The Dbl-related Protein, Lfc, Localizes to Microtubules and
Mediates the Activation of Rac Signaling Pathways in Cells*
Judith A.
Glaven
,
Ian
Whitehead§,
Shubha
Bagrodia
,
Robert
Kay¶, and
Richard A.
Cerione
From the
Department of Molecular Medicine, College of
Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, the § Department of Pharmacology and the Lineberger
Comprehensive Cancer Center, University of North Carolina, Chapel Hill,
North Carolina 27599, and the ¶ Terry Fox Laboratory, British
Columbia Cancer Agency, Vancouver,
British Columbia V5Z 4E6, Canada
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ABSTRACT |
The possibility that the Dbl family member Lfc
can activate Rac1 in cells is investigated in this study. Previously,
we demonstrated that both Lfc and Lsc, like their closest relative Lbc,
can act catalytically in stimulating the guanine nucleotide exchange
activity of RhoA in vitro. Neither Lfc nor Lsc stimulated
the in vitro exchange activity of Cdc42 or Rac1; however,
Lfc was capable of forming a tight complex with Rac1 in
vitro. We show here that Lfc stimulates c-Jun kinase (JNK)
activity in COS-7 cells. This stimulation was blocked by a dominant
negative mutant of Rac1 and somewhat less effectively by dominant
negative RhoA, but not by dominant negative Cdc42. Overexpression of
Lfc in NIH 3T3 cells induced the formation of actin stress fibers and
membrane ruffles, consistent with the activation of both RhoA and Rac1
signaling pathways, whereas overexpression of Lsc led exclusively to
well developed stress fibers. Using a recently developed assay for measuring the cellular activation of Rac, we did not find that expression of Lfc increased the levels of GTP-bound Rac1. However, an
examination of the cellular localization of Lfc showed that it was
localized to microtubules, similar to what has been reported for
activated Rac1, the mixed lineage kinase (MLK) and JNK. Moreover, we
have found that the Pleckstrin homology (PH) domain of Lfc specifically
associates with tubulin. Taken together, these findings suggest a model
where the PH domain-mediated localization of Lfc to microtubules
enables the recruitment of Rac to a site proximal to its signaling
targets, resulting in JNK activation and actin cytoskeletal changes.
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INTRODUCTION |
Lfc was initially identified based on its transforming activity
when overexpressed in NIH 3T3 cells (1). The Lfc oncoprotein is a
member of the Dbl family of growth regulatory proteins. Many members of
the Dbl family have been demonstrated to function upstream of the
Rho-related GTP-binding proteins, acting as guanine nucleotide exchange
factors (GEFs)1 by
stimulating the exchange of GTP for GDP and thereby promoting G protein
activation. This rapidly growing family of regulatory proteins includes
greater than 20 members to date (reviewed in Ref. 2). The fact that
there exist so many GEFs for the Rho subfamily suggests that there will
be multiple pathways leading to the activation of an individual
GTP-binding protein. All members of the Dbl family possess a Dbl
homology (DH) domain in tandem with a Pleckstrin homology (PH) domain,
and it has been demonstrated that both domains are required for the
transforming activities of oncogenic members of the family. The DH
domain typically represents the limit motif for binding the G protein
and stimulating nucleotide exchange (3), whereas the PH domain appears
to be essential for mediating the appropriate cellular localization of
the protein (1, 4).
Additionally, members of the Dbl family contain a number of other
structural motifs that indicate a role in signal transduction. These
domains presumably function to mediate protein/protein and protein/lipid interactions and serve to link members of the Dbl family
to upstream regulation (2). This has been most carefully worked out for
the Dbl family member, Vav, which is a Rac-GEF that is regulated by
tyrosine phosphorylation and by the phospholipids, phosphatidylinositol
4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) (5-7). Lfc contains a
cysteine-rich domain similar to the diacylglycerol binding domain found
in protein kinase C which may function to couple Lfc with upstream
generation of lipids (1).
