(Received for publication, April 7, 1995; and in revised form, June 6, 1995 )
From the
Rho family GTPases appear to play an important role in the
regulation of the actin cytoskeleton, but the mechanism of regulation
is unknown. Since phosphoinositide 3-kinase and phosphatidylinositol
4,5-bisphosphate have also been implicated in actin reorganization, we
investigated the possibility that Rho family members interact with
phosphoinositide kinases. We found that both GTP- and GDP-bound Rac1
associate with phosphatidylinositol-4-phosphate 5-kinase in vitro and in vivo. Phosphoinositide 3-kinase also bound to Rac1
and Cdc42Hs, and these interactions were GTP-dependent. Stimulation of
Swiss 3T3 cells with platelet-derived growth factor induced the
association of PI 3-kinase with Rac in immunoprecipitates. PI 3-kinase
activity was also detected in Cdc42 immunoprecipitates from COS7 cells.
These results suggest that phosphoinositide kinases are involved in Rho
family signal transduction pathways and raise the possibility that the
effects of Rho family members on the actin cytoskeleton are mediated in
part by phosphoinositide kinases. The actin cytoskeleton plays a critical role in a number of
cellular processes including motility, chemotaxis, and cell division (1, 2, 3, 4) . To function properly,
these processes require precise spatial and temporal control of actin
filament organization and assembly. Members of the Rho family of small
GTP-binding proteins have been implicated in this regulation. Rac1
appears to mediate growth factor-induced membrane ruffling caused by
actin reorganization at the plasma membrane, and RhoA promotes the
formation of actin stress fibers and focal
adhesions(5, 6) . Cdc42Sc has also been shown to
control actin organization necessary for bud site assembly in Saccharomyces cerevisiae(7, 8) . The
mechanism by which Rho family members mediate their effects on the
actin cytoskeleton is unclear. Two potential biochemical targets for Rho family members have been identified. Manser et al.(9) demonstrated that the tyrosine kinase
p120 Phosphoinositide kinases
and their products have also been implicated in the regulation of the
cytoskeleton. Studies using platelet-derived growth factor (PDGF) Since both Rho family members and
phosphoinositide kinases are involved in the regulation of the actin
cytoskeleton, it is possible that these proteins are acting in the same
signaling pathway. Evidence for a connection between Rho family members
and phosphoinositide kinases is accumulating in the literature. For
instance, Cdc42 has recently been shown to bind and weakly activate PI
3-kinase in vitro(14) . In addition, Rho may regulate
PI 3-kinase in platelet lysates since C3 ADP-ribosyltransferase, a
specific inhibitor of Rho, partially blocks GTP
GST and GST fusion proteins (10 µg of each) were loaded
with nucleotide as described above and then incubated with homogenate
or fibroblast lysate for 2 h at 4 °C with constant rocking. The
beads were washed twice with 1-ml volumes of ice-cold PBS, 1% Nonidet
P-40 and twice with 1 ml of TNM (10 mM Tris-HCl, pH 7.5, 100
mM NaCl, and 5 mM MgCl
Figure 1:
Association of PI kinases with members
of the Rho family of small G proteins. GST fusion proteins of Rac1,
RhoA, Cdc42, or GST only bound to glutathione-Sepharose beads were
loaded with GTP
In contrast to the
association of PtdIns-P kinase with Rac1, PI 3-kinase associated with
both Rac1 and Cdc42 in a GTPdependent fashion (Fig. 1, lanes2 and 6). The presence of PI 3-kinase is
indicated by the synthesis of PtdIns-P
Figure 2:
The Rac1-associated PtdIns-P kinase is a
PtdIns-4-P 5-kinase. GST-Rac1 bound to glutathione-Sepharose beads was
loaded with GTP
Figure 3:
Characterization of the Rac-associated
PtdIns-4-P 5-kinase. GST-Rac1 loaded with GTP
Figure 4:
Association of PtdIns-4-P 5-kinase and PI
3-kinase with Rac1 immunoprecipitates. A, Rac1 was
immunoprecipitated from quiescent (lane2) or
PDGF-stimulated (lane3) Swiss 3T3 cells and assayed
for PtdIns-P (PIP) kinase activity as described under
``Experimental Procedures.'' Immunoprecipitations using
non-immune (NI) serum and Rac1 antibody pretreated with the
peptide antigen were done as negative controls (lanes1 and 4, respectively). PIP
In contrast to the PtdIns-4-P 5-kinase,
no significant PI 3-kinase activity was detected in Rac
immunoprecipitates from serum-starved cells (Fig. 4B, lane2). Addition of PDGF to cells induced the
association of PI 3-kinase with Rac immunoprecipitates (Fig. 4B, lane3). The presence of PI
3-kinase activity was blocked by preincubating Rac antibodies with the
peptide antigen (Fig. 4B, lane4).
