(Received for publication, May 23, 1995; and in revised form, November 7, 1995)
From the
The Ras-related Rho family are involved in controlling
actin-based changes in cell morphology. Microinjection of Rac1, RhoA,
and Cdc42Hs into Swiss 3T3 cells induces pinocytosis and membrane
ruffling, stress fiber formation, and filopodia formation,
respectively. To identify target proteins involved in these signaling
pathways cell extracts immobilized on nitrocellulose have been probed
with [-
P]GTP-labeled Rac1, RhoA, and
Cdc42Hs. We have identified two 55-kDa brain proteins which bind Rac1
but not RhoA or Cdc42Hs. These 55-kDa proteins were abundant, had pI
values of around 5.5, and could be purified by Q-Sepharose
chromatography. The characteristics on two-dimensional gel analysis
suggested the proteins comprised
- and
-tubulin. Indeed,
-tubulin specific antibodies detected one of the purified 55-kDa
proteins. Rac1 bound pure tubulin (purified by cycles of polymerization
and depolymerization) only in the GTP-bound state. The GTPase negative
Rac1 point mutants, G12V and Q61L, did not significantly affect the
ability of Rac1 to interact with tubulin while the
``effector-site'' mutant D38A prevented interaction. These
results suggest that the Rac1-tubulin interaction may play a role in
Rac1 function.
The Ras superfamily of small molecular weight GTP-binding
proteins (p21s) are molecular switches that control a variety of
cellular processes, including growth and proliferation, protein
trafficking, and changes in morphology (Hall, 1990). p21 proteins
possess intrinsic GTPase and GDP/GTP exchange activity which is
affected by regulatory proteins such as n-chimaerin (GTPase
activating protein, GAPs(); Diekmann et al.(1991))
and DBL (GDP/GTP exchange factor; Hart et al.(1991)) and
allows them to cycle between ``on'' (GTP-bound) and
``off'' (GDP-bound) states. Members of the Rho family, Rac1,
RhoA, and Cdc42Hs are implicated in inducing morphological changes
associated with actin polymerization (Hall, 1992). More specifically,
Rac1 induces cortical actin polymerization seen as the process of
membrane ruffling and lamellipodia formation (Ridley et al.,
1992), RhoA induces stress fiber formation possibly by catalyzing the
formation of focal adhesions (Paterson et al., 1990; Ridley
and Hall, 1992), and Cdc42Hs induces the formation of peripheral actin
microspikes and filopodia (Kozma et al., 1995). Rac1 also
activates pinocytosis (Ridley et al., 1992) and superoxide
formation by the neutrophil NADPH oxidase (Abo et al., 1991).
A common theme that runs through all these events is the requirement
for Rho proteins to translocate between the cytosol and the
membrane/cytoskeleton followed by the formation of protein complexes
with specific functions.
Target proteins that act downstream in
these p21 signaling pathways have been isolated. In mammalian cells,
Raf (Moodie et al., 1993; Van Aeist et al., 1993;
Votjek et al., 1993; Zhang et al., 1993; Warne et
al., 1993) and phosphatidylinositol 3`-kinase (Rodriguez-Viciana et al., 1994) are two such proteins that act directly
downstream of Ras in the mitogenic pathway. Cdc42Hs and Rac1-binding
proteins, p120(Manser et al., 1993),
p65
(Manser et al., 1994), and
p67
(Diekmann et al. 1994; Prigmore et al., 1995), have been identified in brain and in
neutrophils by probing cell extracts and recombinant proteins with
[
-
P]GTP-labeled p21 proteins.
p120
and p65
are protein
kinases that may play a role in Rac1 and Cdc42Hs induced morphology
changes while p67
is a component of the
neutrophil NADPH oxidase.
In the present study, we were interested
in isolating binding proteins specific for individual members of the
Rho family. Brain cell extracts were separated on SDS-PAGE, transferred
to nitrocellulose, and probed with
[-
P]GTP-labeled p21 proteins, Rac1, RhoA,
and Cdc42Hs. Two 55-kDa proteins were identified as Rac1-specific
binding proteins and purified by column chromatography. The biochemical
properties of these proteins suggested that they may be
- and
-tubulin and this was confirmed by probing purified tubulin with
Rac1 and using tubulin-specific antibodies. Rac1 interacted with
purified tubulin only in the GTP-bound state and the Rac1
``effector site'' mutation D38A prevented interaction. These
results suggest that the Rac1-tubulin interaction may have
physiological significance.