Determining the regulation and cellular localization of the growing
family of Rho-GEFs will be important for understanding how Rho-related
GTP-binding proteins mediate multiple cellular activities. Activated
RhoA, Rac1, and Cdc42 regulate both gene transcription and the actin
cytoskeleton, contributing to the control of cell morphology, motility,
and growth (reviewed in Refs. 8 and 9). Through their interactions with
multiple targets, the Rho family of GTP-binding proteins is able to
coordinate these diverse cellular functions. A number of targets have
been identified including two related families of serine/threonine kinases, the p21-activated kinases (Paks) and the mixed lineage kinases
(MLKs), which have been shown to play specific roles in regulating gene
transcription and the actin cytoskeleton (10-15). It has previously
been reported that Rac1 and Cdc42 stimulate the enzymatic activity of
the mitogen-activated protein kinases (MAPKs), JNK and p38 (16).
Additionally, both the Paks and MLKs have also been shown to stimulate
JNK activity (11, 15, 17). The JNKs in turn phosphorylate and regulate
the activity of proteins that control the expression of specific gene
products involved in regulating growth and morphology (18, 19). The JNK
family members have also been referred to as stress-activated protein kinases (SAPKs) because of the ability of ultraviolet radiation, osmotic shock, or inflammatory cytokines such as interleukin-1 and
tumor necrosis factor-
to stimulate their activity (20, 21).
We have previously shown that Lfc, which functions specifically as a
GEF for RhoA in vitro, binds tightly to Rac1 in a
nucleotide-independent manner (22). Additionally, Lfc strongly
activates JNK in COS-7 cells. These findings suggest that Lfc may have
a broader specificity in cells that would include promoting a
Rac1-mediated pathway which leads to JNK activation. This led us to
examine the cellular activity of Lfc. Thus far, Vav, Tiam-1, and SOS
(7, 23, 24) are Dbl family members that with the help of cellular
co-factors (e.g. lipid second messengers) have been shown to
stimulate Rac activation in cells. In the present studies, we show that
Lfc is another Dbl family member that has a positive effect on Rac signaling. However, in this case, we do not detect a direct
Lfc-stimulated activation of Rac1. Rather Lfc, through direct
interactions with microtubules as mediated by its PH domain, may serve
to mark the cellular site for a Rac-signaling complex that leads to JNK
activation and/or actin cytoskeletal changes.
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EXPERIMENTAL PROCEDURES |
Plasmids--
Plasmids pAX142 HAD7-Lfc and HAD6-Lfc (1) and
D7-Lsc (30) have been described previously. The GST-Lfc (PH) was
generated by PCR from the D7-Lfc cDNA. Primers for PCR encoded the
restriction sites HindIII and XbaI to subclone
the PH domain fragment into the Escherichia coli expression
vector pGXKG. The PCR generated fragment encompassed the entire
PH domain plus a small amount of flanking sequence (amino acid sequence
450-573). Generation of all constructs encoding Rac1, Cdc42, and their
dominant negative mutants have been described previously (10, 22, 26).
These were subcloned into the expression vector pCDNA3, which
has been engineered to contain either the HA- or Myc-tag for
expression in mammalian cells. The generation of
J3MPak3 has been described (17); plasmid
J3MPak3(K297R) expresses Myc-tagged kinase-defective Pak3.
Kinase-defective Pak3 was made by mutating Lys at position 297 in the
ATP-binding site to Arg using the PCR overlap method. The pCMV6 Dbl was
generated by ligating a BamHI fragment encoding oncogenic
Dbl from plasmid pc11dbl (a gift from Dr. Sandra Eva, Institute
Giannina Gaslini, Genova, Italy) into the BamHI site of
pCMV6. cDNA encoding the GTPase defective and dominant negative mutants of Rho were a gift from Dr. C. Der (University of North Carolina) and were subcloned into pCDNA3, which contains the
Myc-tag, for expression in mammalian cells. The Flag-tagged human JNK1 has been described previously (17). Plasmids (pCDNA3) expressing MLK3 and MLK3(K114R) were gifts from Dr. J. S. Gutkind (National Institutes of Health, Bethesda, MD). All PCR-generated products were
sequenced to confirm that no errors were introduced.
