* Institute of Medical Chemistry and Biochemistry and Institute of Medical Biology and Human Genetics, University of
Innsbruck, A-6020 Innsbruck, Austria
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Abstract |
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Expression of transforming Ha-Ras L61 in
NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers.
The Ras-induced dissolution of actin stress fibers is
blocked by the specific PKC inhibitor GF109203X at
concentrations which inhibit the activity of the atypical
aPKC isotypes and
, whereas lower concentrations
of the inhibitor which block conventional and novel
PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-
and aPKC-
, as well
as antisense constructs encoding RNA-directed against
isotype-specific 5' sequences of the corresponding mRNA, abrogates the Ha-Ras-induced reorganization
of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-
was unable to counteract
Ras with regard to the dissolution of actin stress fibers.
Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-
and
aPKC-
lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN)
Rac-1 N17 and addition of the phosphatidylinositol 3'-kinase (PI3K) inhibitors wortmannin and LY294002
are in agreement with a tentative model suggesting
that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-
acts upstream of PI3K and Rac-1,
whereas aPKC-
functions downstream of PI3K and
Rac-1.
This model is supported by studies demonstrating
that cotransfection with plasmids encoding L61Ras and
either aPKC- or aPKC-
results in a stimulation of the
kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-
was abrogated by coexpression of DN Rac-1 N17.
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Introduction |
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EXPRESSION of transforming Ha-Ras leads to profound morphological alterations which include a
disassembly of F-actin stress fibers (Bar-Sagi and
Feramisco, 1986; Ridley and Hall, 1992
; Prendergast and
Gibbs, 1993
; Dartsch et al., 1994
). Similar effects have
been described in Src-transformed cells (Felice et al., 1990
;
Chang et al., 1995
). Ras has been shown to induce changes
in cytoskeletal actin through members of the Rho family
(Ridley et al., 1992
; Rodriguez-Viciana et al., 1994
). This
family comprises the RhoA, RhoB, and RhoC proteins,
Rac-1 and Rac-2, TC10, two CDC42Hs proteins (also
known as G25K), RhoG (Hall, 1990
; Shinjo et al., 1990
;
Ridley and Hall, 1992
; Ridley, 1995
), and Rnd1 and Rnd-3/
RhoE (Nobes et al., 1998
). Expression of a dominant-negative (DN)1 mutant of Rac-1 (Asn-17 Rac1) has been
shown to inhibit focus formation and tumorigenesis induced by oncogenic Ras (Qui et al., 1995a
,b; Prendergast et
al., 1995
). Similar findings have been described after expression of a DN Asn-19 RhoB (Prendergast et al., 1995
).
Expression of Asn-19 RhoB did not interfere with foci formation by Raf-1, indicating that the Ras-RhoB pathway is
independent of Raf-1 (Prendergast et al., 1995
). The compound SCH 51344 has been described as a suppressor of
Ha-Ras-mediated transformation (Kumar et al., 1995
) by
antagonizing the actin fiber reorganization without interference with the growth factor-induced activation of
MEK, p44ERK1, or p90RSK (Kumar et al., 1995
). The data
obtained with the DN Rac and Rho mutants and SCH
51344 reverting transformation by Ras emphasize the significance of the Ras-induced reorganization of the actin cytoskeleton for the transformation by the oncogene.
The detailed molecular mechanism by which Ras affects the actin cytoskeleton via Rac and Rho is still insufficiently understood. Microinjection of recombinant,
activated RhoA induces actin stress fibers in the absence
of added growth factors (Paterson et al., 1990). Serum-induced stress fiber formation can be blocked by the
Clostridium botulinum exoenzyme C3, an inhibitor of Rho
(Rubin et al., 1988
; Aktories et al., 1989
; Chardin et al.,
1989
). Actin stress fibers are linked to integrins at the inner surface of the plasma membrane through a multimolecular protein complex called focal adhesion (Burridge et al.,
1988
). Evidence for an implication of enzymes of the protein kinase C (PKC) family in focal adhesion formation
has been reported (Chun and Jacobson, 1992
; Vuori
and Ruoslahti, 1993; Mogi et al., 1995
). Activation of
PKC isoenzymes causes a stimulation of cell attachment,
spreading, and enhanced tyrosine phosphorylation of focal
adhesion kinase, pp125 FAK, a constituent of the focal adhesion complex (Smith-Sinnett et al., 1993
). FAK is tyrosine phosphorylated and its tyrosine kinase activity
enhanced upon integrin-mediated interaction with the extracellular matrix (Guan et al., 1991
; Kornberg et al., 1992
; Zachary and Rozengurt, 1992
). Enhanced tyrosine phosphorylation of FAK is also observed after exposure to several growth factors (Burridge et al., 1992
; Sinnett-Smith et
al., 1993; Rankin and Rozengurt, 1994
). Thus, FAK may
represent a point of convergence where growth factor
induced signals meet signals from activated integrins
(Zachary and Rozengurt, 1992
).
Stimulation of cells by some growth factors like platelet
derived growth factor (PDGF), epidermal growth factor
(EGF), or insulin has been shown to induce a reorganization of actin filaments by mediating actin polymerization
at the plasma membrane, where actin filaments form a
compact meshwork resulting in the formation of membrane ruffles and lamellipodia (Mellström et al., 1988; Ridley and Hall, 1992
; Rankin and Rozengurt, 1994
). Actin
filament organization underlying membrane ruffles appears to be mediated by Rac as microinjection of a DN
Asn-17 Rac-1 inhibits PDGF-induced membrane ruffles,
whereas the constitutively active (CA) Val-12 Rac-1 induces
membrane ruffling and the formation of focal complexes (Ridley and Hall, 1992
; Qiu et al., 1995a,b). Evidence for
an implication of a LIM kinase catalyzed phosphorylation
of cofilin in Rac-mediated reorganization of actin cytoskeleton has been presented (Arber et al., 1998
; Yang et
al., 1998
). An additional form of actin filament organization is found in microspikes and filopodia where small
bundles of actin filaments are attached to focal complexes
at the tips of the filopodia (Nobes and Hall, 1995
). Actin
filament organization in filopodia appears to be regulated
by CDC 42Hs (Nobes and Hall, 1995
).