Treatment of Rac immunoprecipitates from PDGF-stimulated cells with
wortmannin abolished the formation of PtdIns-P (Fig. 4B,
lane 5), confirming the presence of PI 3-kinase in the
immunoprecipitates. Negligible PI kinase activity was detected in the
control immunoprecipitate with non-immune serum (Fig. 4B, lane1). These results
demonstrate that Rac interacts specifically with both PtdIns-4-P
5-kinase and PI 3-kinase in vivo and that the PI 3-kinase
association is regulated by PDGF. Only a small fraction of the total
PtdIns-4-P 5-kinase and PI 3-kinase in Swiss 3T3 cells is capable of
binding to Rac. At saturating concentrations of GST-Rac, about 0.3% of
the total PtdIns-4-P 5-kinase activity and 0.6% of total PI 3-kinase
activity associate with Rac (not shown). Approximately one-third of the
PtdIns-4-P 5-kinase activity and one-half of the PI 3-kinase activity
that binds to exogenously added GST-Rac can be immunoprecipitated with
antibodies to Rac. The association of only a small fraction of
PtdIns-4-P 5-kinase with Rac suggests that either a minor isoform or a
modified, more prevalent isoform of PtdIns-4-P 5-kinase binds to Rac.
The same may also be true for PI 3-kinase.
Figure 5:
Association of PI 3-kinase with Cdc42
immunoprecipitates. Cdc42 was immunoprecipitated from Cos cell lysates
and analyzed for PI 3-kinase activity (lane2).
Control immunoprecipitates were done with non-immune (NI)
serum (lane1), and Cdc42 antibodies were pretreated
with peptide antigen (lane3). Cdc42
immunoprecipitates were also treated with 100 nM wortmannin (Wort., lane4). PIP,
phosphatidylinositol phosphate.
The finding that Cdc42
immunoprecipitates from COS7 cells contain PI 3-kinase activity in the
absence of stimulation is unexpected given that the interaction between
Cdc42 and PI 3-kinase is GTP-dependent. Since Swiss 3T3 cells and COS7
cells contain an equivalent amount of Cdc42 as judged by immunoblotting
(not shown), these results suggest that Cdc42 is constitutively
activated in COS7 cells. There are several possible explanations for
this: an exchange factor might be activated, a GTPase-activating
protein might be inhibited, or less Cdc42 might be bound to Rho GDP
dissociation inhibitor. We have shown that Rac specifically binds to
a type I PtdIns-4-P 5-kinase and that both Rac and Cdc42 interact with
PI 3-kinase. To our knowledge, this is the first demonstration of
enzymatic activities associated with Rho family members in
vivo. The in vivo association of Rac with PI 3-kinase was
regulated by PDGF, suggesting that this interaction may be important in
the PDGF-induced Rac signaling pathway leading to membrane ruffling. An
obvious implication of our work is that PI 3-kinase is an effector for
Rac and Cdc42. However, we do not detect an effect of Rac on PI
3-kinase activity using either immunoprecipitated or purified PI
3-kinase (data not shown). Additionally, preliminary experiments in
which Rac is injected into wortmannin-treated cells suggest that PI
3-kinase is upstream of Rac.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
complexes specifically with GTP-bound
Cdc42Hs in vitro. Similarly, the serine/threonine kinase
p65
binds to both Cdc42 and Rac1 in a
GTP-dependent manner in vitro(10) . These proteins,
however, have not been shown to associate with Rho family members in vivo, and a connection between these kinases and the actin
cytoskeleton has not yet been established.
