From the Department of Biochemistry and Molecular
Biology and the § Walther Oncology Center, ¶ Division
of Nephrology, Indiana University School of Medicine,
Indianapolis, Indiana 46202
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ABSTRACT |
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Rho family GTPases regulate multiple cellular
processes, including cytoskeletal organization, gene expression, and
transformation. These effects are achieved through the interaction of
GTP-bound proteins with various downstream targets. A series of
RhoA/Rac1 and Rho/Ras chimeras was generated to map the domain(s) of
RhoA involved in its association with two classes of effector kinase, represented by PRK2 and ROCK-I. Although the switch 1 domain was required for effector binding, the N terminus of Rho (residues 1-75)
was interchangeable with that of Rac. This suggested that the region of
Rho that confers effector binding specificity lay further C-terminal.
Subsequent studies indicated that the "insert domain"(residues
123-137), a region unique to Rho family GTPases, is not the
specificity determinant. However, a determinant for effector binding
was identified between Rho residues 75-92. Rac to Rho point mutations
(V85D or A88D) within loop 6 of Rac promoted its association with PRK2
and ROCK, whereas the reciprocal Rho(D87V/D90A) double mutant
significantly reduced effector binding capacity. In vivo
studies showed that microinjection of Rac(Q6IL/V85D/A88D) but not
Rac(Q6IL) induced stress fiber formation in LLC-PK epithelial cells,
suggesting that loop 6 residues conferred the ability of Rac to
activate ROCK. On the other hand, the reciprocal Rho (Q6IL/D87V/D90A) mutant was defective in its ability to transform NIH 3T3 cells. These
data suggest that although Rho effectors can utilize a Rho or Rac
switch 1 domain to sense the GTP-bound state of Rho, unique residues
within loop 6 are essential for determining both effector binding
specificity and cellular function.
The Ras superfamily of GTPases is involved in the regulation of
multiple cellular processes, including signal transduction to induce
growth and differentiation (Ras), membrane trafficking (Rab), nuclear
transport (Ran), and cytoskeletal regulation (Rho) (1-3). The best
characterized members of the Rho family of GTPases are RhoA, Rac 1, and
CDC42Hs, which regulate stress fiber formation, lamellipodia formation
and membrane ruffling, and filopodia formation, respectively (2). Rac
and CDC42 also regulate transcription via activation of the JNK and p38
mitogen-activated protein kinase cascades, whereas all three Rho family
members can activate the serum response factor (4, 5) and promote cell
cycle progression (2, 6).
Ras superfamily proteins act as molecular switches, alternating between
inactive GDP-bound and active GTP-bound states. GTP binding induces a
conformational change in two domains of Ras, referred to as switch 1 and switch 2 (7). In the case of Ha-Ras, effector protein binding and
activation occurs primarily through association with the switch 1 domain located in loop 2 and Studies by Hall and colleagues (16, 17) indicated that in contrast to
Ras, the effector-binding domain of Rho extends beyond switch 1. For
example, a Rac73Rho chimera, lacking Rho N-terminal sequences could
still induce stress fiber formation (16). Therefore, we set out to
determine which regions of Rho are involved in its interaction with the
downstream effectors PRK2 and p160 ROCK (18). PRK2 is a
serine/threonine protein kinase isolated by us and others (5, 19-22)
that shares a Rho binding domain,
HR1,1 with PRK1/PKN, PRK3,
Rhotekin, and Rhophilin (see Ref. 18 and Fig. 2). The exact function of
these proteins is currently unknown. Meanwhile, the Rho kinases
(ROK Using Rho/Rac chimeras, we demonstrate here that multiple domains of
Rho are involved in effector binding. Although switch 1 domain residues
common to both Rho and Rac are required for binding Rho-effectors,
unique residues in loop 6 of Rho confer specific interaction with Rho
targets. In vivo data demonstrated that loop 6 of Rho not
only distinguishes between Rho and Rac effectors but also is required
for their physiological activation. Deletion of the Rho insert domain
(residues 123-137) severely disrupted Rho-induced transformation but
did not significantly disrupt PRK2 or ROCK-I binding to Rho in
vitro. The loss of transformation resulting from insert domain
deletion may have been due to requirement of the insert helix for
activation of an additional Rho target. However, we cannot rule out the
possibility that its deletion resulted in disruption of the adjacent
loop 6 structure.
Materials--
[ Construction of Plasmids--
All the Rho mutants and Rho/Rac
chimera constructs used in this study are summarized in Fig.
1. Rho and Rac mutants are offset by two
codons due to the longer N terminus of RhoA. The
Rac(Val12)143Rho, Rac73Rho143Rac, and Rho(G14V/T37A)
constructs have been described previously (16) and were provided by A. Hall (University College, London, United Kingdom). Rho(F39A),
Rho(D87V/D90A), Rac(V85D/A88D), Rac(V85D), and Rac(A88D) mutants were
generated by site-directed mutagenesis using PCR. Rho122Rac139Rho
(Rho Expression of GST Fusion Proteins--
GST-fused Rho GTPases
were expressed in the BL21(DE3)lysE strain of Escherichia
coli. Following a 3-h induction with 0.2 mM isopropyl-1-thio- Rho Overlay Assay--
Bead-bound Rho proteins were loaded with
[ Rho Precipitation Assay--
The GTP binding capacity of each
Rho mutant was determined as above, using [ PRK2 Precipitation Assay--
COS cells were transfected with
pCMV2-FLAG-Prk2 using LipofectAMINE (Life Technologies, Inc.). After 2 days, cells were washed three times with cold PBS and lysed in 20 mM Tris, pH 7.4, 50 mM NaCl, 1 mM
EDTA, 0.5% Nonidet P-40, 10% glycerol, 1 mM
dithiothreitol, 1.9 mg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride. 2 µg of active bead-bound GTPases was
preloaded with GTP Exchange Assay--
WT RhoA was preloaded with
[ Microinjection and Fluorescence Microscopy--
LLC-PK cells
were maintained in 1:1 Dulbecco's modified Eagle's medium/Ham's F-12
medium supplemented with 10% fetal calf serum. For microinjection
experiments, cells were plated on coverslips and grown to >90%
confluence. Cells were then co-microinjected with the indicated Rac
proteins (60 µg/ml), botulinum exoenzyme C3 (10 µg/ml), and
fluoroscein-conjugated dextran as
described.2 C3 exoenzyme was
also prepared as described.2 After 45 min, cells were fixed
and stained with rhodamine-conjugated phalloidin, and F-actin was
visualized using a Bio-Rad MRC 1024 confocal microscope.