CDC 42-, Rac-1-, and Rho-induced focal complexes are
morphologically distinct but share a variety of constituents
like vinculin, paxillin, and pp125 FAK (Nobes and Hall,
1995). Evidence for a hierarchical relationship between
CDC 42, Rac and Rho, in which activation of CDC 42 leads to a sequential activation of Rac and Rho has been
presented (Nobes and Hall, 1995
). The detailed mechanisms, however, regulating the assembly and the spatial
organization of the different structures of actin filaments
are still insufficiently understood. In view of the similarities between Rho- and Rac-induced focal complexes and
the well-documented implication of PKC in the assembly
of integrin-containing focal adhesions, a similar role of
representatives of the PKC family in the formation of Rac-regulated focal complexes appears conceivable. An implication of enzymes of the PKC family in cytoskeleton organization is supported by a series of published observations
(for review see Keenan and Keleher, 1998). The interleukin (IL)-2-mediated alteration of the cytoskeleton has recently been demonstrated to require atypical aPKC-
(Gomez et al., 1997
). Transforming Ras has been shown to activate PKC (Morris et al., 1989
). Evidence for an implication of atypical aPKC-
in the v-Ras-mediated activation and nuclear translocation of mitogen-activated protein kinase has been presented (Bjorkoy et al., 1997
).
Induction of c-fos by oncogenic Ras has recently been
shown to require the coordinated activities of PKC-
, PKC-
,
and PKC-
(Kampfer et al., 1998
). However, whether the Ras-mediated reorganization of the actin cytoskeleton is
PKC dependent, which PKC isotypes are involved, and
what their function in the Ras-mediated restructuring of
the cytoskeleton is, has remained unclear.
In this paper evidence is presented for an implication of
the two atypical PKC-, and PKC-
isotypes in the Ras-mediated reorganization of the actin cytoskeleton. The
data support a tentative model for a signaling pathway in
which aPKC-
acts downstream of Ras but upstream of
phosphatidylinositol-3' kinase (PI3K) and Rac-1, whereas
aPKC-
functions downstream of Rac-1.
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Materials and Methods |
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Reagents and Plasmids
Dulbecco's modified Eagle's medium (DME) and restriction enzymes for
molecular biological approaches were purchased from . Fetal calf serum and L-glutamine were obtained from BioWhittaker.
Leupeptin, myelin basic protein (M-1891), tetramethylrhodamine-isothiocyanate (TRITC)-labeled phalloidin, and aprotinin are products from
. Lipofectin transfection reagents and Opti-Mem I medium were
purchased from Life Technologies. [-32P]ATP (10 mCi/ml, 3000 Ci/
mmol) and Hyperfilm-MP were obtained from . PCR primers
used for subcloning strategy were obtained from ARK Scientific.
Subcloning strategy and oligonucleotides used for antisense constructs
had been described elsewhere (Kampfer et al., 1998). Orientation of insertion was determined by restriction analysis and sequencing. Subcloning
strategy, mutagenic primers, as well as selection primers for the generation of kinase-defective, DN, as well as CA mutants of PKC isotypes had
been described elsewhere (Baier-Bitterlich et al., 1996
; Überall et al.,
1997
; Kampfer et al., 1998
). All cDNAs for PKC isotypes, green fluorescence protein (GFP), and the cDNA for Rac-1 N17 were subcloned into the expression vector pEF-1neo. GF109203X, LY294002, wortmannin, and Pansorbin beads were obtained from .
Cell Culture and Transient Transfection Procedures
NIH3T3 fibroblasts were kept at logarithmic growth phase in DME supplemented with 10% heat-inactivated fetal calf serum, lysophosphatidic acid (100 ng/ml), and 2 mM L-glutamine in a humidified atmosphere containing 5% CO2. To obtain transient transfectants, NIH3T3 cells (5 × 104
cells per well) were seeded in 100-mm-diam wells containing circular glass
coverslips (eight per well) and were transfected for 8 h with 1 µg pEF-1neoGFP expression plasmid, 1.5 µg pSR-II L61 Ha-Ras (encoding a
constitutively active Ras leucine L61 mutant), and 20 µg of plasmids
encoding for kinase-defective, DN cPKC-
K368R, atypical aPKC-
K275W, and aPKC-
K275W mutants. Alternatively, NIH3T3 fibroblasts
were cotransfected with plasmids encoding pEF-1neoGFP plus a CA
aPKC-
A119E, (CA) aPKC-
A119E, and (CA) cPKC-
A25E mutants,
or vector controls (pEF-1neo), respectively.
Fluorescence Imaging
48 h after the transfection procedure cells were washed twice with PBS (140 mM NaCl, 2.7 mM KCl, 4.6 mM Na2HPO4 · 12 H2O, 1.3 mM NaH2PO4 · H2O), fixed with 3.5% formaldehyde (wt/vol in PBS, 10 min) at room temperature and after extensive washing, permeabilized for 5 min with ice-cold acetone. After permeabilizing, the coverslips were washed twice with PBS and incubated for 1 h with FACS® buffer (500 ml PBS, 1 g sodium azid, and 4% fetal calf serum). For visualizing the F-actin structure, assembly cells fixed onto glass coverslips were overlaid with 100 µl (0.1 g/ml) TRITC-conjugated phalloidin per coverslip for 1 h. Afterwards the stained cells were washed six times with PBS and with distilled water and mounted in Mowiol containing 0.1% (wt/vol) p-phenylenediamine.
Mounted cells were viewed on an BX50 fluorescence microscope, and images of green fluorescence-positive cells were done by using a RGB-mode video real-color camera (Optronics Engineering DEI-470). Image processing was carried out with the image processing software MetaMorph (). The green and red fluorescence images were recorded separately by changing the excitation wavelength (from 480 to 550 nm), exported into Adobe Photoshop, and then printed on a color laser copier system (Agfa 707).
Measurement of Total F-actin Fiber Length
Total F-actin fiber length was calculated after digitalizing TRITC-phalloidin stained F-actin by using the MetaMorph image processing software. The edges of the cells were detected by the aid of a convolution kernel comparing the brightness of the neighboring pixels. After thresholding and separating from the background specimen, fiber lengths were determined and expressed as the percentage of the mean fiber length compared with the fiber length of mock-transfected fibroblasts.
Partial Purification of 6× His-tagged PKC Isotypes and PKC In Vitro Assays
African green monkey kidney fibroblasts (COS-1, 106/100-mm dish) were
transfected with 15 µg of circular plasmid DNA per dish by lipofectin reagents, as described by the manufacturer. 48 h posttransfection, cells were
lysed in 1 ml lysis buffer (150 mM NaCl, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES, pH 7.59], 1% Nonidet P-40 (vol/
vol), 50 µg/ml each aprotinin and leupeptin, and 1 mM phenyl-methylsulfonyl fluoride). Lysates were purified by using a Ni2+-resin batch procedure, and equal amounts of recombinant PKC isotypes were subjected to
an enzymatic PKC assay as described elsewhere (Baier-Bitterlich et al.,
1996; Überall et al., 1997
; Kampfer et al., 1998
). Enzyme activities of recombinant PKC-isotypes
,
, and
are expressed as cofactor-dependent
phosphorylation of the [A25S] synthetic PKC peptide (RFARKGSLRQKNVY; presenting the pseudosubstrate sequence of PKC-
with an
alanine to serine substitution). The concentrations of substrate peptide
and cofactors used are: 50 µg/ml [A25S], 280 µg/ml PtdSer, 10 µM TPA,
and 1 mM CaCl2. Expression of the fusion tag peptide COOH-terminal of
PKC-
and PKC-
and PKC-
did not affect the kinase activity in vitro
(Überall et al., 1997
).