Transient Transfections of Plasmid
DNA--
LipofectAMINE-mediated transient transfection of COS-7 cells
and NIH 3T3 cells were carried out according to the manufacturer protocol (Life Technologies, Inc). After 5 h, the medium was
replaced with Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum for COS-7 cells or calf serum for NIH 3T3 cells. For
immune complex kinase assays, cells were lysed at 36-48 h after
addition of DNA. For fluorescence studies, cells were trypsinized 24 h after the addition of DNA and replated onto chamber slides (LABTEK) for
24-36 h.
Cell Lysis, Immunoprecipitation, and Kinase Assays--
Cells
were serum starved for 2 h prior to lysis for kinase assays. Cells
were washed with cold phosphate-buffered saline, lysed in 40 mM Hepes, pH 7.4, 1% Nonidet P-40, 100 mM
NaCl, 1 mM EDTA, 25 mM NaF, 1 mM
sodium orthovanadate, 20 mM
-glycerophosphate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin and centrifuged at 12,000 × g for 25 min at 4 °C. Protein expression
was confirmed by Western blot analysis. For immune complex kinase
assays, lysates were normalized for the amount of kinase, Flag-JNK or
Myc-Pak3. Flag-tagged JNK was immunoprecipitated with monoclonal
antibody M5 (Kodak Scientific Imaging Systems) prebound to protein
G-Sepharose. Myc-tagged Pak3 was immunoprecipitated with monoclonal
anti-Myc antibody (10E-9, ATCC) prebound to protein A-Sepharose.
Immunoprecipitates were washed three times in lysis buffer and divided
into two equal aliquots. One aliquot was subjected to Western blot
analysis, and the second was used in the kinase assay. JNK kinase
immunoprecipitates were washed twice in 10 mM
MgCl2 and 40 mM Hepes, pH 7.4, and incubated
with 3 µg of GST-Jun substrate, 5 µCi [
-32P]ATP,
and 20 µM ATP in 25 mM Hepes, pH 7.4, 20 mM
-glycerophosphate, 0.1 mM sodium
orthovanadate, and 2 mM dithiothreitol (final volume = 30 µl) for 20 min at 22 °C. Pak3 kinase immunoprecipitates
were washed twice in 40 mM Hepes, pH 7.4, and 20 mM MgCl2 and mixed with 5 µg of myelin basic
protein (MBP) (Sigma), 10 µCi [
-32P]ATP, and 20 µM ATP in a final volume of 30 µl for 10 min at 22 °C. The kinase reactions were terminated by the addition of EDTA
containing Laemmli sample buffer, and resolved by SDS-PAGE. The
incorporation of 32P was visualized by autoradiography.
Rac1 Activation Assay--
The p21 (Cdc42/Rac1) binding domain
(PBD) of Pak3 was expressed in E. coli as a GST fusion
protein and immobilized by binding to glutathione-Sepharose beads. The
immobilized GST-PBD was used to precipitate activated Rac1 from
transfected COS-7 cell lysates. Cells were washed in cold
phosphate-buffered saline and lysed in 20 mM Hepes, pH 7.4, 100 mM NaCl, 0.5% Nonidet P-40, 10 mM MgCl2, 10 mM
-glycerophosphate, 10%
glycerol, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Lysates were
cleared by centrifugation at 12,000 × g for 25 min at
4 °C, frozen in liquid nitrogen, and stored at
80 °C. The
expression of proteins was confirmed by Western blotting of an aliquot
prior to affinity precipitation, and lysates used for affinity
precipitation were normalized for HA-Rac1 levels. Affinity
precipitations were carried out for 1 h at 4 °C, washed three
times in lysis buffer, and analyzed by Western blotting. HA-tagged Rac1
was detected with monoclonal antibody 12CA5 (Berkley Antibody Co). The
primary antibody was detected with horseradish peroxidase-coupled sheep
anti-mouse antibody using the enhanced chemiluminescence detection
reagent ECL (Amersham Pharmacia Biotech).