NIH 3T3 Cell Transfection--
For transformation assays, NIH
3T3 cells were transfected with 2 µg of empty pZIP vector or vector
encoding Rho(Q63L), Rho(Q63L/D87V/D90A) or Rho The HR1a Domain of PRK2 Is Sufficient for Its Interaction with
Rho--
PRK2 and the related kinase PKN/PRK1 possess three tandem
copies of a 60-70-amino acid sequence at their N terminus that has been referred to as homology region 1 (HR1) (Ref. 19 and Fig. 2a). Single HR1 units are also
present in other Rho-binding proteins (Rhophilin and Rhotekin) and
appear to represent a common Rho binding motif (18). Another
Rho-binding motif, RBD (Fig. 2b), that is present in ROCK
does not share significant primary sequence homology with HR1 (23).
Both domains, however, are highly charged and predicted to contain
significant The Switch 1 Domain of Rho Influences Effector
Binding--
Introduction of residues from Rho into the switch 1 domain of Rac has previously been shown to disrupt various Rac
functions (12). However, we found that a Rac73Rho chimera (containing residues 1-73 of Rac1) still bound to HR1 and RBD, suggesting that
either a Rac or a Rho switch 1 domain could support Rho-effector interaction (see Fig.
3). In
contrast, introduction of a Rho T37A mutation into Rho disrupted its
ability to bind the PRK2-HR1 domain. A similar observation was
previously reported for PKN (30). However, this mutation may disrupt
the interaction of Thr37 with Mg2+ and the
The Insert Domain Is Not Required for Rho Effector
Interaction--
The highly charged insert domain has been postulated
to be an effector binding determinant and has been reported to be
required for Rac-induced activation of the neutrophil NADPH oxidase
(15, 32, 33). Because the HR1 and RBD sequences are also highly charged
and could possibly associate with the insert domain via ionic
interaction, we exchanged the entire insert domain of Rho with that of
Rac. As seen in Fig. 4, the
Rho123Rac138Rho chimera (Rho
Although introduction of the Rac insert domain did not lead to loss of
Rho-effector binding, this sequence possesses a very similar
conformation and charge distribution to that of Rho, having highly
charged residues on the outer surface of the helix and long-chain
hydrophobic residues on the inner side facing loop 6 (34, 35). So, just
as residues 1-73 of Rac could replace residues 1-75 of Rho, it was
possible that the insert domains may also be interchangeable. To
determine whether the insert domain is involved in Rho-effector
binding, we removed this region, replacing it with the corresponding
sequence from loop 8 of Ras. This swap was equivalent to that
previously made in CDC42 by McCallum et al. (36), which also
removes prolines 138 and 141 to minimize disruption of the global
structure. This Rho Rac/Rho Chimeras Define a Novel Effector-binding Domain in
RhoA--
To identify regions of Rho outside of the classic switch
domains that confer effector binding specificity, we used an additional series of Rho/Rac chimeras. Although a Rac143Rho chimera was
ineffective, Rac73Rho143Rac still bound to both HR1 and RBD (Fig. 4).
This suggested that a region between residues 75 and 143 of Rho is sufficient for effector binding. This notion is supported by the observation that Rac73Rho143Rac induced stress fiber formation in Swiss
3T3 cells as effectively as Rho (16). The significant decrease in
binding of HR1 and RBD to Rac73Rho143Rac, as with Rho Rac Residue 78 Weakly Influences Rho-Effector
Binding--
Although we previously found no interaction between
full-length PRK2 and Rac1-GTP in precipitation experiments (5), it has
been reported that Rac can weakly bind to this kinase (20). However,
this binding discrepancy may have been due to the use of a Rac F78S
mutant (34) in the latter study. Because residue 78 is located within
the residue 76-92 region defined above, we compared the ability of
Rac(WT) and Rac(F78S) to bind to Rho effectors. As shown in Fig. 5,
b and c, the Rac(F78S) mutant bound, albeit weakly, to PRK2-HR1 and ROCK-RBD under conditions where Rac(WT) did
not. Although this may help resolve differences in the literature regarding Rho-effector binding specificity (5, 12, 13, 20, 29, 37), the
weak signal suggested that other residues within the 76-92 region must
contribute to PRK2 and ROCK binding.
Loop 6 Confers Rho Protein Effector Binding Specificity--
A
region spanning residues 74-90 has previously been shown to be
required for Rac binding to the Rac GAP, BCR (16). Therefore we next
looked at the ability of a Rac73Rho90Rac chimera to bind to HR1a and
RBD. Introducing residues 76-92 of Rho into Rac conferred HR1 and RBD
binding capability on Rac (Fig. 5a). The reciprocal Rho75Rac92Rho chimera was unstable, making it difficult to assess its
effector binding properties.
Comparison of Rho residues 76-92 with the equivalent sequence of Rac
(residues 74-90) indicated several differences (Fig. 6a). Most notably, the
negatively charged Asp residues 87 and 90 of RhoA loop 6 were
hydrophobic Val and Ala residues in Rac. Because loop 6 is a surface
exposed region of Rho, it represents a potential second
effector-binding site. Therefore, we swapped these loop 6 residues
between Rac and Rho. Introduction of a V85D/A88D double mutation into
Rac (RacDD) conferred strong HR1 and RBD binding activity (Fig.
6b). Furthermore, the introduction of the reciprocal
(D87V/D90A) mutation into loop 6 of Rho (RhoVA) resulted in a partial
loss of binding to RBD and almost complete loss of binding to HR1 (Fig.
6b). This data suggested that loop 6 is at least one of the
determinants of Rho-effector binding specificity. To establish whether
a single Asp residue was responsible for effector binding, the residue
85 and 88 mutations were introduced individually into Rac. Both of
these mutants were capable of conferring HR1 and RBD binding, although
less effectively than RacDD. Interestingly, the RhoVA mutant bound to
RBD more effectively than to HR1 (Fig. 6b). A similar
binding difference was also observed with Rho75Rac (Fig.