Figure 1:
Detection of
p21-[-
P]GTP-binding proteins from rat brain
cytosol. The filters were probed with: lane 1, Rac1; lane
2, Cdc42Hs; lane 3, RhoA. Arrow corresponds to
55-kDa Rac1-specific binding proteins. Aliquots containing 100 µg
of protein from rat brain extracts were separated by SDS-PAGE, blotted
onto nitrocellulose, and probed with p21 proteins labeled with
[
-
P]GTP as described under ``Materials
and Methods.''
Figure 2:
Purification of the 55-kDa Rac1-binding
proteins. Lane 1, rat brain cytosol. Lane 2,
flow-thorough. Lanes 3-7, 0.1, 0.2, 0.3, 0.4, and 0.5 M NaCl elutions, respectively. Proteins from rat brain cytosol
were loaded onto a Q-Sepharose column and elution carried out with a
step gradient of NaCl. Fractions were separated by SDS-PAGE and
analyzed by silver staining and
Rac1-[-
P]GTP binding as described under
``Materials and Methods.''
Figure 3:
Two-dimensional SDS-PAGE analysis of the
55-kDa Rac1-binding proteins. 25 µg of rat brain cytosol protein or
5 µg of the purified 55-kDa proteins were resolved by
two-dimensional SDS-PAGE as described under ``Materials and
Methods.'' Gels were analyzed by Coomassie Blue staining (left
panels), Rac1-[-
P]GTP binding (middle panels), and immunoblotting with
-tubulin
specific antibodies (right panels) as indicated on the figure.
Isoelectric focusing (left to right) and molecular
weight separation (top to bottom).
Figure 4:
Rac1 binds to tubulin purified by the
Shelanski method. Lane 1, rat brain cytosol. Lanes 2 and 3, supernatant and pellet from first polymerization. Lanes 4 and 5, pellet and supernatant from second
polymerization. Lane 6, pellet from third polymerization.
Coomassie Blue staining (top) and
Rac1-[-
P]GTP probed (bottom).
Tubulin was purified by 3 cycles of polymerization and depolymerization
(Shelanski et al., 1973), see ``Materials and
Methods'' for details and samples
analyzed.
Figure 5:
Rac1 binds tubulin isolated from liver and
testes. Lane 1, rat brain cytosol. Lanes 2-5,
20, 10, 5, and 2 µg of the 55-kDa purified proteins from rat brain
cytosol. Lane 6, liver proteins eluted in 0.5 M NaCl
fraction. Lane 7, testes proteins eluted in 0.5 M NaCl fractions. Soluble extracts from rat tissues were loaded onto
Q-Sepharose columns and eluted with a step gradient of NaCl. The 0.5 M NaCl fractions were separated by SDS-PAGE and analyzed by
Coomassie Blue staining (top panel),
Rac1-[-
P]GTP binding (middle
panel), and
-tubulin antibody (bottom
panel).
Figure 6:
Specificity of Rac1 binding to rat brain
cytosol and purified tubulin. A, 100 µg of rat brain
cytosol protein (lanes 1, 3, 5, and 7) and 20 µg
of purified rat brain tubulin (lanes 2, 4, 6, and 8)
were separated by SDS-PAGE, blotted onto nitrocellulose, and probed
with Rac1-GTP or GDP. Probes used were as follow: lanes 1 and 2, Rac1-[-
P]GTP. Lanes 3 and 4, Rac1-GDP (produced by allowing
-
P group to be hydrolyzed). Lanes 5 and 6, Rac1-[
-
P]GTP. Lanes 7 and 8, Rac1-[
-
P]GDP. B, 100 µg of rat brain cytosol protein (lanes 1, 3, and 5) and 20 µg of purified rat brain tubulin (lanes 2, 4, and 6) were separated by SDS-PAGE,
blotted onto nitrocellulose, and probed with Rac1 point mutants. Lanes 1 and 2, Rac1-G12V. Lanes 3 and 4, Rac1-Q61L. Lanes 5 and 6, Rac1-D38A. C, 50 µg of tubulin (lanes 1 and 1`) and
tubulin digested with chymotrypsin at 25 °C for 20 min (lanes 2 and 2`). The polypeptides were separated on 12% SDS-PAGE
and analyzed by Coomassie Blue staining (lanes 1 and 2) and Rac1-[
-
P]GTP binding (lanes 1` and 2`).