Immunofluorescence--
For morphological studies, cells were
serum starved for 16 h, fixed with 3.7% formaldehyde for 10 min,
and permeabilized with 0.1% Triton X-100 for 5 min. Actin cytoskeleton
was stained by incubation with 3.3 µM Texas
Red-conjugated phalloidin (Molecular Probes) for 1 h followed by
three washes with phosphate-buffered saline. For immunolocalization
studies, cells were fixed and permeabilized as above. HA-D7 Lfc was
detected using anti-HA11 IgG monoclonal antibody (Babco, Berkeley, CA)
at 1:200 dilution for 1 h followed by Bodipy-conjugated
anti-IgG-specific secondary antibody (Molecular Probes, Eugene, OR) for
1 h. Microtubules were stained with anti-tubulin IgM monoclonal
antibody (a gift from Dr. G. Bloom, Southwestern Medical Center,
Dallas, TX) for 1 h, followed by rhodamine-conjugated anti-IgM-specific secondary antibody (Southern Biotechnology
Associates, Inc., Birmingham, AL) for 1 h. The secondary
antibodies did not show nonspecific labeling, or cross-reactivity.
Cells were observed and photographed under a Zeiss fluorescence microscope.
Affinity Precipitation Assay for Tubulin Binding--
The
expression and purification of GST-DHPH Lfc (GST-D10 Lfc) from
Sf21 insect cells has been previously described (22). The GST-PH
fusion protein was expressed in E. coli and an overnight culture that was grown from a single colony was used to inoculate a
1-liter culture that was grown at 37 °C, while shaking, to an A560 nm of 0.6 (~4 h). Protein expression was
then induced by the addition of 200 µM isopropyl
-D-thiogalactoside for 1 h. Bacteria were harvested
by centrifugation and frozen at
80 °C. The pellets were thawed in
20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 2 mM dithiothreitol, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride. Bacteria were lysed on ice by adding 0.5 mg/ml lysozyme followed by 10 mg/ml of DNase I (Boehringer Mannheim)
and 5 mM MgCl2. The lysates were cleared by
centrifugation for 30 min at 30,000 rpm. Proteins were purified by
glutathione-agarose affinity chromatography. The glutathione-agarose bound GST fusion proteins were used in affinity precipitations from
Jurkat T-cell lysates (20 mM Hepes, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 mM sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Precipitations were carried out at 4 °C for 2 h and washed in cell lysis buffer three times, and then the resuspended samples were
subjected to Western blot analysis. Precipitated tubulin was detected
with monoclonal anti-tubulin primary antibody (Amersham Pharmacia
Biotech), and the primary antibody was detected with horseradish
peroxidase-coupled sheep anti-mouse antibody using the
chemiluminescence detection reagent ECL (Amersham Pharmacia Biotech).
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RESULTS AND DISCUSSION |
It has been reported previously that both Rac1 and Cdc42 can
activate the JNK mitogen-activated protein kinase cascade in COS-7
cells, whereas RhoA does not (16). We have observed that the
overexpression of Lfc in COS-7 cells leads to JNK activation. In Fig.
1, we show a schematic representation of
full-length Lfc and Lsc, and the different forms of the recombinant
proteins used in this study. Using an in vitro kinase assay
to determine the activity of immunoprecipitated Flag-tagged JNK from
lysates, we find that Lfc and Dbl can activate JNK in COS-7 cells to an
extent similar to the activation induced by exposing cells to
ultraviolet radiation, whereas Lsc shows no detectable stimulation
(Fig. 2A). The Lfc-mediated
stimulation of JNK activity was not dependent on the diacylglycerol
binding domain since the D6-Lfc and D7-Lfc constructs both yielded
strong JNK activation (Fig. 2B). The lack of stimulation by
Lsc indicates that although RhoA can activate JNK in some cell types,
as reported by Teramoto et al. (25), it does not activate
JNK in COS-7 cells. Thus, we set out to determine whether Rac1 mediates
the Lfc-stimulated activation of JNK in COS-7 cells.