Magnetic Separation of Transient Transfected COS-1
Cells and PKC- Immunocomplex Kinase Assay
COS-1 cells cotransfected with pMACS4 (Miltenyi Biotech, 1.5 µg), and therefore expressing a truncated CD4 surface marker, were washed twice with PBS and then incubated with PBE (PBS with 5 mM EDTA and 0.5% skimmed milk powder) for 10 min at 37°C to detach cells from the cell culture dish. Suspended cells were incubated for 45 min, 4°C with MACS4 magnetic microbeads (diluted 1:10 in PBE) at 4°C and the positive-transfected cells were separated from the nontransfected background over magnetic columns. Magnetically separated COS-1 cells were washed with cold PBS and lysed on ice in 500 µl of lysis buffer A (50 mM Tris-HCl, pH 7.3, 50 mM NaCl, 5 mM Na4P2O7 · 10 H2O (NaPP), 5 mM EDTA, 2% Nonidet P-40, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 50 mM NaF, and 100 mM Na3VO4) for 10 min and lysates clarified by centrifugation at 10,000 g for 5 min 4°C.
Aliquots of cell equivalents containing equal amounts of protein were
subjected to an immunoprecipitation (IP) procedure using a corresponding PKC- antibody purchased from . IPs were
recovered by using Pansorbin beads. PKC-
molecules bound to 35 µl of
Pansorbin beads, were resuspended in 20 µl kinase buffer, and then mixed
with 9 µg myelin basic protein ( M-1891). The kinase reactions were
initiated by the addition of 2 µCi [
-32P]ATP (10 mCi/ml, 3,000 Ci/mmol)
and incubation of the tubes by frequent vortexing at 30°C for 30 min.
Phosphorylation of myelin basic protein was terminated by the addition of
5 µl of 5× SDS sample buffer and boiling the samples for 5 min. Probes
were analyzed by SDS-PAGE (10%) and transferred to polyvinylene difluoride (PVDF) membranes (). Computer-assisted calculation
of PKC-
enzyme activities was done after scanning the corresponding
PVDF membranes by using the Scanner Controller Sci System.
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Results |
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The Reorganization of Actin Cytoskeleton Induced by Transforming Ras Is Antagonized by the Specific PKC Inhibitor GF109203X
NIH3T3 cells transiently transfected with the transforming
Ha-Ras L61 oncogene exhibit dramatic morphological differences compared with their nontransfected counterparts.
Normal NIH3T3 fibroblasts are spreading on the extracellular matrix, appear flat, and exhibit bundles of actin stress
fibers traversing the cell (Fig. 1 A). Cells expressing the
Ras oncogene are more spindle-shaped, exhibit frequently
long protrusions, and are characteristically devoid of actin
stress fibers (Fig. 1 B). Similar morphological effects of
transforming Ras have been described by other authors (Bar-Sagi and Feramisco, 1986; Ridley and Hall, 1992
;
Prendergast and Gibbs, 1993
; Dartsch et al., 1994
; Rodriguez-Viciana et al., 1994
).
|
As shown in Fig. 1 D, the PKC-specific inhibitor
GF109203X (Toullec et al., 1991) is capable of reversing
the dissolution of actin stress fibers by Ras. This effect is
observed in the presence of 6 µM of the inhibitor (Fig. 1
D). 200 nM of the compound which had been shown to be
sufficient for blocking c- and n-type PKC isotype kinase
activity in cell-free extracts (Fig. 2 A) (Überall et al.,
1997
), is, however, without an effect on the Ras-mediated stress fiber dissolution (Fig. 1 C). 6 µM GF109203X corresponds roughly to the IC50 of the compound against the
atypical PKC-
(Fig. 2 B, see also Martiny-Baron et al.,
1993
), and as shown in Fig. 2 C, exerts a similar effect on
PKC-
, suggesting that if a PKC is involved in the Ras-induced reorganization of the actin cytoskeleton, it should
be an atypical (a-), rather than a conventional (c-) or novel
(n-) type PKC isoform.
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Reversion of Ras-induced Alteration of Actin
Cytoskeleton by Kinase-defective, DN Mutants of
Atypical PKC-, and PKC-
as well as by PKC-
- and
PKC-
-specific Antisense Constructs
As shown in Fig. 3, B and D, expression of aPKC-
K275W as well as PKC-
K275W mutants, which contain
an inactive ATP-binding site, is able to revert the Ras-induced depolymerization of actin stress fibers. For these
experiments NIH3T3 cells were transiently cotransfected
with vectors encoding Ha-Ras L61 and kinase-defective, DN aPKC-
K275W or aPKC-
K275W mutants, respectively, using a green fluorescence protein expression vector as a transfection marker. Ras-induced reorganization
of F-actin cytoskeleton was not affected by an expression
of a kinase-defective, DN mutant of PKC-
K368R (Fig. 3
F). Expression levels of cPKC-
K368R, aPKC-
K275R,
and aPKC-
K275R were found to be in a similar range (data not shown).
|
The effects observed after expression of the DN versions of PKC- and PKC-
were checked by PKC-
- and
PKC-
specific antisense constructs. For this purpose,
cells were transfected with constructs encoding isotype-specific 5' sequences targeted to the corresponding mRNA.
As described previously, generation of these antisense
RNA sequences leads to an almost complete depletion of
the endogenous aPKC-
and -
(Kampfer et al., 1998
).
Fig. 3, C and E demonstrate that isotype-specific depletion of aPKC- and aPKC-
yields the same results as expression of DN mutants of these isotypes. In agreement
with the results obtained with the DN PKC-
mutant,
PKC-
antisense did not affect the Ras-mediated alterations of F-actin organization (Fig. 3 G). Neither cPKC-
nor aPKC-
/-
sense constructs did affect the Ras-mediated disassembly of F-actin fibers (data not shown).
A quantitative evaluation of Ras-mediated stress fiber depolymerization and the effects of (DN) aPKC-
K275W, (DN) aPKC-
K275W, aPKC-
, and aPKC-
antisense on Ras-induced actin fiber organization was performed by measuring F-actin fiber length with the aid of the
MetaMorph image processing software (Fig. 4, A and B).
|
The data shown in Figs. 3 and 4 clearly indicate that the
Ras-mediated alteration of actin cytoskeleton is antagonized by an isotype-specific inhibition or depletion of
aPKC- and aPKC-
. These findings strongly suggest that
the Ras-mediated reorganization of the actin cytoskeleton
is mediated by these two atypical aPKC isotypes.