5a), suggesting that RBD was less dependent on loop 6 for Rho binding.
To confirm that the binding pattern observed with isolated Rho binding
domains reflected interactions of the full-length proteins, we
expressed FLAG-tagged PRK2 in COS cells as described previously (5) and
used GST-Rho/Rac proteins to precipitate it from cell lysates.
Consistent with the isolated HR1 data, PRK2 bound to all Rho/Rac
chimera containing the Rho loop 6 region (Fig. 7).
HR1a Inhibits Rho Guanine Nucleotide Exchange--
If Rho
effectors interact with loop 6 of Rho, they will occupy the groove
created between the switch 1 and insert domains (Fig.
8), occluding the GTP-binding pocket.
Consequently, effector binding would reduce the GTP exchange rate of
Rho. To test this hypothesis, we measured the rate of
[ Loop 6 of Rho Is Required for Stress Fiber Formation and Cellular
Transformation--
Although the physiological role of PRK2 is not yet
certain, it is well established that ROCK mediates Rho-induced stress
fiber formation (2, 23, 38). Therefore, to determine whether the
interaction of Rho loop 6 with RBD resulted in ROCK activation, serum-starved LLC-PK epithelial cells were microinjected with activated
Rac(Q61L) or Rac(Q61L)DD, and stress fiber formation was determined.
Cells were co-injected with botulinum exoenzyme C3 to prevent any
secondary activation of endogenous Rho by Rac. Microinjection of
Rac(Q61L)DD resulted in the appearance of numerous actin stress fibers
(Fig. 10). In contrast, Rac(Q61L) was
only capable of inducing membrane ruffles. This suggested that the addition of two Rho loop 6 residues into Rac enabled it to function as
Rho to activate ROCK, leading to stress fiber formation in vivo. Due to the low yield of GST-Rho(Q63L)VA in E. coli, it was not possible to determine whether mutation of loop 6 of Rho would prevent its activation of ROCK. However, if loop 6 of Rho
is required for effector activation, then the DD Rac Residue 61 and Rho Residue 63 also Influence Rho-Effector
Binding--
At the outset of these studies we used Rac(Q61L) and
Rho(Q63L) activating mutations equivalent to those found in activated Ras (39) because these proteins bound to Rho effectors more efficiently
than WT Rac and Rho. However, Rac(Q61L)-GTP with no additional
mutations was found to bind to both Rho effectors (Figs. 5 and
12). It was also found that Rac(Q61L,
V85D)-GDP and Rac(Q61L, A88D)-GDP bound to HR1 very efficiently in
contrast to their wild type counterparts (Fig. 12). Similarly, a
Rho(Q63L) mutant compensated for the loss of binding of RhoVA to HR1.
However, G12V mutations, equivalent to those found in oncogenic Ras,
bound to effectors less efficiently than WT
mutants/chimeras.3 This suggested that Rac- and
Rho-effector interactions are best assessed using a WT protein
background. Although Rac(Q61L) interacted with the ROCK-RBD in
vitro (Fig. 5c), it did not induce stress fiber
formation after microinjection into the LLC-PK cells (Fig. 10). This
suggested that additional sequences, within loop 6, were required for
activation of ROCK in vivo. However, we have not ruled out
the possibility that Rho interacts differently with the isolated RBD
in vitro than with full-length ROCK in vivo.
It has been well established that the key molecular determinants
for Ras-effector protein binding are the switch 1 domain (residues
32-40) and surrounding sequences, together with the switch 2 domain
(for review, see Ref. 3). The switch 1 domain is also important for
dictating Rac-effector specificity because Rac to Rho point mutations
in the extended effector domain (12) or mutation of the core switch 1 region (13, 14) selectively disrupted signaling to various downstream
effectors. However, a complete swap of residues 1-75 of Rho for Rac
residues 1-73 did not disrupt the ability of Rho to induce stress
fiber and focal adhesion complex formation in Swiss 3T3 cells (16). In the present study, we set out to determine what sequences in Rho are
required to confer effector binding specificity to two protein kinases
that contain distinct Rho-binding domains (18). We found that Rac
residues 1-73 could replace the N terminus of Rho for binding to both
PRK2-HR1 and ROCK-RBD. In contrast, mutating switch 1 residues that are
conserved between Rho and Rac, e.g. Thr37 and
Phe39 (this study), Tyr34 (40),
Val38, Asp40/Glu40, and
Tyr42 (41) disrupted Rho-effector binding. Therefore, it
appears that whereas Rac relies on unique N-terminal residues for
effector interaction, Rho utilizes the residues that it shares with Rac for this purpose. Although PRK2 and ROCK must interact with the switch
1 domain to sense whether the GTPase is in its active GTP-bound state,
an additional region(s) in the C-terminal two-thirds of Rho must be
required to confer its effector binding specificity. Because residues
1-75 encompass both switch 1 and 2, the specificity determinant
appears to reside in a region of Rho that does not change conformation
upon GTP binding. This is consistent with the ability of Rho-GDP to,
albeit weakly, bind to PRK1 and PRK2 in vitro (5, 20,
28).
In addition to the switch 1 domain, a region located between residues
143 and 175 of Rac is essential for its association with the NADPH
oxidase component, p67 (16). The insert domain, a 13-amino acid
sequence that replaces loop 8 of Ras with an amphipathic helix, may
also be required for Rac-induced oxidase activation (15, 32, 33, 42).
Furthermore, a region encompassing residues 74-90 has been shown to be
required for Rac to interact with the GAP domain of BCR (16). Using a
series of Rho/Rac chimeras, we found that sequences C-terminal to
residue 92 were not required for HR1 or RBD interaction. However,
introduction of Rho residues 76-92 into Rac conferred on it
Rho-effector binding ability. A similar observation was made by
Fujisawa et al. (43) while the present report was under
review. This sequence encompasses loop 5, All Rho/Rac chimeras that lacked an intact Rho loop 6 were unable to
associate with HR1/HR1a, indicating that loop 6 is indispensable for
PRK2 binding. However, the Rho75Rac (Fig. 5a) and RhoVA
(Fig. 6b) chimeras, which do not contain Rho loop 6 sequences, still weakly bound to RBD. This suggests that loop 6 is not
as critical a binding determinant for RBD. Nonetheless, loop 6 must
contribute to RBD binding because the V85D and A88D mutations result in
a gain of Rac binding and also confer upon Rac the ability to induce stress fiber formation in LLC-PK cells.
