 10 min (A-F) or
5 ng/ml platelet-derived growth factor for 30 min (G and
H), fixed, and stained. Actin filaments were visualized with
TRITC-phalloidin (A and G); phosphotyrosine was
visualized with antibody 4G10 followed by rhodamine B-conjugated goat
anti-mouse antibody (C); vinculin was visualized with
antibody VIN-11-5 followed by rhodamine B-conjugated goat anti-mouse
antibody (E). FITC-dextran were coinjected to identify
microinjected cells (B, D, F, and
H), as indicated by the arrowheads (A,
C, E, and G). The bar
represents 20 µm.
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Overexpression of p122 Induces Morphological Changes in Adherent
Cells--
To examine the potential functions of whole p122 molecules
in mammalian cells in vivo, we constructed an expression
vector, pGFP-WT, encoding a GFP fused to full-length p122. The plasmid was introduced into a variety of cell types, Swiss 3T3, MDCK, and
BHK21, by liposome-mediated transfection. The p122-GFP fusion products
were expressed in these cells and easily identified under fluorescence
microscopy. After 24 h, all cells expressing p122 fusion proteins
became profoundly rounded, refractile in cell shape, and left beaded
dendritic process-like structures (i.e. protrusions)
attached to the substrate (Fig. 3). In
contrast, cells transfected with a plasmid encoding GFP alone displayed diffuse fluorescence throughout the cytoplasm and nucleus and exhibited
a regular phenotype on morphologic examinations (data not shown). These
results indicate that the p122 protein induces morphological rounding
not only in fibroblasts but also in other adherent cell types.

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Fig. 3.
Morphological alterations induced by the
expression of p122 in various adherent cells. Swiss 3T3 cells
(A and B), MDCK cells (C and
D), and BHK21 cells (E and F) were
transiently transfected with pGFP-WT vector expressing a GFP fused to
p122. After 24 h, cells expressing the fusion proteins were
examined by fluorescence microscopy and photographed using a
FITC-compatible filter (A, C, and E).
Identical cell fields are shown under phase-contrast microscopy
(B, D, and F). The bar
represents 20 µm.
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The GAP Domain of p122 Is Responsible for the Morphological Changes
and Actin Organization--
Next, we constructed expression vectors
encoding various p122 deletion mutants (Fig.
4A) and expressed the
truncated proteins in BHK21 cells (Fig. 4B). The expression
of pGFP-534
C (Fig. 4B, c), encoding the first
534 amino acids of p122, displayed diffuse staining throughout the
cytoplasm and did not cause the morphological changes as seen with
pGFP-117
C (data not shown). The expression of pGFP-798
N (Fig.
4B, i) and pGFP-949
N (data not shown),
encoding part of the C-terminal region of p122, also did not produce
the changes. On the other hand, the expression of pGFP-863
C and
pGFP-617
N (Fig. 4B, e and g)
induced morphological rounding and detachment as shown with pGFP-WT
(Fig. 4B, a). These results indicate that the GAP
domain of p122 is necessary for the rounding and detachment of these
cells.

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Fig. 4.
Effect of p122 and its deletion mutants on
morphological alterations and the organization of actin stress
fibers. A, schematic representation of GFP fused to
p122 and the individual mutants produced by eukaryotic expression
vectors. Shaded boxes denote GFP regions, whereas
solid boxes denote GAP domains. The numbers refer
to the positions of amino acids in p122. Pluses and
minuses are arbitrary designations to indicate the relative
activity of the GFP fusion proteins in producing the morphological
alterations. B, BHK21 cells were transiently transfected
with plasmids pGFP-WT (a and b), pGFP-534 C
(c and d), pGFP-863 C (e and
f), pGFP-617 N (g and h), or
pGFP-798 N (i and j). After 4 h
(a and b) or 24 h (c-j), the
cells were examined under fluorescence microscopy and photographed
using a FITC-compatible filter (a, c,
e, g, and i). In identical cell
fields, F-actin was visualized with TRITC-phalloidin (b,
d, f, h, and j). The
bar represents 20 µm. WT, wild type.
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Because these morphological alterations were likely to be accompanied
by changes in actin organization, we visualized F-actin fibers by
rhodamine-conjugated phalloidin staining. As shown in Fig.
4B, b, stress fibers were absent from cells
expressing the p122-GFP fusion protein after 4 h of transfection.