(
)receptor mutants and the PI 3-kinase inhibitor
wortmannin have demonstrated that PI 3-kinase is involved in growth
factor-induced membrane ruffling (11, 12, 13) . In addition,
PtdIns-4,5-P
, the product of PtdIns-4-P 5-kinase, has been
shown in vitro to bind actin regulatory proteins such as
profilin and gelsolin and promote actin filament
polymerization(2) .
S stimulation of PI
3-kinase(15) . GTP-bound Rho also appears to enhance
PtdIns-4,5-P
synthesis in fibroblasts(16) .
However, to our knowledge there is no evidence in the literature for
binding of Rho family members to any enzymatic activity in
vivo. We have therefore investigated the possibility that Rho
family members interact specifically with phosphoinositide kinases in vivo and in vitro.
Materials
Antibodies against human Rac1 and
Cdc42 were obtained from Santa Cruz Biotechnology, Inc.
Phosphoinositides and glutathione-Sepharose 4B beads were purchased
from Sigma. [-
P]ATP was purchased from
DuPont NEN.
Preparation and Nucleotide Loading of GST Fusion
Proteins
Glutathione S-transferase fusion proteins of
Rac1, RhoA, and Cdc42 (kindly provided by Dr. Larry Feig, Department
of Biochemistry, Tufts University) were expressed in bacteria and
purified with glutathione-Sepharose beads as described
previously(17) . The fusion proteins were stored in 5 mM HEPES, pH 7.0, 150 mM NaCl, 1 mM DTT with 50%
glycerol at -70 °C. The proteins were determined to be active
based on their ability to bind GTP in a filter binding assay (5) . The fusion proteins were loaded with nucleotide by
incubating proteins in 20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT with a 10-fold
excess of either GTPS or GDP
S for 15 min at 30 °C.
Following incubation, MgCl
was added to a final
concentration of 5 mM.
Association of Phosphoinositide Kinase Activity from Rat
Liver Homogenate and Cell Lysate with Rho Family Members
Rat1
cells maintained in Dulbecco's modified Eagle's medium with
10% fetal calf serum were washed twice with PBS and lysed in lysis
buffer (50 mM Hepes (pH 7.4), 150 mM NaCl, 5 mM MgCl, 1% Nonidet P-40, 10% glycerol, 1 mM DTT, 1 µg/ml each of leupeptin,
[4-(2-aminoethyl)-benzenesulfonyl fluoride, HCl], and
pepstatin, 0.5 mM ZnCl
, and 0.5 mM orthovanadate). Crude cell extract was clarified by centrifugation
(14,000 rpm for 10 min). Rat liver homogenate was prepared essentially
as described(18) . Bovine serum albumin was added to both rat
liver homogenate and fibroblast lysate at a final concentration of 2
mg/ml.
). Lipid kinase
assays were then performed on the beads using PtdIns-4-P or crude brain
phosphoinositides as lipid substrates as described
previously(18) . Lipids were extracted and separated by thin
layer chromatography(19, 20) . Phosphoinositides were
visualized by autoradiography and quantitated using a molecular imager
(Bio-Rad). The effects of spermine and phosphatidic acid on PtdIns-P
kinase activities were assayed as described(21, 22) .
For the determination of the effect of spermine on PtdIns-P kinase
activities, the concentration of MgCl
was decreased to 2
mM.
High Pressure Liquid Chromatography Analysis
The
PtdIns-P kinase associated with GST-Rac1 was assayed as described
above. The product was separated by thin layer chromatography and then
deacylated and analyzed by HPLC as described(18) . Standards
were made from tritiated PtdIns-4-P and PtdIns-4,5-P (DuPont NEN), which were deacylated and included with the
P-labeled product in the HPLC run.