Expression of CA Mutants of Atypical aPKC- and
aPKC-
Mimic the Effect of Transforming Ras L61 on
Actin Cytoskeleton
It has previously been demonstrated that substitution of
an alanine by a glutamate within the pseudosubstrate domain of PKC isotypes generates CA mutants with reduced
cofactor requirements. Biochemical and biological properties of these mutants had been described in preceding publications (Baier-Bitterlich et al., 1996; Überall et al., 1997
;
Kampfer et al., 1998
).
If Ha-Ras uses atypical PKC isotypes for the reorganization of the actin cytoskeleton as suggested by the data
shown in Figs. 3 and 4, expression of CA aPKC- and
(CA) aPKC-
mutants should affect actin stress fibers like
transforming Ha-Ras. As shown in Fig. 5, B and D, this is
indeed the case. A CA mutant of PKC-
A25E did not
show any significant effect on stress fiber rearrangements (data not shown).
|
Stress fibers reappear after treatment of the aPKC-
A119E- or aPKC-
A119E-expressing cells with the PKC
inhibitor GF109203X demonstrating that the alterations of
the actin cytoskeleton in cells expressing the constitutively
active versions of these atypical PKC isotypes are indeed
caused by a PKC activity (Fig. 5, C and E).
Evidence That Atypical aPKC- Acts Upstream and
aPKC-
Acts Downstream of Rac-1
An early event after expression of oncogenic Ras is the
generation of membrane ruffles and a reorganization of
the actin cytoskeleton, i.e., stress fibers disappear whereas
F-actin accumulates at the cell periphery (Bar-Sagi and
Feramisco, 1986; Ridley et al., 1992
; Rodriguez-Viciana et al.,
1994
). Evidence for an implication of Rac-1 in Ras-mediated reorganization of the actin cytoskeleton has been presented (Ridley et al., 1992
; Rodriguez-Viciana et al., 1994
).
The data described so far suggest that the atypical PKC
isotypes
and
are also involved in this process. It appeared interesting, therefore, to investigate whether they
act within the Ras/Rac pathway and if they do, whether
their position within this pathway can be identified.
As should be expected, DN N17 Rac-1 inhibits the Ras-mediated disassembly of stress fibers (Fig. 6, A and B). To
obtain some information whether PKC- and PKC-
cooperate with Rac-1 in the same pathway, cells were
cotransfected with combinations of either CA aPKC-
A119E and N17 Rac-1 or CA aPKC-
A119E and N17
Rac-1.
|
As shown in Fig. 6 C, N17 Rac-1 is able to overcome
stress fiber disassembly induced by CA aPKC- A119E
(compare with Fig. 5 for the effect of aPKC-
A119E in
the absence of N17 Rac-1), indicating that aPKC-
acts
upstream of Rac-1. Stress fiber disassembly by aPKC-
A119E, however, is not affected by N17 Rac-1 (Fig. 6 D),
suggesting that aPKC-
acts either downstream or independent of Rac-1. A quantitative evaluation of the stress
fiber alterations shown in Fig. 6 is presented in Fig. 4 B
and Fig. 10.
|
Effects of Transforming Ras and CA Rac on aPKC-
and aPKC-
The data presented so far suggest that Ras mediates the
effects on the cytoskeleton via a pathway containing
aPKC--Rac-1 and aPKC-
. If this model is correct, Ras
should activate aPKC-
and aPKC-
whereas Rac should
be able to stimulate aPKC-
. Unfortunately, this question
could not be addressed in NIH3T3 cells due to the low
transfection efficiencies in this cell type. Therefore, these studies were performed with COS cells. As shown in Fig. 7
A, cotransfection of COS cells with plasmids encoding
Ras L61 and 6× His-tagged aPKC-
leads to a significant
activation of the kinase activity of aPKC-
. Coexpression
of Ras L61 and aPKC-
results in a marked stimulation
of aPKC-
as demonstrated in Fig. 7 B. Furthermore, cotransfection of a plasmid encoding CA V12Rac with a construct encoding aPKC-
also revealed an activation of
aPKC-
by Rac (Fig. 7 C). Surprisingly, V12Rac in addition to aPKC-
also activated aPKC-
(data not shown).
However, this finding is not in conflict with data or models
presented so far. Possible interpretations for this effect
will be presented in the Discussion. Our conclusion that
Ras activates aPKC-
by a Rac-1-dependent mechanism is
supported by the fact that expression of DN N17Rac
blocks Ras-mediated stimulation of aPKC-
(Fig. 7 D).
N17Rac does not inhibit Ras-mediated activation of PKC-
(data not shown).
|
Effect of PI3K Inhibitors on Actin Cytoskeleton
Reorganization Induced by Oncogenic Ras or CA
Mutants of aPKC- and aPKC-
PI3K has been shown to be implicated in the Ras-induced
reorganization of actin cytoskeleton (Rodriguez-Viciana
et al., 1994). In agreement with these findings, treatment
of Ras-expressing cells with the PI3K inhibitors wortmannin or LY294002 counteracts the effects of Ras on the actin cytoskeleton (Fig. 8). Both inhibitors also antagonize
the disassembly of actin stress fibers induced by constitutively active aPKC-
A119E, whereas the cytoskeletal reorganization mediated by the aPKC-
A119E mutant was
not affected (Fig. 9). These data suggest that aPKC-
acts
upstream of PI3K whereas aPKC-
functions either downstream or independent of PI3K. A quantitative evaluation
of the effects of the PI3K inhibitors is presented in Fig. 4 B
and Fig. 10.
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![]() |
Discussion |
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The data presented here demonstrate that transforming
Ha-Ras uses atypical aPKC- and aPKC-
for the rearrangement of actin cytoskeleton. This conclusion is based
on the observation that (a) the Ha-Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the
activity of the atypical aPKC isotypes
and
. (b) Coexpression of transforming Ha-Ras L61 with kinase-defective, DN mutants of aPKC-
and aPKC-
, as well as
antisense constructs encoding RNA directed against isotype-specific 5' sequences of the corresponding mRNAs,
abrogate the Ha-Ras-induced reorganization of the actin
cytoskeleton. (c) Finally, transfection of cells with constructs encoding CA mutants of atypical aPKC-
and
aPKC-
mimic the effect of oncogenic Ha-Ras on actin cytoskeleton reorganization.
With regard to the effects of the PKC inhibitor
GF109203X, it may appear surprising that concentrations
of the inhibitor which, in vitro, reduce the activity of
aPKC- and aPKC-
to ~50% cause an almost complete
reversal of the Ras-mediated disassembly of F-actin stress
fibers. It should be emphasized, however, that neither the
biological activators nor the intracellular substrates of
atypical PKC isozymes have been sufficiently identified.