Loop 6 and switch 1 are on the same molecular surface of RhoA. It
therefore seemed likely that these two loops cooperated to promote
effector binding. A large, negatively charged groove is located between
the insert and switch 1 domains of Rho-GTP (Fig. 8) that includes
aspartate residues 13, 87, 90, and 124 (35, 44). This groove could
accommodate a positively charged helix present in HR1 or RBD (which are
highly charged and predicted to have considerable During the completion of this study, it was reported that loop 6 of Rho
also confers sensitivity to p190 Rho GAP (46). Introduction of Rho
residue Asp90 into CDC42 resulted in a gain of Rho GAP
sensitivity, whereas the reciprocal swap of residue Ser88
from CDC42 into Rho reduced its responsiveness to p190 (46). Therefore,
similarly to Ras, it appears that Rho GAPs and effector proteins
interact with common domains of their cognate GTPases. In contrast, Bae
et al. (40) reported that mutation of the Asp residues in
loop 6 to the equivalent CDC42 residues did not disrupt the ability of
Rho to activate phospholipase D (40). Instead, a D76Q mutation in loop
5 partially attenuated choline generation. There is no apparent
homology between phospholipase D and the HR1 or RBD domains, suggesting
that there are at least three Rho-binding structures.
It is not clear how the F78S mutation in The insert domain of Rac is not required for binding to the phagocyte
NADPH oxidase component p67, but its presence may be essential for
oxidase activation, possibly by interacting with cytochrome
b558 (15, 32, 33). Additionally it has been proposed that
although switch 1 might dictate GTP- versus
GDP-dependent binding of effectors, the insert domain might
be responsible for conferring effector specificity (35, 47). Although
swapping the RhoA insert domain with Ras loop 8 resulted in partial
loss of effector binding, it did not impair binding to full-length PRK2. Furthermore, exchanging the Rho insert domain for that of Rac
(Rho The recently solved solution structure of CDC42-GDP revealed a close
contact between residues 120-129 (the insert helix) and residues
84-89 (loop 6) of CDC42 (47). It was also reported that a hydrophobic
patch is formed between loop 6 (Pro89) and the insert domain (residues
127, 131, and 138) of Rho (44). It is therefore possible that deletion
of the insert domain may destabilize the conformation of loop 6, making
it unable to effectively interact with and/or activate Rho effectors.
Alternatively, the insert domain may mediate the association of Rho-GTP
with a novel effector(s) that is essential for certain Rho functions.
Deletion of the insert domain of CDC42 in a similar manner to our
Rho Our initial interpretation of data was complicated by the use of
activating Leu61/Leu63 mutants. For example,
Rac(Q61L) was found to bind to Rho effectors even in the absence of Rho
sequences, whereas a Rho(Q63L) mutation compensated for the loss of
binding by RhoVA. Q61L mutants have previously been shown in various
Ras family GTPases to have higher affinity for their targets and to
functionally override the effects of other mutations. For example, 1)
Ras(Q61L) has a significantly higher affinity for p120 GAP than Ras(WT)
or Ras(G12V) (49); 2) a Rac(D38A, Q61L) double mutant restored the
ability of a Rac(D38A) effector-binding domain mutant to induce NADPH
oxidase activation and membrane ruffling (50); 3) Ras(Q61L) has also
been reported to bind to Raf even in a GDP-bound state (51), suggesting
that this mutation influences the global conformation of Ras, thus leading to altered effector binding properties. Therefore caution should be taken in interpreting Rho interaction data, and if possible, wild type proteins should be used in in vitro assays. It
should be noted that the presence of a Rac(Q61L) mutation alone did not appear to result in ROCK-induced stress fiber formation in
vivo despite binding to the isolated RBD domain.
In summary, the interaction of Rho with its downstream effectors, PRK2
and ROCK, appears to involve multiple binding sites. Sequences
conserved between the switch 1 domains of Rac and Rho rather than their
unique residues contribute to the binding of Rho-GTP to both PRK2 and
ROCK-I. However, unique residues in loop 6 of Rho enable these
effectors to distinguish it from other Rho family GTPases. The insert
domain may provide an additional feature for recognition of other,
unknown, effectors. These domains are in close proximity in the Rho
three-dimensional structure and may represent the effector-binding
surface (Fig. 8). Therefore, we propose that upon GTP binding, the
switch 1 domain moves closer to loop 6, enabling effectors to bind
concomitantly with both domains. Upon GTP hydrolysis, the cooperation
between switch 1 and loop 6 is lost, resulting in only low affinity,
GDP-dependent effector binding. An alternative hypothesis
is that the negatively charged residues in loop 6 induce the switch
domains of Rac or Rho to adopt a Rho-like conformation to promote
specific interact with Rho targets. In both models, Rho loop 6 is the
key effector binding specificity determinant. Because PRK2 and ROCK-I
both bound to the same Rho-Rac chimeras, it appears that both classes of Rho-binding domain have evolved to recognize similar molecular determinants. Stronger binding of RBD versus HR1 to Rho loop
6 mutants suggests that Rho-ROCK-I interaction relies less heavily on
loop 6. However, because Rac(Leu61)DD could induce stress
fiber formation, it appears that an interaction with Rho loop 6 is
required to promote ROCK activation. A more detailed picture of
Rho-effector interaction awaits the determination of a co-crystallized structure.
INTRODUCTION
Top
Abstract
Introduction
References
-strand 2 (residues 32-40) (3).
Indeed, point mutations within this region of Ras can differentially
disrupt its interaction with various downstream targets (8-11).
Mutations in the Rac switch 1 domain similarly perturb specific
effector protein interactions (12-14). However, additional regions of
Rac have been implicated in effector binding and/or activation. These
include an
-helix located between residues 123-137 that is unique
to Rho family members (henceforth referred to as the "insert
domain") and a region located between residues 143-175 (15, 16).