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Fig. 1.
A schematic representation of Lfc and
Lsc. The full-length Lfc and Lsc proteins are shown along with the
constructs used for expression in mammalian cells in the transfection
studies described here. DAG, diacylglycerol.
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Fig. 2.
Activation of JNK by Lfc in
vivo. A, COS-7 cells were transiently
co-transfected with plasmid pcDNA3-Flag-JNK (0.4 µg) together
with an empty expression vector or with an expression vector encoding
HA-D7 Lfc, HA-D7 Lsc, or Dbl (1 µg). Cells were lysed at 36-48 h
post-transfection, and JNK activity was measured by immune complex
kinase assays using [ -32P]ATP and c-Jun as substrates.
Proteins were separated by SDS-PAGE and analyzed by autoradiography to
show phosphorylation of substrate (top panel) and Western
blot analysis to show relative amounts of Flag-JNK in the kinase
reactions (bottom panel). B, a comparison of the
ability of HA-D7 Lfc and HA-D6 Lfc (which lacks the
diacylglycerol-binding domain) to stimulate JNK activity in transient
transfections and immune complex kinase assays (as described in Fig.
2A).
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Ability of Dominant Negative Mutants of the Rho Subfamily to
Inhibit Lfc-stimulated JNK Activity--
We next examined whether the
activation of JNK in COS-7 cells could be inhibited by dominant
negative mutants of the Rho family of small GTP-binding proteins. Based
on in vitro binding studies, which indicated that Lfc could
form stable complexes with both RhoA and Rac1, we would anticipate that
dominant negative versions of RhoA (RhoA(T19N)) and Rac1 (Rac(S17N))
would be capable of binding Lfc and inhibiting its activation of JNK.
As shown in Fig. 3, Rac(S17N) blocked
Lfc-stimulated JNK activity, whereas comparable levels of RhoA(T19N)
showed a significantly weaker inhibition. At higher levels of
expression, RhoA(T19N) was then able to provide an inhibitory effect.
Dominant negative Cdc42 (Cdc42(T17N)) was not able to inhibit
Lfc-stimulated JNK activity under any condition.

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Fig. 3.
Dominant negative mutants of Rac1 and RhoA
block the Lfc-stimulated JNK activity. COS-7 cells were
transiently co-transfected with Flag-JNK (0.4 µg of DNA) together
with an empty expression ( ) vector or with an expression vector (1 µg of DNA) encoding HA-D7 Lfc (+), dominant negative Rac1N17,
RhoAN19, and Cdc42N17 (2 or 4 µg of DNA, each). Cells were lysed at
36-48 h post-transfection, and JNK activity was measured by immune
complex kinase assays using [ -32P]ATP and c-Jun as
substrates. Proteins were separated by SDS-PAGE and analyzed by
autoradiography to show phosphorylation of substrate (top
panel) and Western blot analysis to show relative amounts of
Flag-JNK in the kinase reactions (bottom panel).
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Co-expression of Dominant Negative Forms of Pak3 and MLK3 Interfere
with Lfc-stimulated JNK Activity--
The Paks, which serve as targets
for Cdc42 and Rac but not RhoA, have been shown to mediate the
activation of JNK in certain cell types (10, 11, 17). Thus, we examined
whether a kinase-defective mutant of Pak3 (Pak3(K297R)), by interfering
with Rac1-stimulated signaling, would inhibit Lfc-mediated JNK
activation. Fig. 4A (where KDP
represents the Pak3 mutant) shows that this was the case. However, it
was interesting that we did not find that Lfc stimulated Pak3 activity
in these cells. We examined this by transiently co-expressing
Myc-tagged Pak3 with either Lfc, Lsc, or Dbl in COS-7 cells. These
studies showed that Lfc did not activate Pak3, under conditions where
Dbl stimulated Pak3 activity (Fig. 4B). This suggests that
the direct stimulation of Rac (and Pak3) activity by Lfc is not the
underlying mechanism by which Lfc mediates JNK activation (also, see
below). MLK3 is another putative protein kinase target for Cdc42 and
Rac that has recently been shown to be a strong activator of JNK (15).