The expression of pGFP-863
C (Fig. 4B, f) and
pGFP-617
N (Fig. 4B, h) induced the disassembly
of stress fibers, whereas the expression of pGFP-534
C and
pGFP-798
N did not affect actin organization (Fig. 4B,
d and j). These observations indicate that the
GAP domain of p122 regulates actin organization.
To further confirm the specificity of GAP, we used a constitutively
active mutant of Rho, V14-RhoA. This mutant V14-RhoA causes a large
decrease in intrinsic GTPase activity and is unresponsive to all GAP
proteins. Because BHK21 cells are not suitable for nuclear injection of
plasmids, we used MDCK cells and examined the effect of V14-RhoA on the
morphological changes caused by p122. Microinjection of both pGFP-WT
and pEXV-myc-VAL14-RhoA (kindly provided by A. Hall of University
College London via S. Narumiya of Kyoto University) into MDCK cells
resulted in a flat morphology (Fig. 5,
A-C), whereas cells expressing p122 alone was prospectively rounded (Fig. 5, D-F). These results indicate that active
V14-RhoA can neutralize the effect of p122 on the morphological
changes, suggesting that p122 is a Rho-specific GAP.

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Fig. 5.
Effects of dominant-active V14-RhoA on
p122-induced morphological alterations. MDCK cells were
microinjected with plasmids pEXV-myc-VAL14-RhoA and pGFP-WT
(A-C) or pGFP-WT (D- F) into the nucleus. After
24 h, cells expressing GFP fusion proteins were examined under
fluorescence microscopy (A and D). V14-RhoA was
stained with anti-myc antibody 9E10 followed by rhodamine B-conjugated
goat anti-mouse antibody (B and E). Identical
cell fields are shown under phase-contrast microscopy (C and
F). The arrowheads indicate the microinjected
cells. The bar represents 20 µm.
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Effect of Substitutions in the GAP Domain of p122 on Cell
Morphology and Actin Organization--
Crystal structure analyses of
p50rhoGAP have suggested that Arg-85 and Asn-194 are important for
binding to Rho proteins and enhancing GTPase activity (15). The
positions of these amino acids correspond to Arg-668 and Asn-779 of
p122 (Fig. 6B). Moreover, sequence alignment of the GAP domains of various GAPs reveals conserved
lysine and arginine residues. Based of these findings, we replaced
Arg-668 with Glu (R668E), Lys-706 with Glu (K706E), Arg-710 with Glu
(R710E), or Asn-779 with Val (N779V) (Fig. 6A) and
transfected these mutants independently into BHK21 cells. As shown in
Fig. 7, A and B,
cells expressing of R668E, K706E, or R710E underwent no morphological
alterations, whereas cells expressing N779V showed an altered
morphology and detachment. Furthermore, no obvious alterations of
F-actin organization in R668E, K706E, or R710E-transfected cells as
compared with nontransfected cells were detected (Fig. 7A,
b, d, and f). In contrast,
overexpression of N779V induced a loss of stress fibers (Fig.
7A, h). These results suggest that arginine
residues 668 and 710 and lysine residue 706 of p122 are involved in
stimulating the GTPase activity for Rho.

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Fig. 6.
Construction of p122 mutants and sequence
comparison of RhoGAP family members with p122. A,
schematic representation of p122 mutants used in this study. Point
mutations introduced into the wild type (WT) are indicated.
B, sequence alignment of four members of the RhoGAP family
was performed using the MacDNASIS program (26). The sequences of
the various cDNAs can be found in the following references: p122
(13) (D31962), rhoGAP (27) (Z23024), bcr (28) (Y00661), and p85 (29)
(M61745). The consensus sequence is shown with uppercase
letters, indicating identical residues found in all members, and
lowercase letters, indicating residues conserved in at least
three members. The numbers represent the positions of amino
acid mutations.
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Fig. 7.
Effect of p122 mutants on the morphological
changes and the organization of actin stress fibers in BHK21
cells. A, cells were transfected with plasmids
pGFP-R668E (a and b), pGFP-K706E (c
and d), pGFP-R710E (e and f), or
pGFP-N779V (g and h). After 24 h, the cells
were examined under fluorescence microscopy and photographed using a
FITC-compatible filter (a, c, e, and
g). In identical cell fields, F-actin was stained with
TRITC-phalloidin (b, d, f, and
h). The bar represents 20 µm. B,
values represent the percentage of cells showing the rounded phenotype
among total GFP-positive cells counted. The data were collected in
three independent experiments (approximately 300 cells for each
transfectant). WT, wild type.