/ROCK-II/Rho-kinase and ROK
/ROCK-I/p160 ROCK) that mediate
Rho-induced stress fiber formation possess a distinct Rho-binding
structure (23), referred to herein as RBD. Although HR1 and RBD do not
share primary sequence homology, they are both highly charged and are
predicted to contain significant
-helical content. It was therefore
of interest to determine whether or not the same domains of Rho
recognized both classes of effector-binding motif.
EXPERIMENTAL PROCEDURES
-32P]GTP (3,000 Ci/mol) was
purchased from Amersham Pharmacia Biotech. Vent DNA polymerase and all
restriction enzymes were purchased from New England Biolabs. PCR
cloning vector pCR Blunt was from Invitrogen. M2-FLAG and hemaglutinin
antibodies were from Sigma and Berkeley Antibody Co., respectively.
Rac) was created by PCR using long oligonucleotides that
replaced the Rho insert domain (codons 123 to 138) with that of Rac.
Rho
Ras mutations were also created using this strategy, replacing
codons 122-141 of Rho with codons 121-127 of Ha-Ras. The Rho75Rac and
Rac73Rho chimeras were made by swapping the N terminus of Rho/Rac,
using the PvuII site at codon 59 (residues 59-73 are
identical between Rho and Rac). Site-directed mutagenesis was used to
generate wild type Rho75Rac92Rho and Rac73Rho90Rac using
Rho75Rac92Rho(Leu63) and Rac73Rho90Rac(Leu61)
(16) as PCR templates. Rho92Rac and Rac90Rho were constructed by
swapping the N-terminal 59 amino acids of Rho75Rac92Rho and Rac73Rho90Rac, taking advantage of the codon 59 PvuII common
to the chimeras. All mutants were subcloned into pGEX-2T (Amersham Pharmacia Biotech) to generate GST fusion protein or into pZIP (24) or
pCGN for expression in mammalian cells. Codons 905-1046 of p160 ROCK
were PCR-amplified from a human small intestine Marathon-ready cDNA
library (). Codons 1-284 (HR1), 36-113
(HR1a), 133-208 (HR1b), and 210-284 (HR1c) were amplified from PRK2
by PCR. All constructs were subcloned into pGEX-2T and pMal-c (New
England Biolabs) to generate GST and maltose-binding protein (MBP)
fusion proteins, respectively. pCMV2-FLAG-PRK2 has been described
previously (5). All cDNAs created for this study by PCR were
confirmed by dideoxynucleotide sequencing.
View larger version (12K):
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Fig. 1.
Summary of Rho/Rac chimeras used in this
study. Closed bars represent Rho sequences; open
bars represent Rac or Ras sequences. Nonchimeric mutants are
indicated with hatched boxes. Rho and Rac mutants are offset
by two codons due to the longer N terminus of RhoA.
-D-galactopyranoside at 37 °C, cells
were pelleted and resuspended in lysis buffer (50 mM Tris,
pH 7.6, 100 mM NaCl, 5 mM MgCl2,
100 µM GDP, 0.5% Nonidet P-40, 1 mM
dithiothreitol, 1.9 mg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride). After sonication to lyse the cells, the
supernatant was tumbled with glutathione-agarose beads (Sigma) at
4 °C overnight. Beads were then washed three times with lysis buffer
and stored at 4 °C. GST-fused HR1 and RBD were purified similarly
except for using 1 mM EDTA instead of MgCl2 and
GDP in the lysis buffer. MBP fusion proteins were also purified in a
similar manner except for using amylose beads (New England Biolabs) and
the following lysis buffer: 20 mM Tris, pH 7.4, 200 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 1 mM dithiothreitol, 1.9 mg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Rac proteins for
microinjection studies were cleaved from GST using 10 units of thrombin
in 50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 2.5 mM CaCl2,
1 mM dithiothreitol at 4 °C overnight. After removing
thrombin with p-aminobenzamidine beads (Sigma), the
supernatants were dialyzed against 15 mM Tris, pH 7.6, 150 mM NaCl, 5 mM MgCl2, 0.1 mM dithiothreitol at 4 °C for 12 h and concentrated
using an Amicon Centricon filter, as described (25). The active Rac
protein concentration was estimated by [32P]GTP loading
(26).
-32P]GTP by incubation for 20 min at 30 °C in 50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM EDTA, 5 µM GTP, 10 µCi of
[
-32P]GTP, and 1 mM dithiothreitol.
MgCl2 was added to 10 mM to stop the reaction,
and free nucleotide was removed by washing beads three times with the
loading buffer containing 10 mM MgCl2 in place
of GTP/EDTA. Proteins were then eluted by incubating beads with 100 µl of 20 mM glutathione, 100 mM Tris-HCl, pH
8.0, 50 mM NaCl, 5 mM MgCl2, 10%
glycerol, and 1 mM dithiothreitol on ice for 20 min. HR1a
and RBD proteins were run on SDS-polyacrylamide gel electrophoresis
gels, transferred to polyvinylidene difluoride membrane, and blocked in
1% bovine serum albumin, 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, and 0.05%
Tween-20 for 2 h. The membrane was then incubated for 15 min at
4 °C with 25 pmol of [
-32P]GTP-bound Rho
(determined by scintillation counting) in blocking buffer supplemented
with 10 mM MgCl2 and 100 µM GDP.
Following extensive washing in binding buffer, the membrane was
autoradiographed at -80 °C to visualized the binding of Rho to its effectors.
-32P]GTP.
2.5 µg of active GST-fused GTPases was then preloaded with unlabeled
GTP
S or GDP and eluted off glutathione-agarose beads as described
above. Subsequently, the proteins were incubated with 5 µg of
MBP-fused HR1 or 1 µg of MBP-fused RBD immobilized on amylose beads
in 50 mM Tris, pH 7.6, 50 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol, and 50 µM GTP at 4 °C
for 1 h. Beads were washed three times in the above buffer (except
for 0.1% Triton X-100), and bound proteins were separated by
SDS-polyacrylamide gel electrophoresis. The binding of GST-Rho/Rac to
HR1 and RBD was then determined by Western blotting using
"in-house" anti-GST polyclonal antibody and ECL (Amersham Pharmacia
Biotech) detection.