Fig. 5 shows that like the kinase-defective Pak3, a kinase-defective form of MLK3 (MLK3(K114R)) (Fig. 5, designated KDMLK), inhibits Lfc-stimulated JNK
activity. Thus far, we have not been able to determine whether Lfc
stimulates MLK3 activity because this protein kinase is constitutively
active when expressed in cells, and so it has not been possible to
conclude whether MLK is an essential participant in the Lfc-mediated
signaling pathway leading to JNK activation.

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Fig. 4.
Dominant negative mutant of Pak3 blocks
Lfc-stimulated JNK activity, but Lfc does not activate Pak3.
A, COS-7 cells were transiently co-transfected with Flag-JNK
(0.4 µg) together with an empty expression ( ) vector or with an
expression vector encoding HA-D7 Lfc (+), (1 µg), and 2 or 4 µg of
J3MPak3 K297R (KDP, kinase dead) or 2 and 4 µg of J3MPak3
(WTP, wild type). Cells were lysed at 36-48 h
post-transfection, and JNK activity was measured by immune complex
kinase assays using [ -32P]ATP and c-Jun as substrates.
Proteins were separated by SDS-PAGE and analyzed by autoradiography to
show phosphorylation of substrate (top panel) and Western
blot analysis to show relative amounts of Flag-JNK in the kinase
reactions (bottom panel). B, COS-7 cells were
transiently transfected with Myc-Pak3 (0.5 µg) together with an empty
expression vector or with an expression vector encoding HA-D7 Lfc,
HA-D7 Lsc, or Dbl (1 µg). Cells were lysed at 36-48 h
post-transfection, and PaK activity was measured by immune complex
kinase assays using [ -32P]ATP and MBP as substrates.
Proteins were separated by SDS-PAGE and analyzed by autoradiography to
show phosphorylation of substrate (top panel) and Western
blot analysis to show relative amounts of Myc-Pak3 in the kinase
reactions (bottom panel).
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Fig. 5.
Dominant negative mutant of MLK3 blocks
Lfc-stimulated JNK activity. COS-7 cells were transiently
co-transfected with Flag-JNK (0.4 µg) together with an empty
expression ( ) vector or with an expression vector encoding HA-D7 Lfc
(+) (1 µg) and 2 µg of MLK3 K114R (KDMLK, kinase dead)
or 2 µg of MLK3 (WTMLK, wild type). Cells were lysed at
36-48 h post-transfection, and JNK activity was measured by immune
complex kinase assays using [ -32P]ATP and c-Jun as
substrates. Proteins were separated by SDS-PAGE and analyzed by
autoradiography to show phosphorylation of substrate (top
panel) and Western blot analysis to show relative amounts of
Flag-JNK in the kinase reactions (bottom panel).
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Detection of Rac1 Activation in Vivo--
Our initial guanine
nucleotide exchange measurements performed in vitro
indicated that Lfc stimulated GDP-GTP exchange on RhoA but not on Rac1
(22), suggesting that Lfc did not act as a GEF for Rac1. However,
similar in vitro results have been obtained for Vav, Tiam-1,
and Sos, but it was subsequently shown that cellular co-factors were
necessary to enable each of these Dbl-related molecules to act as GEFs
for Rac1 in cells (7, 23, 24). Thus, we took advantage of a recently
developed assay for Rac activation (26) to determine whether Lfc could
increase the levels of GTP-bound Rac1 in cells. Specifically, the
p21-binding domain (PBD) of Pak3 was expressed as a GST-fusion protein
and immobilized on glutathione-Sepharose beads and then was used as an
affinity reagent to precipitate GTP-bound (activated) HA-Rac1 from
transfected COS-7 cell lysates. As shown in Fig.