Immunoprecipitation Experiments
For growth factor
stimulation experiments, confluent Swiss 3T3 cells were serum-starved
with Dulbecco's modified Eagle's medium plus 0.1% fetal
calf serum for 24 h and stimulated with 40 ng/ml PDGF for 10 min. Cells
were lysed and clarified as described above, and Cdc42 or Rac was
immunoprecipitated(23) . In some cases, antibodies were
preincubated with 10-fold (by weight) excess peptide antigen for 2 h at
25 °C. The immune complexes were washed twice with 1-ml volumes of
ice-cold PBS, 1% Nonidet P-40 and twice with 1-ml volumes of TNM (10
mM Tris-HCl, pH 7.5, 100 mM NaCl, and 5 mM MgCl). After washes, selected samples were incubated
with 100 nM wortmannin for 20 min at 25 °C. PI kinase
assays were then performed on the immunoprecipitates and analyzed as
described above.
Association of Rho Family Members with Phosphoinositide
Kinases in Vitro
To determine whether phosphoinositide kinases
might directly associate with Rho family GTPases, we used GST fusion
proteins of Rac1, RhoA, and Cdc42 expressed in bacteria. The purified
proteins, bound to GSH beads, were incubated with rat liver cytosol and
assayed for PI kinase activities using crude brain phosphoinositides.
As shown in Fig. 1, a PtdIns-P kinase activity associated with
both the GTP- and GDP-bound forms of Rac1 (lanes2 and 3), resulting in the synthesis of
phosphatidylinositol bisphosphate. A small amount of PtdIns-P kinase
activity also associated with RhoA (lanes4 and 5) and Cdc42 (lanes6 and 7). The
PtdIns-P kinase activity complexed with Rho and Cdc42 was 10- and
15-fold less, respectively, than that associated with Rac, as
quantified by a molecular imager. Negligible activity was found in the
GST control (lane1). Recently, Rho has been shown to
stimulate PtdIns-4,5-P synthesis in fibroblast
cells(16) . Since we do not detect a significant association
between Rho and a PtdIns-P kinase, our results suggest that Rho
stimulates PtdIns-P kinase activity indirectly.
S or GDP
S and then incubated with rat liver
cytosol. The beads were washed and assayed for PI kinase activities
using crude brain phosphoinositides as described under
``Experimental Procedures.''
P-Labeled
phosphoinositides generated by purified PI 3-kinase (PI3K) are
included as standards (lane8). The migration
positions of the phosphoinositide standards are indicated. PIP, phosphatidylinositol phosphate; PIP2,
phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol
trisphosphate.
in the
phosphoinositide kinase assays. Again, a negligible amount of PI
3-kinase bound to GST alone or to RhoA. Similar results were obtained
when the association experiments were done using Rat 1a fibroblast
lysates as the source of PI kinases (data not shown). We also detected
a GTP-dependent association of Rac and Cdc42 with PI 3-kinase by
Western blotting proteins associated with GST fusions of Rac, Rho, and
Cdc42 with p85 antibodies (data not shown).
Characterization of the Rac-associated PtdIns-P
Kinase
The two known isomers of PtdIns-P,
PtdIns-3,4-P
and PtdIns-4,5-P
, are synthesized
by phosphorylation of PtdIns-4-P by PI-3 kinase and PtdIns-4-P
5-kinase, respectively. To determine the type of PtdIns-P kinase that
associates with Rac, the PtdIns-P
product was deacylated
and analyzed by HPLC. The product was identified as PtdIns-4,5-P
based on comigration with a
H-labeled
PtdIns-4,5-P
standard (Fig. 2). The Rac-associated
PtdIns-P kinase is therefore a PtdIns-4-P 5-kinase. At least two forms
of PtdIns-4-P 5-kinase exist in mammalian cells. The best characterized
form is a 53-kDa protein, termed type II, that has been purified to
homogeneity from human red blood cell membranes and cytosol and from
bovine brain cytosol(21, 24, 25) . A second
class, termed type I, is found in most
tissues(21, 22) . The two enzymes can be distinguished
by the ability of spermine and phosphatidic acid to stimulate type I
PtdIns-4-P 5-kinases (but not type II)(21, 22) . When
the effects of these compounds were tested on the Rac-associated
PtdIns-4-P 5-kinase, we found that the PtdIns-4-P 5-kinase was
significantly stimulated by both spermine and phosphatidic acid (17-
and 18-fold, respectively), indicating that it is a member of the type
I family of PtdIns-4-P 5-kinases (Fig. 3).