S as described above. Equal amounts of cell lysate
supplemented with 10 mM MgCl2 and 50 µM GTP were incubated with each GTPase at 4 °C for
2 h. Beads were washed three times with 20 mM Tris, pH
7.4, 50 mM NaCl, 10 mM MgCl2, 0.1%
Nonidet P-40, 10% glycerol, and 1 mM dithiothreitol. The
binding of PRK2 to immobilized GTPases was determined by Western blotting with M2 anti-FLAG antibody.
-32P]GTP as described above. 25 pmol of active Rho
was then incubated with 400 µg of GST (as control) or different
amounts of GST-HR1a (0.5-12.5 nmol/16-400 µg) on ice for 1 h
to allow ligand binding. Subsequently, 500 µl of GTP exchange buffer
(20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, and
50 µM GTP) was added to each sample, and exchange
reactions were incubated at 37 °C. Aliquots were removed at the
indicated time points, and reactions were terminated by addition of 500 µl of ice-cold GTP exchange buffer. Protein-bound nucleotide was then
captured on BA85 nitrocellulose filters (Schleicher & Schuell), which
were washed three times with 1 ml of cold GTP exchange buffer prior to
scintillation counting.
Ras(Leu63)
along with 1 µg of additional pZIP vector or pZIP-Raf(Y340D), as
indicated, using the calcium phosphate precipitation procedure (27).
Cells were maintained in regular growth medium, and transforming foci
were fixed and stained with crystal violet after 12-16 days of
culture. To create stable cell lines, NIH 3T3 cells were transfected with 0.5 µg of pCGN plasmids containing the above mutants and selected on growth medium supplemented with 200 µg/ml hygromycin B. At least 100 colonies were pooled and immunoblotted for HA-tagged Rho
expression approximately 3 weeks posttransfection.
RESULTS
-helical content. We therefore established in
vitro binding assays to determine whether these two distinct
structures interacted with common domains of Rho. Fig. 2c
shows that interaction of RhoA-GTP with the HR1 region of PRK2 occurs
primarily via the N-terminal-most repeat, HR1a. Weaker interaction was
detectable with the HR1b but not with the HR1c domain. This is in
agreement with Flynn et al. (28), who similarly found that
Rho predominantly bound to the HR1a domain of PRK1/PKN. Binding of Rho
to both HR1a and HR1b was determined to be GTP-dependent.
RhoA-GTP also bound to the RBD of p160 ROCK (Fig. 2d).
Although ROCK-RBD has been reported to interact with Rac (13, 14),
under our assay conditions (Fig. 2d) and those described by
Leung et al. (29), there was no interaction of RBD with WT
Rac1-GTP. We therefore generated a series of Rho/Rac chimeras to map
which domains of Rho are required for its specific interaction with
PRK2 and ROCK-1.
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Fig. 2.
Rho-GTP specifically interacts with the HR1a
domain of PRK2 and the RBD of ROCK. a, diagram
indicating the presence of HR1a, HR1b, and HR1c putative Rho-binding
domains, at the N terminus of PRK2. HR2, catalytic domain and a
Nck-binding domain (5, 19) are also shown. b, diagram
showing the RBD of ROCK-I, located between the coiled-coil and
pleckstrin homology (PH) domain (18). The catalytic domain
and cysteine-rich region are also shown. c, the indicated
MBP-HR1 fusion proteins were immobilized on amylose beads and incubated
with GST-Rho that had been loaded with GTP or GDP. Co-precipitation of
Rho by MBP fusion proteins was then determined by Western blotting with
anti-GST antibody. NS indicates a nonspecific band present
in all but the HR1a lanes. Results are representative of at
least two experiments. d, both HR1a and RBD bound to
Rho-GTP S but not Rac-GTP
S in the Rho precipitation assay.
-phosphate of GTP that is necessary for the switch 1 domain to adopt
its active conformation (7, 26, 31). Therefore, we also created a
mutation in Phe39 that, like Thr37, is
conserved between Rac and Rho. The F39A mutation totally abolished the
ability of Rho to bind to both HR1 and ROCK-RBD, suggesting that the
switch 1 domain does indeed contribute to effector binding but does not
determine the specificity of Rho-effector interaction. The GTP
dependence of Rho-effector binding (Fig. 2) was also supportive of the
involvement of switch 1. However, because Rac does not normally
interact with PRK2 or ROCK (5, 29), then the C-terminal two-thirds of
Rho must determine effector binding specificity.
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Fig. 3.
Mutation of conserved residues within the Rho
switch 1 domain but not its replacement by Rac sequences can disrupt
binding to HR1a and RBD. a, using the Rho overlay assay
(see under "Experimental Procedures"), GST-RBD and GST-HR1a were
found to bind to [ -32P]GTP-labeled RhoA but not Rac1.
Mutation of Rho switch 1 residues 37 and 39 or mutations that exchanged
the C-terminal two-thirds of Rho with Rac disrupted Rho-effector
binding. b, to confirm these findings, interaction of
mutants was assessed using the Rho precipitation assay. The same
GST-Rho/Rac mutants described in a were incubated with
amylose-immobilized MBP-HR1, and binding of Rho proteins was determined
by Western blotting. Weak binding of effectors with Rho(T37A) and
Rho75Rac were more evident in this assay due to the sharper resolution
of Rho protein bands. NS indicates a nonspecific band
appearing in all lanes. Data are representative of at least three
independent experiments.
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Fig. 4.
Rho/Rac chimeras localize the Rho-effector
binding determinant to residues 76-122. To address binding of Rho
effectors to the insert domain, this region (residues 123-138) was
swapped with the equivalent residues of Rac (Rho Rac) or Ras loop 8 (Rho
Ras). Although the Rho
Ras mutant did not bind as efficiently,
both chimeras associated with HR1a and RBD in the overlay assay. A
Rac73Rho143Rho containing the central portion of Rho bound to HR1a and
RBD, but a Rac143Rho chimera containing the C terminus of Rho did not.
Together, these data narrow the Rho-specific binding determinant to
residues 76-122. The numbers below each lane show
(phosphorimager) quantitation of the Rho-effector binding data and
indicate the ability of each chimera to bind GTP (a measure of protein
viability). Data are representative of at least three experiments.