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p122 Stimulates the PIP2-hydrolyzing Activity of
PLC
1 in Vivo--
We have previously demonstrated that p122
functions as a specific activator of PLC
1 but not PLC
1 and
PLC
1 in vitro (13). To further investigate the effect of
p122 on the PIP2-hydrolyzing activity of PLC
1 in
vivo, intracellular Ca2+ mobilization was visualized
by fluorescence microscopy. Because Swiss 3T3 or BHK21 cells are not
suitable for loading the nonratiometric Ca2+ indicator,
fluo-3, we examined Ca2+ signals in MDCK cells
microinjected with recombinant p122-N (Fig. 8A, n = 11) or
p122-C (Fig. 8B, n = 6) proteins. Cells
showed a brisk elevation of intracellular Ca2+ levels in
central areas within 20 s after the microinjection with p122-C
(Fig. 8B). The intracellular Ca2+ then decreased
to the basal level within 5 min (Fig. 8C). In contrast,
microinjection of p122-N had no effect on this Ca2+
response (Fig. 8A). The expression of PLC
1 in MDCK cells
was confirmed by Western blot analysis (data not shown). These results suggest that the C terminus of p122 encompassing the GAP domain can
enhance the PIP2-hydrolyzing activity of PLC
1 in
vivo, resulting in the inositol 1,4,5-triphosphate generation,
which predominantly induces Ca2+ release from intracellular
stores.

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Fig. 8.
Microscopic analysis of the intracellular
Ca2+ increase in MDCK cells loaded with fluo-3. Cells
microinjected with recombinant proteins, p122-N (A) or
p122-C (B) are indicated by arrowheads. The
gallery shows selected images from the time series at the indicated
time after microinjection. The difference 20 s minus 0 s
images (20-0) are shown at the right. The scale
bar shows the fluorescence intensity map. The peak intensity in
the center of the cell (20-0 in B) reflects the
greater thickness in the central region of the cell and possibly the
different fluorescent properties of the indicator, fluo-3, in the
cytoplasm and the nucleus (30). C, time courses of
fluorescence intensity marked with the area in microinjected cells
(a and b) were shown. The arrow
indicates the time at which recombinant proteins were microinjected.
The data were collected in independent experiments (n = 11 for p122-N, n = 6 for p122-C).
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DISCUSSION |
Here we demonstrated that the overexpression of p122 induces in
adherent cells morphological changes and detachment from the substrate.
The LPA-induced formation of stress fibers and focal adhesions is
suppressed by microinjecting the GAP domain of p122 into cells, whereas
platelet-derived growth factor-induced ruffle formation is not affected
(Fig. 2). Cell rounding is also detected within 24 h after p122
transfection and the formation of stress fibers and focal adhesions is
suppressed during the course of these changes. These changes induced by
p122 are attenuated by cotransfection with the constitutive active
mutant of Rho, V14-RhoA. We previously reported that p122 shows strong
GAP activity for RhoA but not for Rac or Cdc42 in vitro
(13). Considering that Rho controls the formation of stress fibers and
focal adhesions rather than membrane ruffling and filopodia formation,
it appears certain that p122 functions as a GAP for Rho in
vivo and is involved in the regulation of cytoskeletal disassembly.
Although stress fibers and focal adhesions have been widely studied in
many types of cells, their regulations in vivo are not yet
clear. The formation of focal adhesions is considered to be associated
with integrin clustering, which is induced by exogenous stimuli such as
formylpeptides and aggregation (16). This clustering is suppressed by a
Rho inhibitor, C3 transferase (16). Stress fibers, in concert with
focal adhesions, also regulate cell adhesion and motility. In MDCK
cells, scatter factor/hepatocyte growth factor stimulates motility and
spreading, whereas active Rho inhibits these reactions, presumably by
promoting filament assembly and adhesion (17). These reveal that p122
is involved in the regulation of cell adhesion and motility through
reorganization of cytoskeletal components. In this context, the
intracellular mechanisms underlying the regulation and localization of
p122 are intriguing.