6, when using this assay, we were unable
to detect an increase in the levels of GTP-bound Rac1 in cells that
were co-transfected with Lfc and HA-Rac1. However, we were able to
detect an increase in GTP-bound Rac1 when Dbl was co-expressed with
HA-Rac1, consistent with prior results indicating that Dbl can
stimulate the activation of Rac in cells (26).

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Fig. 6.
Lfc does not detectably activate Rac1
in vivo. Shown is an assay for Rac1 activation
in vivo. COS-7 cells were transiently co-transfected with
HA-Rac1 (1 µg) together with an empty expression vector ( ) or with
an expression vector encoding HA-D7 Lfc, HA-D7 Lsc, or Dbl (1 or 2 µg). Cells were lysed at 36-48 h after transfection and affinity
precipitated (AP) with immobilized GST-PBD. Proteins were
separated by SDS-PAGE and Western blotted and probed with anti-HA
antibody (top panel). Bottom panel represents
10% of the total cell lysate (TCL) used in the binding
assay.
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Morphological Changes Induced by Lfc in NIH 3T3
Cells--
Rearrangements in the actin cytoskeleton have been shown to
be regulated by members of the Rho subfamily of GTP-binding proteins (reviewed in Ref. 9). Moreover, it has been shown that each of the Rho
family members induces distinct morphological phenotypes. By taking
advantage of these morphological changes, we set out to determine
whether Lfc promotes a Rac1-like morphology. Cells were stained with
Texas Red-conjugated phalloidin (Molecular Probes) to visualize the
actin cytoskeleton in NIH 3T3 cells co-transfected with an expression
vector encoding HA-D7 Lfc or HA-D7 Lsc and pEGFP (an expression vector
containing green fluorescent protein) (Fig.
7). Similar types of experiments were
performed using indirect immunofluorescence with an anti-HA monoclonal
antibody (HA11, Babco) to identify cells expressing HA-D7 Lfc or HA-D7
Lsc. We also examined the actin rearrangements induced by transient
expression of GTPase-defective RhoA (RhoA(G14V)) and Rac1 (Rac(Q61L)).
As shown in Fig. 7, cells expressing the GTPase-defective RhoA mutant have enhanced actin stress fibers (Fig. 7C) as compared with
control NIH 3T3 cells (Fig. 7A). NIH 3T3 cells expressing
GTPase-defective Rac1 (Fig. 7B) have an enhanced actin
meshwork at their periphery, resulting in membrane ruffles. We show in
Fig. 7E that Lsc strongly induces actin stress fibers and a
morphology similar to that observed when over-expressing the activated
RhoA mutant. Lfc, however, appears to not only promote the formation of
actin stress fibers but also induces lamellipodia and membrane
ruffling, indicative of signaling through both activated RhoA and Rac1
(Fig. 7D).

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Fig. 7.
Lfc induces the formation of lamellipodia and
actin stress fibers. Serum-starved NIH 3T3 cells transiently
expressing GTPase-defective Rac(Q61L), GTPase-defective RhoA(Q63L),
Lfc, or Lsc were analyzed by indirect immunofluorescence analysis for
actin cytoskeletal organization with Texas Red phalloidin. Vector
control transfected cells (A) show very few organized actin
filaments. Expression of activated Rac (B) induces
lamellipodia, whereas activated Rho (C) strongly induces
stress fiber formation. Expression of Lfc (D) leads to the
formation of lamellipodia and stress fibers while Lsc (E)
induces stress fiber formation, similar to that seen with activated
RhoA.