The Ras point mutations,
G12V, Q61L, and D38A, have been used to determine specificity of
interaction with potential targets. The G12V and Q61L mutants are
GTPase negative and therefore trapped in the on state while D38A does
not interact with Ras effectors such as Raf (Warne et al.,
1993). Similarly, Rac1-G12V and Rac1-Q61L are GTPase negative (see
``Materials and Methods'') and Rac1-D38A does not induce
membrane ruffling ()(Xu et al., 1994) or activate
neutrophil oxidase (Xu et al., 1994; Diekmann et al.,
1994). These Rac1 proteins were loaded with
[
-
P]GTP and used to probe both rat brain
cytosol and purified tubulin (Fig. 6B). The signal for
tubulin was not significantly affected by the GTPase negative mutations
but was eliminated by the D38A mutation. Interestingly, the signal
generated in rat brain cytosol from the PAK proteins was not eliminated
but rather enhanced by the D38A mutation.
To determine the region
binding Rac1 we subjected tubulin to limited proteolysis with
chymotrypsin. This protease cleaves -tubulin specifically (leaving
-tubulin intact) generating two polypeptides of 30 and 16 kDa
(N-terminal and C-terminal, respectively). Rac1 was found to bind to
the intact
-tubulin and the N-terminal polypeptide of
-tubulin but not its C-terminal (Fig. 6C).
Figure 7:
Localization of Rac1 in Swiss 3T3 cells.
Swiss 3T3 cells were stained with either anti--tubulin antibodies (A) or anti-Rac1 antibodies (B) as described under
``Materials and Methods.''
Figure 8:
Effect of tubulin on the Rac1-p190
interaction. Rac1-GTPase activity, with no additions (), with 4
µg of tubulin (
), and with 10 µg of tubulin (
).
Rac1-GTPase activity with 25 ng of p190 GAP domain in the absence
(
) or presence of 4 µg of tubulin (
). Results are
expressed as a % of bound GTP (cpm) present at zero time. Rac1 was
loaded [
-
P]GTP as described under
``Materials and Methods'' and GTP hydrolysis followed by
filtration on nitrocellulose. Similar results were obtained in one
other experiment.
Figure 9:
Effect of Rac1 on tubulin polymerization.
Tubulin (approximately 36 µg), with no additions (), with 5
µg of Rac1 (
), with 10 µM taxol with GTP (
)
and 10 µM taxol with 5 µg of Rac1-GTP (
).
Tubulin polymerization was induced with taxol and the optical density
followed as described under ``Materials and
Methods.''
Rac1 is an activator of
membrane ruffling and pinocytosis in Swiss 3T3 cells (Ridley et
al., 1992). Translocation of Rac1 between the
membrane/cytoskeleton and cytosol is likely to be required for Rac1 to
activate these events. It is therefore possible that the function of
the Rac1-tubulin interaction may be to allow Rac1 to use the
microtubule network for the process of translocation. To examine this
possibility we treated Swiss 3T3 cells with colchicine to disrupt the
microtubule network and then monitored pinocytosis by lucifer yellow
uptake and membrane ruffling by phalloidin staining of actin. In serum
starved control cells (without colchicine treatment) fetal calf serum
stimulated pinocytosis seen by diffuse cytosolic lucifer yellow
staining. Interestingly, lucifer yellow uptake in cells treated with
colchicine was very different. Almost all cells contained intense
particulate staining throughout the cytoplasm (data not shown).
Furthermore, colchicine-treated cells did not undergo membrane ruffling
on stimulation with fetal calf serum, platelet-derived growth factor,
or phorbol myristyl acetate. ()
Growth factors activate Rac1, RhoA, and Cdc42Hs to induce
specific changes in the actin-based cell morphology of mammalian cells.
Platelet-derived growth factor induces Rac1-mediated membrane ruffling
and lamellipodia formation (Ridley et al., 1992),
lysophosphatidic acid induces RhoA-mediated stress fiber formation
(Ridley and Hall, 1992) and bradykinin induces Cdc42Hs-mediated
peripheral actin microspike formation and filopodia formation (Kozma et al., 1995). The mechanism(s) by which these factor-induced
changes in morphology involving Rho family proteins occurs is unclear.
As one step in elucidation of the p21 mechanism of action, proteins
have been isolated that interact directly with Rac1 and Cdc42Hs and
therefore may be components of these morphological pathways.
p120 and p65
are protein kinases that bind
to Cdc42Hs and Rac1/Cdc42Hs, respectively (Manser et al.,
1993, 1994). Rac1 and Cdc42Hs also bind to p67
(Diekmann et al., 1994; Prigmore et al., 1995).