It is thought that GAPs stimulate the GTP hydrolysis of monomeric G
proteins by supplying basic amino acid residues to the active center of
G proteins, resulting in a stabilization of the increasing negative
charge in the center (18, 19). Almost all GAPs contain two conserved
arginine residues and one conserved lysine residue (Arg-668, Lys-706,
and Arg-710 in p122), and mutations in any of these basic residues
significantly reduces GAP activity (20). Moreover, the recently
determined crystal structure of RhoA in its GDP-bound form with
p50rhoGAP in the presence of AlF4
,
which formed a transition-state analogue, suggested that Arg-85 and
Asn-194 in p50rhoGAP act to stabilize the transition state of the
GTPase reaction (21). In particular, Arg-85 appears to be important in
GAP-activated GTP hydrolysis through interaction with the
-
bridging oxygen and Gln-63 in RhoA. In addition, Lys-122 and Arg-126
seem to be involved in an extensive hydrogen-bonding network with
Asp-65 in RhoA. In this study, we introduce mutations at the putatively
indispensable arginine, lysine, and asparagine sites in the GAP domain
of p122 (Arg-668, Lys-706, Arg-710, and Asn-779). Replacing any of
Arg-668, Lys-706, or Arg-710 with glutamate markedly diminishes the
morphological changes and cell detachment caused by p122. These results
indicate that residues Arg-668, Lys-706, and Arg-710 are essential for
the function of p122 as a Rho-specific GAP in vivo, possibly
by stabilizing the transition state of GTP hydrolysis. The importance
of a charged residue at Arg-668 is also supported by Müller
et al. (22). They demonstrated that mutation of arginine to
methionine in the GAP domain of myr 5 (Arg-1695) corresponding to
Arg-668 of p122 or Arg-85 of p50rhoGAP abolished the GAP activity both
in vitro and in vivo (22). On the other hand, it
is interesting that changing Asn-779 (equivalent to Asn-194 of p50) to
valine does not affect the activity of p122, which is inconsistent with
above crystal analysis. Asn-779 may not be essential for the biological
activity of p122.
In addition to its Rho GAP activity, p122 is able to enhance the
PIP2-hydrolyzing activity for PLC
1 in vitro
(13). Using the Ca2+-sensitive dye fluo-3, we demonstrate
in this report that p122-C, but not p122-N, when injected into MDCK
cells, leads to an increase in fluo-3 fluorescence. Therefore, the rise
in Ca2+ is consistent with the hypothesis that p122 leads
to an increase in PLC
1 activity, leading to increased hydrolysis of
PIP2 and the generation of inositol 1,4,5-triphosphate,
which induces Ca2+ release (Fig. 8) (23, 24). In addition,
it is also possible that the hydrolysis of PIP2 is enhanced
by active PLC
1, resulting in the release of actin-binding proteins
from the membrane or PIP2 micelles. Free actin-binding
proteins bind to G-actins, a process that accelerates the disassembly
of actin fibers. These sequential reactions are quite similar to those
induced by Rho GAPs. It is conceivable that the PLC
1-enhancing
activity of p122 acts synergistically with its GAP activity in causing
the morphological changes and detachment. In fact, Tribioli et
al. (25) previously reported that overexpression of p115, which is
a protein with characteristics of a RhoGAP predominantly expressed in
hematopoietic cells, in fibroblasts inhibited stress fiber formation
but did not induce morphological changes. Furthermore, myr 5 required more than 40 h after transfection to induce a loss of stress
fibers and focal adhesions concomitant with cell rounding (22) as
compared with our results that p122 induces these alterations within
4 h (Fig. 4B, a and b). Further
careful studies using various mutants are required to assess the exact
contribution of the PLC
1-enhancing part of p122 on these changes.
 |
ACKNOWLEDGEMENTS |
We thank Dr. A. Hall for providing the
eukaryotic expression vector, pEXV-myc-VAL14-RhoA, Dr. K. Matsuoka for
providing Swiss 3T3 cells, H. Ohashi and J. Yamaki for technical
assistance, and K. Suyama and S. Takano for photographic assistance.
 |
FOOTNOTES |
*
This research was supported in part by a grant-in-aid for
Scientific Research and a grant for Cancer 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. Tel.:
81-24-548-2111; Fax: 81-24-548-3041; E-mail:
yoshihom{at}cc.fmu.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
PI, phosphatidylinositol;
PIP2, PI 4,5-bisphosphate;
GAP, GTPase-activating protein;
PLC, phospholipase C;
MDCK, Madin-Darby
canine kidney cells;
BHK, baby hamster kidney cells;
FITC, fluorescein
isothiocyanate;
PCR, polymerase chain reaction;
PBS, phosphate-buffered
saline;
GFP, green fluorescent protein;
TRITC, tetramethylrhodamine B
isothiocyanate;
LPA, lysophosphatidic acid.
 |
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