Rac) still bound efficiently to HR1a and
RBD, suggesting that the insert domain did not dictate Rho effector
binding specificity.
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Fig. 5.
A sequence encompassing residues 76-92 is
responsible for specific interaction of Rho versus Rac
with HR1 and RBD. a, as shown in Fig. 3, Rho75Rac had
reduced effector binding ability, whereas Rac73Rho still bound to HR1
and RBD in the Rho precipitation assay. A Rho92Rac chimera also bound
strongly to effectors, but the complementary Rac90Rho chimera had no
detectable interaction with HR1 or RBD, narrowing the Rho-effector
binding determinant to residues 76-92. This was confirmed using a
Rac73Rho90Rac chimera. Data are representative of 3-5 experiments.
b, although Rac did not associate with HR1, a previously
described F78S mutant (34) was found to weakly bind to it in a
GTP-dependent manner. Data are representative of two
experiments. c, in the Rho precipitation assay,
GTP S-bound Rac(Q61L) and Rac(F78S) associated with MBP-RBD.
Ras mutant still interacted with PRK2 and
ROCK-RBD (Fig. 4), indicating that the insert domain is not essential for PRK or ROCK family binding. Although
Rho
Ras did not bind to effectors quite as effectively as WT Rho
(~60% binding), it was not as efficiently expressed in E. coli and bound fewer mol of GTP/mg of total protein (Fig. 4),
suggesting that the decreased binding may just reflect recombinant protein instability.
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Fig. 6.
Rho residues 87 and 90 in loop 6 confer
specific interaction with HR1 and RBD. a, alignment of
RhoA residues 76-92 with the equivalent sequence of Rac. Identical
residues are indicated by asterisks, and unique residues are
in boldface. The most divergent residues, 87 and 90, are
shown in larger type. The bar indicates the positions of
loop 5, -strand 4, loop 6, and
-helix 3 in the Rho-GTP
S
three-dimensional structure (44). b, using the Rho
precipitation assay, double mutation of Rho aspartate residues 87 and
90 to those present in Rac resulted in a dramatic decrease in binding
to both Rho effectors. Introducing these aspartate residues into
positions 85 and 88 of Rac, together or alone, resulted in a gain of
RBD and HR1 binding. There was no binding to Rac or the MBP control.
Data are representative of at least three experiments.
Ras, was very
likely due to the poor GTP binding capacity of Rac73Rho143Rac (Fig. 4).
Because the Rho
Rac and Rho
Ras chimeras that still bound Rho
targets encompassed residues 123-138, we concluded that the
Rho-effector-binding domain lay between residues 75 and 123. This was
confirmed using a Rho122Rac chimera, which bound to both
effectors.3 Additionally,
because a Rho92Rac but not a Rac90Rho chimera could efficiently
interact with HR1a and RBD (Fig. 5a), the
Rho-effector-binding sequence was further narrowed down to Rho residues
76-92.
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Fig. 7.
Loop 6 of Rho is also required for
association with full-length PRK2. Full-length, FLAG-tagged PRK2
was expressed in COS cells and precipitated from cell lysates using the
indicated GTP S-bound GTPases immobilized on glutathione-agarose
beads. PRK2 was detected using anti-FLAG antibody. The far right
lane (Lysate) indicates the position of PRK2 Western
blotted from the cell lysate. As observed for the isolated HR1 and HR1a
domains, full-length PRK2 bound to Rac chimeras containing Rho loop 6 residues. Binding did not require the Rho insert domain. Data are
representative of at least two experiments.
-32P]GTP release from Rho in the presence and absence
of excess GST-HR1a. As shown in Fig. 9,
the presence of HR1a inhibited the rate of GTP exchange in a
dose-dependent manner. Despite having a higher affinity for
Rho-GTP than HR1, preliminary data indicated that the ROCK-RBD could
not inhibit GTP release under similar conditions.3
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Fig. 8.
Three-dimensional representation of Rho A
showing proximity of Rho-effector interaction domains.
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Fig. 9.
The HR1a domain of PRK2 inhibits GTP release
from RhoA. To examine the effect of HR1a-Rho interaction on
nucleotide exchange, RhoA was loaded with [ -32P]GTP
and incubated with GST (400 µg) or various concentrations of GST-HR1a
at 37 °C (up to 400 µg). Nucleotide exchange was monitored at the
indicated times using a nitrocellulose filter binding assay. Increasing
the GST-HR1a concentration resulted in a dose-dependent
inhibition of GTP release. Representative of two experiments performed
in duplicate.
VA mutation of
Rho(Q63L) should result in a loss of transforming potential. Therefore, we examined the ability of Rho(Q63L) and Rho(Q63L)VA to cooperate with
the partially activated Raf(Y340D) mutant to induce transforming foci
in NIH 3T3 cells. Whereas Rho(Q63L) synergized with Raf(Y340D) to
generate foci, Rho(Q63L)VA was considerably less effective (Fig.
11a). Interestingly, the
Rho
Ras(Q63L) mutant, which lacks the insert domain, was also unable
to cooperate with Raf(Y340D) to induce transformation, even though it
retained the ability to bind to both PRK2 and ROCK-I. It was not
possible to determine the expression of the Rho
Ras in NIH 3T3 cells
because the only available Rho antibody is directed to the (deleted)
insert domain. Therefore, to confirm the expression of Rho mutants in
NIH 3T3 cells, HA epitope-tagged constructs were generated in the
vector pCGN, and stable cell lines were established. Western blotting of cell lysates with anti-HA antibody indicated that the Rho mutants were expressed at similar levels (Fig. 11b).
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Fig. 10.
Rho loop 6 residues confer Rac with the
ability to induce stress fiber formation in porcine epithelial
cells. LLC-PK kidney epithelial cells were microinjected with
buffer containing activated Rac(Q61L) or Rac(Q61L)DD as indicated.
Cells were co-injected with C3 ADP-ribosyltransferase to prevent
endogenous Rho activation and fluorescein-conjugated dextran to detect
injected cells. After 45 min, cells were fixed and stained with
rhodamine-conjugated phalloidin to detect F-actin, and injected cells
were visualized by confocal microscopy. Upper panels show
rhodamine-phalloidin staining; lower panels identify
fluorescein-dextran-injected cells. Introduction of Rac(Q61L) induced
the formation of peripheral membrane ruffles (middle
column), whereas Rac(Q61L)DD, containing the double Asp mutation,
induced extensive stress fiber formation (right column).