Thus, the reaction mixtures used for the determination of
the enzyme activity of the two kinases certainly differ from
the in vivo conditions and this may affect the sensitivity to
the inhibitor. Furthermore, both aPKC-
and aPKC-
are
affected by the inhibitor to about the same extent, i.e.,
~50%. Since both enzymes are required for the Ras-mediated restructuring of actin cytoskeleton, the simultaneous
inhibition of both enzymes may result in an additive effect.
Finally, the intracellular concentration of the inhibitor is
not known. It is possible, therefore, that locally higher
concentrations than 6 µM have been achieved.
Expression of a kinase-defective, DN mutant of PKC-
or PKC-
depletion by intracellular generation of PKC-
antisense RNA did not affect the Ras-induced alterations
of the actin cytoskeleton. In a previous paper we had
demonstrated that the phosphorylation of MARCKS in
NIH3T3 cells under biological conditions is mediated by
PKC-
and that the phorbol ester-induced phosphorylation of MARCKS is markedly inhibited by expression of
the kinase-defective, DN PKC-
K368R mutant (Überall
et al., 1997
). Thus, the inability of PKC-
K368R to interfere with the Ras-induced effects on actin cytoskeleton is
not explained by an insufficient intracellular expression
level. Furthermore, the phosphorylation of MARCKS, which has been discussed as an important regulator of actin organization (Rosen et al., 1990
), does not appear to be
involved in the Ras-induced reorganization of the actin cytoskeleton.
The Ras-induced reorganization of actin cytoskeleton
has been shown to be mediated by Rac-1 and PI3K (Joneson and Bar-Sagi, 1997). The data presented here suggest
that aPKC-
acts upstream of PI3K and Rac-1, whereas
aPKC-
functions either downstream or independent of
Rac-1. This conclusion is based on the following findings:
(a) the effects of the constitutively active mutant of aPKC-
which mimics Ras with regard to the alterations of actin cytoskeleton are inhibited by the PI3K blockers wortmannin and LY294002 and also by expression of DN N17 Rac-1;
and (b) expression of N17 Rac-1 does not interfere with
the disassembly of actin stress fibers induced by the CA
aPKC-
A119E mutant. The sequence Ras-aPKC-
-PI3K-Rac-1 would be in accordance with the model suggested by Rodriguez-Viciana et al. (1994)
, who propose a pathway in which the Ras-mediated activation of Rac-1 is mediated by PI3K. Our findings would add atypical aPKC-
as an upstream element of this sequence, a model consistent with data demonstrating a physical interaction of
aPKC-
with p21ras (Diaz-Meco et al., 1994
).
The studies presented here demonstrate that in intact
cells, Ras is capable of activating aPKC-. Furthermore,
it is shown here that in addition to the stimulation of
aPKC-
, Ras also enhances the kinase activity of aPKC-
.
The latter effect is suppressed in cells expressing DN
N17Rac. We conclude, therefore, that Ras activates
aPKC-
by a Rac-1-dependent mechanism. This conclusion is supported by the finding that expression of constitutively active V12Rac enhances the kinase activity of
aPKC-
. Surprisingly, V12Rac was also found to be capable of activating aPKC-
. However, this observation is not
in conflict with the other data presented in this paper, and
does also not contradict a model suggesting that the effects
of Ras on actin cytoskeleton are mediated by a pathway
comprising aPKC-
-Rac-aPKC-
. The fact that in COS
cells V12Rac can activate aPKC-
indicates that an activation of Rac-1 by a Ras-independent pathway, e.g., via
CDC 42, may lead to a Rac-mediated activation of aPKC-
and aPKC-
. This is obviously in contrast to the pathway
activated by oncogenic Ras where aPKC-
acts upstream
and aPKC-
downstream of Rac as outlined above. A conclusion which is also supported by the observation that in COS cells DN N17Rac does not interfere with Ras-mediated activation of aPKC-
.
As the mechanism by which Ras affects actin filament
structures are incompletely understood, it can be only
speculated with regard to the function of aPKC- or
aPKC-
in this pathway. The disassembly of actin stress fibers has been correlated to the accumulation of inactive,
GDP-charged RhoA (Qui et al., 1995a
,b). Activation of
RhoA by an exchange of the GDP by a GTP was found to
stimulate stress fiber formation (Ridley and Hall, 1992
).
These findings suggest that the Ras-induced disassembly
of actin stress fibers is the result of a conversion of the active, GTP-charged Rho to the inactive GDP-bound form.
Models for a biochemical linkage between the Ras and
Rho proteins have been proposed (Boguski et al., 1992
;
Nobes and Hall, 1994
; Chant and Stowers, 1995
). In one
simple model, the Ras/Rho pathway includes p120GAP,
the Ras GTPase-activating protein which may also act as
a Ras effector (Chant and Stowers, 1995
) and p190, a
p120GAP-associated protein which exhibits a Rho-specific
GTPase-activating domain (Settleman et al., 1992
; Foster
et al., 1994
). Phosphorylation by aPKC-
and/or aPKC-
may enhance the activity of p190Rho-GAP, resulting in an increased conversion of RhoGTP to RhoGDP and stress fiber dissolution. Tyrosine phosphorylation of p190 by c-Src
has been shown to be correlated with EGF-induced stress fiber disassembly (Chang et al., 1995
). Evidence for a
PKC-catalyzed serine/threonine phosphorylation of p190,
however, is still lacking. Alternatively, aPKC-
and/or
aPKC-
may enhance the RhoGDP level by inhibiting
Rho-GDI, or exchange factors (GEF) acting on Rho, like
SmgGDS, Rap1, Dbl, Ect2, and Ost (Hiraoka et al., 1992
;
Miki et al., 1993
; Horii et al., 1994
). However, models in
which the Ras-induced disassembly of actin stress fibers
are described as resulting exclusively from a Ras-mediated
conversion of RhoGTP to RhoGDP are inconsistent with
the findings demonstrating an activation of Rho by Ras as
essential for transformation and mitogenesis by oncogenic
Ras (Prendergast et al., 1995
; Qui et al., 1995a
,b; Olson et
al., 1998
).