In the
present study we have identified two 55-kDa proteins from brain that
bind Rac1 but not RhoA or Cdc42Hs. These are the first Rac1-specific
binding proteins to be isolated. The 55-kDa proteins were shown to be
tubulin by using -tubulin-specific antibodies to probe purified
protein on two-dimensional SDS-PAGE. Rac1 bound
- and
-tubulin, tubulin purified from brain, liver, testes, and spleen,
and tubulin purified by cycles of polymerization and depolymerization.
The characteristics of this binding were as follows: (i) Rac1 bound
tubulin only in its GTP-bound form, (ii) the strength of Rac1 binding
to tubulin (cpm/mg protein) was similar to that seen with Rac1 binding
to the NADPH oxidase component p67
, (iii) the GTPase
negative mutations G12V or Q61L did not significantly affect binding
while the effector site mutation D38A eliminated binding, and (iv) Rac1
bound to the N-terminal (30 kDa) of
-tubulin. Furthermore,
anti-Rac1 antibodies stained microtubules indicating that Rac1 can
interact with both tubulin monomers and polymers. Taken together, these
results show that the interaction between Rac1 and tubulin is specific
and that it may have physiological relevance.
Rac1-D38A does not
induce membrane ruffling (Xu et al., 1994) or activate the
neutrophil oxidase (Diekmann et al., 1994; Xu et al.,
1994) and Cdc42Hs-D38A does not induce peripheral actin microspike
formation, however, both D38A mutants bound the PAK proteins from cell
extracts and recombinant p65. (
)This result
makes it unlikely that the PAK proteins are the sole downstream targets
of Rac1 and Cdc42Hs responsible for the changes in actin-based cell
morphology.
Tubulin does not contain any regions of obvious sequence
identity to the known Rac1 binding proteins: p65,
p67
, the Bcr family of GAPs, or the Rho-GDP dissociation
inhibitor, suggesting the existence of a unique binding domain. Unlike
the other Rac1 interacting proteins tubulin did not affect the GTPase
activity of Rac1 or compete with GAP proteins (p190 was used here) for
interaction with Rac1. This implies that p190 and tubulin can bind to
Rac1 simultaneously and this possibility is supported by the fact that
the D38A mutation, while eliminating tubulin binding, does not affect
the ability of Rac to act as a substrate for p190 GAP domain (Xu et
al., 1994). However, we cannot rule out the possibility that the
binding constants for interaction with Rac1 of tubulin and p190 GAP
domain are such that competition would be difficult to observe. In
preliminary experiments, the K
of Rac1 binding
tubulin was found to be in the micromolar range making the latter
possibility unlikely. Further work will be necessary to resolve this
issue.
What are the physiological implications of Rac1 binding to
tubulin? Rac1 bound to the N-terminal 30 kDa of tubulin while proteins
such as tau and the microtubule-associated proteins, which stimulate
tubulin polymerization, bind to the C-terminal. It is therefore
unlikely that the role of the Rac1-tubulin interaction is to affect
tubulin polymerization (microtubule formation). Indeed, Rac1 did not
influence taxol induced polymerization. The growth factor mediated
induction of membrane ruffling and pinocytosis in Swiss 3T3 cells is
rapid and probably requires Rac1 to translocate between cytosol and
plasma membrane (Ridley et al., 1992). Additionally,
stimulation of neutrophils leads to a rapid translocation of Rac (Benna et al., 1994) from the cytosol to the membrane and
cytoskeleton where components (p67, p47
,
p40
, and cytochrome b) of the NADPH oxidase
form a complex which is capable of generating superoxide. Thus,
cellular translocation is likely to be a key event in Rac1 function. It
is therefore possible that tubulin binding allows Rac1 to use the
microtubule network for translocation. This possibility was tested by
examining the effect of colchicine (and nocadazole) on pinocytosis and
membrane ruffling. Disruption of the microtubule network with these
drugs affected factor induced pinocytosis and inhibited membrane
ruffling.
Two recent studies also support a role for
microtubules in Rac1 function. Gloushankova et al.(1994) have
observed that lamellipodia formation induced by transfection of
N-ras, possibly via activation of Rac1 (Ridley et
al., 1992), in IAR epitheliocytes is inhibited by the microtubule
network disrupting agents taxol and colcemid. Tanaka et
al.(1995) have found that disruption of the microtubule network of
neural growth cones with vinblastine leads to a loss of lamellipodia
and ruffled edges while leaving filopodia intact. On washing out
vinblastine from these neurons lamellipodia and ruffled edges
re-emerge. Further work examining cellular movement of Rac1 upon factor
stimulation in the presence/absence of colchicine is needed to
establish whether microtubules are required for Rac1 function.