S and incubated with Rat 1a cell lysate. The
product of the associated PtdIns-P kinase was deacylated and then
analyzed by HPLC. Migration positions of
glycero-PtdIns-3,4-P
, glycero-PtdIns-4,5-P
, and
inositol (Ins)-1,4,5-P
standards are
indicated.
S or GST alone was
incubated with Rat-1a cell lysates, washed, and assayed for PtdIns-P (PIP) kinase activity with or without 2 mM spermine
or 80 µM phosphatidic acid (PA) as described
under ``Experimental Procedures.'' A representative
experiment from three independent experiments is
presented.
Association of Phosphoinositide Kinases with Rac
Immunoprecipitates
To determine whether the complexes that form
between Rac and phosphoinositide kinases in vitro also occur in vivo, Rac1 immunoprecipitates were assayed for PI kinase
activities. Rac immunoprecipitates from serum-starved Swiss 3T3 cells
contained a significant amount of PtdIns-4-P 5-kinase activity (Fig. 4A, lane2). PDGF stimulation
did not noticeably affect this activity (Fig. 4A, lane 3). The associated PtdIns-P kinase was identified as a
type I PtdIns-4-P 5-kinase based on its activation by spermine and
phosphatidic acid, consistent with it being the same enzyme that
complexed with recombinant Rac (data not shown). The presence of the
PtdIns-4-P 5-kinase in Rac immunoprecipitates was blocked by
preincubating Rac antibodies with the peptide antigen, indicating that
the interaction is specific (Fig. 4A, lane4). Negligible PI kinase activity was detected in control
immunoprecipitates with non-immune serum (Fig. 4A, lane1).
,
phosphatidylinositol bisphosphate. B, Rac1 was
immunoprecipitated from quiescent (lane2) and
PDGF-stimulated (lane3) Swiss 3T3 cells and assayed
for associating PI 3-kinase activity. Control immunoprecipitations were
performed as described in A. To confirm the presence of PI
3-kinase, Rac immunoprecipitates from PDGF-stimulated cells were
treated with wortmannin (Wort., lane5).
Association of PI 3-Kinase with Cdc42
Immunoprecipitates
To determine whether a complex between Cdc42
and PI 3-kinase forms in vivo, Cdc42 immunoprecipitates were
assayed for PI 3-kinase activity. Although we detected a small amount
of PI 3-kinase in Cdc42 immunoprecipitates from PDGF-stimulated Swiss
3T3 cells (not shown), a more marked association of PI 3-kinase
activity was found in Cdc42 immunoprecipitates from COS7 cells (Fig. 5, lane2). The presence of PI 3-kinase
activity in Cdc42 immunoprecipitates was blocked by peptide antigen,
indicating that PI 3-kinase specifically associates with Cdc42 (Fig. 5, lane3). Treatment of Cdc42
immunoprecipitates with wortmannin inhibited the synthesis of PtdIns-P (Fig. 5, lane4), confirming that the PI
kinase activity is PI 3-kinase. Negligible PI kinase activity was
detected in the control immunoprecipitate with non-immune serum (Fig. 5, lane1). The amount of PI 3-kinase
that immunoprecipitated with Cdc42 was approximately 0.1% of the total
PI 3-kinase activity in COS7 cells (data not shown). In comparison,
about 0.4% of the total PI 3-kinase activity in COS cell lysate
precipitated with GST-Cdc42.
(
)In light of these
data, it is interesting to speculate that PI 3-kinase may serve to
localize the Rac/PtdIns-4-P 5-kinase complex to growth factor
receptors. Local synthesis of PtdIns-4,5-P
could then
reorient the actin cytoskeleton by binding to actin-binding proteins
such as gelsolin and profilin and promoting actin filament growth as
proposed by Stossel(2) .
S, guanosine
5`-3-O-(thio)triphosphate; GST, glutathione S-transferase; DTT, dithiothreitol; GDP
S, guanyl-5`-yl
thiophosphate; PBS, phosphate-buffered saline; HPLC, high pressure
liquid chromatography.
We thank Larry Feig for providing the Rac, Rho, and
Cdc42 GST fusion proteins.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.