CA mutants of Rac1 and RhoA enhance the transforming activity of Ras including the oncogene-induced morphological alterations (Khosravi-Far et al., 1995).The
biological meaning of the reorganization of the actin cytoskeleton by transforming Ras as well as the role of
RhoA in Ras transformed cells are not quite clear. As outlined above, activation of RhoA is required for transformation by Ras and expression of a CA mutant of RhoA
enhances the transforming activity of Ras (Khosravi-Far
et al., 1995
). The fact, however, that oncogenic Ras causes
a disassembly of actin stress fibers whereas CA RhoA promotes stress fiber formation indicates that oncogenic Ras
somehow deregulates the normal Rho-mediated effects on
the cytoskeleton. In Ras-transformed cells, the activation of Rho has been shown to be required for a suppression of
p21Waf1/Kip which is upregulated in cells expressing oncogenic Ras (Olson et al., 1998
), thus indicating that Rho
exerts other functions besides regulation of the actin cytoskeleton. This notion is supported by findings indicating
that RhoA can mediate several distinct effector pathways
and that transformation by RhoA and the ability to remodel the cytoskeleton are, to some extent, independent, e.g., transformation was found to correlate with Rho-associated kinase binding rather than stress fiber formation
(Sahai et al., 1998
). Thus, the Ras-mediated disassembly of
actin stress fibers is not necessarily in conflict with the postulated activation of RhoA in Ras-transformed cells. Furthermore, expression of the Rho family member Rnd3/
RhoE has been shown to result in a phenotype which
strikingly resembles the phenotypic alterations caused by oncogenic Ras, i.e., cell rounding, loss of stress fibers, and decreased cell adhesion (Nobes et al., 1998
). Recent results suggest that RhoE may act to inhibit signaling downstream of RhoA, by altering some RhoA-regulated responses, such as stress fiber formation, whereas other
RhoA-mediated effects remain unaffected (Guasch et al.,
1998
).
The biochemical function of aPKC- and aPKC-
in the
Ras/Rac/Rho pathway remains to be elucidated. Evidence
for PKC as an important regulator of cytoskeletal functions has been presented by numerous studies (Keenan
and Kelleher, 1998
). Stress fibers are associated with focal
adhesion complexes where cells interact with the extracellular matrix. The interaction with the extracellular matrix
is mediated by integrin receptors which are integral components of focal adhesion plaques (Schwartz et al., 1995
).
Activation of integrin receptors initiates a signaling cascade which has been shown to depend on stress fibers
(Hynes, 1992
; Rosales et al., 1995
; Wu et al., 1995
) and to
cooperate with growth factor-mediated events (Clark and
Brugge, 1995
; Schwartz et al., 1995
). An implication of
PKC in focal adhesion formation and integrin-mediated
signaling has been described (Clark and Brugge, 1995
;
Schwartz et al., 1995
). By permitting anchorage-independent growth, transforming Ras may override the necessity
for cell attachment. Evidence in support of this concept
has been presented (Kang and Krauss, 1996
). Both Src and
Ras have been shown to act as important elements in integrin-mediated signaling (Schlaepfer et al., 1998
). Expression of oncogenic mutants of either protein may not only
circumvent integrin receptor activation but may also lead
to a disruption of focal adhesions and stress fibers as a result of a persistent activation of downstream elements of
the integrin-regulated pathways, as had been suggested for v-Src (Fincham et al., 1995
; Hildebrand et al., 1993
).
![]() |
Footnotes |
---|
Address correspondence to F. Überall, Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Fritz Preglstrasse 3, A-6020 Innsbruck, Austria. Tel.: (43) 512-507-3508. Fax: (43) 512-507-2872. E-mail: florian.ueberall{at}uibk.ac.at
Received for publication 10 September 1998 and in revised form 24 December 1998.
F. Überall and K. Hellbert contributed equally to this work.
We are grateful to A. Hall (London, UK), H. Mischak (Berlin, Germany),
and J. Moscat (Madrid, Spain) for providing the plasmid pGST-N17Rac,
pRc-CMV-PKC- K275W, pCDNA3-PKC-
K275W-HA-tagged, and a
full-length PKC-
cDNA, to S. Geley (Innsbruck, Austria) for subcloning
pEFneoGFP-S65T, and to M. Karin (San Diego, CA) for plasmid pSR-
II
L61 Ha-Ras. Furthermore, the authors wish to thank E. Preuss (Praesentation Dokumentation Lernsysteme, Innsbruck, Austria) for illustration.
This work was supported in part by grants from the Austrian Fond zur Förderung der wissenschaftlichen Forschung (FWF, P12547-MOB), des Sonderforschungsbereiches (SFB, F201, Biological communication systems, P12104-MED), and the Austrian Federal Bank (project 7399).
![]() |
Abbreviations used in this paper |
---|
a, atypical; c, conventional; CA, constitutively active; DN, dominant-negative; GFP, green fluorescence protein; n, novel; PI3K, phosphatidylinositol-3' kinase; PKC, protein kinase C; PVDF, polyvinylene difluoride.
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References |
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---|
1. | Aktories, K., S. Braun, S. Roesener, I. Just, and A. Hall. 1989. The rho gene product expressed in E. coli is a substrate for botulinum ADP-ribosyltransferase C3. Biochem. Biophys. Res. Commun 158: 209-213 |
2. | Arber, S., F.A. Barbayannis, H. Hanser, C. Schneider, C.A. Stanyon, O. Bernard, and P. Caroni. 1998. Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature. 393: 805-809 |
3. |
Baier-Bitterlich, G.,
F. Überall,
B. Bauer,
F. Fresser,
H. Wachter,
H. Grunicke,
G. Utermann,
A. Altman, and
G. Baier.
1996.
PKC-![]() |
4. | Bar-Sagi, D., and J.R. Feramisco. 1986. Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by Ras proteins. Science. 233: 1061-1068 |
5. |
Bjorkoy, G.,
M. Perander,
A. Overvatn, and
T. Johansen.
1997.
Reversion of
Ras- and phosphatidylcholine-hydrolyzing phospholipase C-mediated transformation of NIH 3T3 cells by a dominant interfering mutant of protein kinase C lambda is accompanied by the loss of constitutive nuclear mitogen-activated protein kinase/extracellular signal-regulated kinase activity.
J.
Biol. Chem
272:
11557-11565
|
6. | Boguski, M.S., R.C. Hardison, S. Schwartz, and W. Miller. 1992. Analysis of conserved domains and sequences motifs in cellular regulatory proteins and locus control regions using new software tools for multiple alignment and visualization. New Biol 4: 247-260 |
7. | Burridge, K., K. Fath, T. Kelly, G. Nuckolls, and C. Turner. 1988. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu. Rev. Cell Biol 4: 487-525 . |
8. | Burridge, K., C.E. Turner, and L.H. Romer. 1992. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracelluar matrix: role in cytoskeletal assembly. J. Cell Biol 119: 893-903 [Abstract]. |
9. | Chang, J.H., S. Gill, J. Settleman, and S.J. Parsons. 1995. c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation. J. Cell Biol 130: 355-368 [Abstract]. |
10. | Chant, J., and L. Stowers. 1995. GTPase cascades choreographing cellular behavior: movement, morphogenesis, and more. Cell 81: 1-4 |
11. | Chardin, P., P. Boquet, P. Maduale, M.R. Popoff, E.J. Rubin, and D.M. Gill. 1989. The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. EMBO (Eur. Mol. Biol. Organ.) J 8: 1087-1092 [Abstract]. |
12. | Chun, J.S., and B.S. Jacobson. 1992. Requirement for diacylglycerol and protein kinase C in HELA cell-substratum adhesion and their feedback amplification of arachidonic acid production for optimum cell spreading. Mol. Biol. Cell 4: 271-281 [Abstract]. |
13. | Clark, E.A., and J.S. Brugge. 1995. Integrins and signal transduction pathways: the road taken. Science. 268: 233-239 |
14. | Cubitt, A.B., R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, and R.Y. Tsien. 1995. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20: 448-455 |
15. | Dartsch, P.C., M. Ritter, D. Haussinger, and F. Lang. 1994. Cytoskeletal reorganization in NIH3T3 fibroblasts expressing the Ras oncogene. Eur. J. Cell Biol 63: 316-325 |