Scale bar in lower left panel represents 20 µm.
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Fig. 11.
Mutation of Rho loop 6 or deletion of the
insert domain disrupts transformation of NIH 3T3 cells.
a, NIH 3T3 cells were transfected with empty pZIP vector or
the indicated Rho mutants in the absence (upper panels) or
presence (lower panels) of Raf(Y340D). Transforming foci
were visualized by staining with crystal violet after 12-16 days in
culture. Representative data are shown from five independent
experiments performed in quadruplicate. b, NIH 3T3 cells
were transfected with the Rho/Rac mutants in the vector pCGN, which
provides an N-terminal HA tag. Following stable selection on hygromycin
B, Rho protein expression levels were determined on a pooled population
of cells using anti-HA antibody.
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Fig. 12.
Rac(Leu61) and
Rho(Leu63) mutations can override other GTPase
mutations. Rho(WT) and Rac(WT) (upper panels) or
activated Rac(Q61L) and Rho(Q63L) mutants (lower panels)
were loaded with GTP (RhoA), GTP S (Rac1), or GDP as indicated and
incubated with MBP-HR1 in the Rho precipitation assay. Several of the
mutationally activated GTPases bound more efficiently than the WT
proteins, and this interaction was not always
GTP-dependent. Data are representative of at least two
experiments.
DISCUSSION
strand 4, loop 6, and the
proximal portion of
helix 3 (Fig. 6a). Only two residues
within the 76-92 region (Rho residues Asp87 and
Asp90) show significant divergence between Rac and Rho and
are both located within the surface exposed loop 6. Swapping these
residues between Rac and Rho resulted in a decrease in Rho binding and a gain of Rac binding to the PRK2-HR1 and ROCK-RBD, strongly suggesting that loop 6 is a specificity determinant for interaction with both
classes of Rho effector.
-helical content).
If so, the effector would close off the GTP-binding pocket of Rho and
prevent nucleotide release. Consistent with this model, we found that
the HR1a domain of PRK2, at as low as 20 molar excess over Rho,
effectively inhibited guanine nucleotide release. However, the
Ras-binding domain of Raf (residues 51-131) can also inhibit GTP
release from Ras, implying that interaction of an effector with the
switch1 domain of Ras may be sufficient to block nucleotide exchange
(45). Although we cannot exclude the possibility that association of
PRK2 with switch 1 alone may also be sufficient to block nucleotide
exchange on Rho, preliminary data indicated that a 20-fold molar excess of RBD over Rho could not block GTP release.3 Furthermore,
the HR1 domain of PKN(30) but not the ROCK-RBD (29) was found to
inhibit the intrinsic GTPase activity of Rho, again suggesting
differential interaction of these two effectors with Rho. Therefore,
due to their stronger interaction with loop 6, PRK2 and PKN may more
effectively occlude the GTP-binding pocket.
-strand 4 of Rac promotes
weak Rho-effector binding. Because residue 78 is partially buried in
the core of Rac, and Ser78 makes contact with
Thr108 and Pro109 in loop 7, located between
3 and
5 (34), this mutation could have several subtle effects on
Rac biochemistry. It has been suggested from computer modeling studies
that the F78S mutation will not significantly perturb Rac structure,
and it did not affect GAP-stimulated GTPase activity (34). Nonetheless,
the weak interaction of Rac(F78S) with PRK2 may explain the discrepancy
in Rac-PRK2 binding data observed in previous studies that may have
used this mutant form of Rac (5, 20). Not knowing the source of Rac
used in other studies, it is not possible to predict whether this also
explains differences in Rac-ROCK interaction (12-14).
Rac) or using a Rho92Rac chimera did not prevent the association
of Rho with either HR1 or RBD. This suggested that the insert domain is
not a specificity determinant for these Rho targets. A similar
conclusion was made regarding Rho activation of phospholipase D (40).
Due to the inability to detect robust activation of PRK2 by Rho using
in vitro kinase or transcription assays and the low yield of
GST-Rho
Ras in E. coli, it was not possible to determine
whether the insert was required for PRK2 activation or stress fiber
formation. However, the insert helix of Rho was essential for it to
induce cellular transformation. So, as previously reported for Rac (15,
32), the insert domain in Rho may be involved in effector activation
rather than being a binding determinant.
Ras chimera also disrupts its ability to induce cell growth and
transformation (48). This effect was independent of JNK and PAK
activation or actin cytoskeletal regulation, suggesting that the insert
domain couples CDC42 to a novel effector signaling pathway. The insert domain also appears to be essential for the mitogenic activity of
Rac (42). Again, deletion of the insert domain did not affect JNK
activation or actin polymerization but instead abrogated mitogenesis by
blocking Rac-induced superoxide generation. Whether Rac- or CDC42-induced transformation pathways are shared with Rho or whether the Rho insert domain interacts with additional target proteins will
require further investigation.
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ACKNOWLEDGEMENTS |
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We are grateful to A. Hall for generously providing Rho constructs, Z. Derewenda for providing the Protein Data Bank coordinates for the Rho-GDP crystal structure prior to general release, D. Timm for assistance with computer modeling, and C. Bi for technical assistance.
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FOOTNOTES |
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* This work was supported by Research Project Grant 97-007-01-BE from the American Cancer Society (to L. A. Q.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Indiana University School of
Medicine, 635 Barnhill Dr., MS-4053, Indianapolis, IN 46202-5122.
Tel.: 317-274-8550; Fax: 317-274-4686; E-mail:
lquillia{at}iupui.edu.
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ABBREVIATIONS |
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The abbreviations used are:
HR, homology region;
GST, glutathione S-transferase;
MBP, maltose-binding
protein;
RBD, Rho binding domain of ROCK-1;
PCR, polymerase chain
reaction;
HA, hemagglutinin;
WT, wild type;
GTPS, guanosine
5'-O-(thiotriphosphate).
2 N. Raman and S. J. Atkinson, submitted for publication.
3 H. Zong and L. A. Quilliam, unpublished observations.
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REFERENCES |
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