16. |
Diaz-Meco, M.T.,
M.M. Municio,
E. Berra,
S. Frutos,
L. Sanzo, and
J. Moscat.
1994.
Evidence for the in vitro and in vivo interaction of Ras with protein kinase zeta.
J. Biol. Chem.
269:
31706-31710
|
17. | Feig, L., and B. Schaffhausen. 1994. The hunt for Ras targets. Nature. 370: 508-509 |
18. | Felice, G.R., P. Eason, M.V. Nermut, and S. Kellie. 1990. pp60src association with the cytoskeleton induces actin reorganization without affecting polymerization status. Eur. J. Cell Biol 52: 47-59 |
19. | Fincham, V.J., J.A. Wyke, and M.C. Frame. 1995. v-Src-induced degradation of focal adhesion kinase during morphological transformation of chicken embryo fibroblasts. Oncogene. 10: 2247-2252 |
20. | Foster, R., K.Q. Hu, D.A. Shaywitz, and J. Settleman. 1994. p190 rhoGAP, the major rasGAP-associated protein, binds GTP directly. Mol. Cell. Biol 14: 7173-7181 [Abstract]. |
21. | Gomez, J., L. Rodriguez-Borlado, C. Martinez, A. Silva, M. Fresno, A.C. Carrera, C. Eicher-Streiber, and A. Rebello. 1997. IL-2 signaling controls actin reorganization through Rho-like protein family, phosphatidylinositol 3-kinase, and protein kinase C-zeta. J. Immunol 158: 1516-1522 [Abstract]. |
22. | Guan, J.T., J.E. Trevithick, and R.O. Hynes. 1991. Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein. Cell Regul 2: 951-964 |
23. |
Guasch, R.M.,
P. Scambler,
G.E. Jones, and
A.J. Ridley.
1998.
RhoE regulates
actin cytoskeleton organization and cell migration.
Mol. Cell. Biol
18:
4761-4771
|
24. | Hall, A.. 1990. The cellular function of small GTP-binding proteins. Science. 249: 635-640 |
25. | Hildebrand, J.D., M.D. Schaller, and J.T. Parsons. 1993. Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions. J. Cell Biol 23: 993-1005 . |
26. | Hiraoka, K., K. Kaibuchi, S. Ando, T. Musha, K. Takaishio, T. Mizuno, L. Menard, E. Tomhave, and J. Didsbury. 1992. Both stimulatory and inhibitory GDT/GTP exchange proteins, smg GDS and rho GDI, are active on multiple small GTP-binding proteins. Biochem. Biophys. Res. Commun 182: 921-930 |
27. | Horii, Y., J.F. Beeler, K. Sakaguchi, M. Tachibana, and T. Miki. 1994. A novel oncogene, ost, encodes a guanine nucleotide exchange factor that potentially links rho and rac signaling pathways. EMBO (Eur. Mol. Biol. Organ.) J 13: 4776-4786 [Abstract]. |
28. | Hynes, R.O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 69: 11-25 |
29. | Joneson, T., and D. Bar-Sagi. 1997. Ras effectors and their role in mitogenesis and oncogenesis. J. Mol. Med 75: 587-593 |
30. |
Kampfer, S.,
K. Hellbert,
A. Villunger,
W. Doppler,
G. Baier,
H.H. Grunicke, and
F. Überall.
1998.
Transcriptional activation of c-fos by oncogenic Ha-Ras in mouse mammary epithelial cells requires the combined activities of
PKC-![]() ![]() ![]() |
31. | Kang, J.S., and R.S. Krauss. 1996. Ras induces anchorage-independent growth by subverting multiple adhesion regulated cell cycle events. Mol. Cell. Biol 16: 3370-3380 [Abstract]. |
32. | Keenan, C., and D. Kelleher. 1998. Protein kinase C and the cytoskeleton. Cell Signal. 10: 225-232 |
33. | Khosravi-Far, R., P.A. Solski, G.J. Clark, M.S. Kinch, and C.J. Der. 1995. Activation of rac1, rhoA, and mitogen-activated protein kinases is required for ras transformation. Mol. Cell. Biol. 15: 6443-6453 [Abstract]. |
34. |
Kornberg, L.,
H.S. Earp,
J.T. Parsons,
M. Schaller, and
R.L. Juliano.
1992.
Cell
adhesion or integrin clustering increases phosphorylation of a focal adhesion-associated tyrosine kinase.
J. Biol. Chem
267:
23439-23442
|
35. | Kumar, C.C., C. Rogers-Prorock, J. Kelly, Z. Dong, J.-J. Lin, L. Armstrong, H.-F. Kung, M.J. Weber, and A. Afonso. 1995. SCH51344 inhibits Ras transformation by a novel mechanism. Cancer Res. 55: 5106-5117 [Abstract]. |
36. |
Martiny-Baron, G.,
M.G. Kazanietz,
H. Mischak,
P.M. Blumberg,
G. Kochs,
H. Hug,
D. Marme, and
C. Schaechtele.
1993.
Selective inhibition of protein kinase C isoenzymes by the indolocarbazole Goe 6976.
J. Biol. Chem
268:
9194-9197
|
37. | Mellström, K., C.H. Heldin, and B. Westermark. 1988. Induction of circular membrane ruffling on human fibroblasts by platelet-derived growth factor. Exp. Cell Res 177: 347-359 |
38. | Miki, T., C.L. Smith, J.E. Long, A. Eva, and T.P. Fleming. 1993. Oncogene ect-2 is related to regulators of small GTP-binding proteins. Nature. 362: 462-465 |
39. | Mogi, A., M. Hatai, H. Soga, S. Takenoshita, Y. Nagamachi, J. Fujimoto, T. Yamamoto, J. Yokota, and Y. Yaoi. 1995. Possible role of protein kinase C in the regulation of intracellular stability of focal adhesion kinase in mouse 3T3 cells. FEBS (Fed. Eur. Biochem. Soc.) Lett 373: 135-140 . |
40. | Morris, J.D.H., B. Price, A.C. Lloyd, A.J. Self, C.H. Marshall, and A. Hall. 1989. Scrape-loading of Swiss 3T3 cells with Ras protein rapidly activates protein kinase C in the absence of phosphoinositide hydrolysis. Oncogene. 4: 27-31 |
41. | Nobes, C.D., and A. Hall. 1994. Regulation and function of the rho subfamily of small GTPases. Curr. Opin. Genet. Dev 4: 71-81 |
42. | Nobes, C.D., and A. Hall. 1995. Rho, rac, and cdc42 GTPase regulates the assembly of multimolecular focal complexes associated with actin stress fibres, lamellipodia, and filopodia. Cell. 81: 53-62 |
43. |
Nobes, C.D.,
I. Lauritzen,
M.G. Mattei,
A. Hall, and
P. Chardin.
1998.
A new
member of the Rho family, Rnd1, promotes disassembly of actin filament
structures and loss of cell adhesion.
J. Cell. Biol
141:
187-197
|
44. | Olson, M.F., H.F. Paterson, and C.J. Marshall. 1998. Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1. Nature. 394: 295-299 |
45. | Paterson, H.F., A.J. Self, M.D. Garrett, I. Just, K. Aktories, and A. Hall. 1990. Microinjection of recombinant p21rho induces rapid changes in cell morphology. J. Cell Biol. 111: 1001-1007 [Abstract]. |
46. | Prendergast, G.C., and J.B. Gibbs. 1993. Pathways of Ras function: connections to the actin cytoskeleton. Adv. Cancer Res 62: 19-64 |
47. | Prendergast, G.C., R. Khosravi-Far, P.A. Solski, H. Kurzawa, P.F. Lebowitz, and C.J. Der. 1995. Critical role of Rho in cell transformation by oncogenic Ras. Oncogene. 10: 2289-2296 |
48. | Qui, R.G., J. Chen, D. Kim, F. McCormick, and M. Symons. 1995a. An essential role for rac in Ras transformation. Nature. 374: 457-459 |
49. | Qui, R.G., J. Chen, D. Kim, F. McCormick, and M. Symons. 1995b. A role of rho in Ras transformation. Proc. Natl. Acad. Sci. USA. 92: 11781-11785 [Abstract]. |
50. |
Rankin, S., and
E. Rozengurt.
1994.
Platelet-derived growth factor modulation
of focal adhesion kinase p125FAK and paxillin tyrosine phosphorylation in
Swiss 3T3 cells. Bell-shaped dose response and cross-talk with bombesin.
J.
Biol. Chem
269:
704-710
|
51. | Ridley, A.J.. 1995. Rho-related proteins: actin cytoskeleton and cell cycle. Curr. Opin. Genet. Dev 5: 24-30 |
52. | Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 70: 401-410 |
53. | Ridley, A.J., and A. Hall. 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibres in response to growth factors. Cell 70: 389-399 |
54. | Rodriguez-Viciana, P., P.H. Warne, R. Dhand, B. Vanhaesebroeck, I. Gout, M.J. Fry, M.D. Waterfield, and J. Downward. 1994. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370: 527-532 |
55. | Rosales, C., V. O'Brien, L. Kornberg, and R. Juliano. 1995. Signal transduction by cell adhesion receptors. Biochim. Biophys. Acta 1242: 77-98 |
56. | Rosen, A., K.F. Keenan, M. Thelen, A.C. Nairn, and A. Aderem. 1990. Activation of protein kinase C results in the displacement of its myristoylated, alanine-rich substrate from punctate structures in macrophage filopodia. J. Exp. Med 92: 1211-1215 . |
57. | Rubin, E.J., D.M. Gill, P. Boquet, and M.R. Popoff. 1988. Functional modification of a 21 kDa G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum. Mol. Cell. Biol 8: 418-426 |
58. |
Sahai, E.,
A.S. Alberts, and
R. Treisman.
1998.
RhoA effector mutants reveal
distinct effector pathways for cytoskeletal reorganization, SRF activation
and transformation.
EMBO (Eur. Mol. Biol. Organ.) J
17:
1350-1361
|
59. |
Schlaepfer, D.D.,
K.C. Jones, and
T. Hunter.
1998.
Multiple Grb2-mediated integrin-stimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-Src- and focal adhesion kinase-initiated tyrosine
phosphorylation events.
Mol. Cell. Biol
18:
2571-2585
|
60. | Schwartz, M.A., M.D. Schaller, and M.H. Ginsberg. 1995. Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell. Dev. Biol 11: 549-599 . |
61. | Settleman, J., C.F. Albright, L.C. Foster, and R.A. Weinberg. 1992. Association between GTPase activators for Rho and Ras families. Nature. 359: 153-154 |
62. | Shinjo, K., J.G. Koland, M.J. Hart, V. Narasimhan, D.I. Johnson, T. Evans, and R.A. Cerione. 1990. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42. Proc. Natl. Acad. Sci. USA. 87: 9853-9857 [Abstract]. |
63. |
Smith-Sinnett, J.,
I. Zachary,
A.M. Valverde, and
E. Rozengurt.
1993.
Bombesin stimulation of p125 focal adhesion kinase tyrosine phosphorylation.
J.
Biol. Chem
268:
14261-14268
|
64. |
Toullec, D.,
P. Pianetti,
H. Coste,
P. Bellevergue,
T. Grand-Perret,
M. Ajakane,
V. Baudet,
P. Boissin,
E. Brousier,
F. Loriolle,
L. Duhamel,
D. Charon, and
J. Kirilovsky.
1991.
The bisindolylmaleimide GFX 109203 X is a potent and
selective inhibitor of protein kinase C.
J. Biol. Chem.
266:
15771-15781
|
65. |
Überall, F.,
S. Giselbrecht,
K. Hellbert,
F. Fresser,
B. Bauer,
M. Gschwendt,
H.H. Grunicke, and
G. Baier.
1997.
Conventional PKC-![]() ![]() ![]() ![]() |
66. |
Vuori, K., and
E. Ruoslathi.
1993.
Activation of protein kinase C precedes ![]() ![]() |
67. | Wu, C., V.M. Keivens, T.E. O'Toole, J.A. McDonald, and M.H. Ginsberg. 1995. Integrin activation and cytoskeletal interaction are essential for the assembly of fibronectin matrix. Cell. 83: 715-724 |
68. | Yang, N., O. Higuchi, K. Ohasi, K. Nagata, A. Wada, K. Kangawa, E. Nishida, and K. Mizuno. 1998. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature. 393: 809-812 |
69. | Zachary, I., and E. Rozengurt. 1992. Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins and oncogenes. Cell 71: 891-894 |