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Cellular Localization of Lfc to the Microtubules--
We used
HA-D7 Lfc in immunofluorescence experiments to examine the cellular
localization of Lfc. Using a monoclonal anti-HA antibody (HA11, Babco)
as the primary antibody, and Bodipy-labeled secondary antibody, we
detected a distinct cellular distribution for Lfc, as reflected by a
filamentous pattern (data not shown). This pattern of staining closely
resembled the microtubule network. We next double stained cells
expressing HA-D7 Lfc to directly examine whether Lfc co-localized with
microtubules. HA-D7 Lfc was detected as described above, and
microtubules were detected using an anti-tubulin antibody followed by
rhodamine-labeled secondary antibody. The results of this indirect
immunofluorescent staining clearly demonstrate that Lfc (Fig.
8A) colocalizes with the
microtubule network (Fig. 8B). A localization to
microtubules has also been reported for Rac1 (27) and more recently for
MLK and JNK (28).

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Fig. 8.
Colocalization of Lfc and microtubules.
NIH 3T3 cells transiently expressing HA-Lfc were fixed, and cells
expressing Lfc were visualized by indirect immunofluorescence using
anti-HA (HA11) and BODIPY-labeled secondary antibody to detect HA-Lfc
(A) and anti-tubulin antibody and a rhodamine-labeled
secondary antibody to visualize microtubules (B).
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Affinity Precipitation of Tubulin from Cell Lysates with GST-Lfc
Fusion Proteins--
The GST-D10 Lfc, which contains just the DH-PH
domains of Lfc, was used as an affinity reagent to identify potential
binding partners. Fig. 9B
(lane 3) shows that a 55-kDa protein co-precipitates with
the GST-Lfc construct. The 55-kDa protein, which can be photo-labeled with [
-32P]GTP (data not shown), was identified as
tubulin, based on Western blot analysis. To further characterize this
interaction, we made a GST fusion construct containing just the PH
domain of Lfc (Fig. 9A). When the GST-Lfc (PH) fusion
protein was expressed in E. coli and used as an affinity
reagent, it was able to bind tubulin in an essentially identical manner
as we originally observed with the DH/PH domain construct (Fig.
9B, fourth lane). The GST-Dbl (PH) domain was
unable to bind tubulin under the same experimental conditions (data not
shown). Taken together, these findings identify tubulin as a putative
binding partner for the PH domain of Lfc and suggest that it is the PH
domain that is responsible for the co-localization of Lfc with
microtubules in cells.

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Fig. 9.
Lfc binds tubulin in vitro.
A, a schematic representation of the GST-fusion proteins
used for affinity precipitation of tubulin from Jurkat T-cell lysates.
GST-Lfc (DH-PH) was purified from Sf-21 insect cells, and
GST-Lfc (PH) was purified from E. coli. B,
GST-fusion proteins of Lfc (DH/PH) and Lfc (PH)
(~5 µg) were immobilized on glutathione-agarose beads and used in
affinity precipitations from Jurkat cell lysates. Proteins were
separated by SDS-PAGE and Western blotted using an anti-tubulin
antibody. GST (~15 µg) bound to glutathione-agarose was used as a
negative control.
|
|
Conclusions--
Lfc serves as a potent nucleotide exchange factor
for RhoA in vitro and is able to induce actin stress fibers
when overexpressed in NIH 3T3 cells. However, the fact that we have
shown that Lfc forms a tight binding complex with Rac1 in
vitro, together with the observation that Lfc stimulates JNK in
COS-7 cells, led us to examine whether Lfc promotes or stabilizes the
activated form of Rac1 in cells. The activation of JNK by Vav, Ost, and
Dbl have all been shown to be mediated by Rac1, such that the dominant negative Rac1(S17N) mutant blocks their ability to activate JNK (16,
29). The data presented here indicate that Rac1 also mediates the
activation of JNK by Lfc in COS-7 cells. Lfc-stimulated JNK activity
was inhibited by expression of the dominant negative form of Rac1, but
not by dominant negative Cdc42. Dominant negative mutants of Pak3 and
MLK3, two targets of Rac1, also inhibited Lfc-stimulated JNK activity.
Relatively higher levels of expression of dominant negative RhoA also
inhibited the Lfc-stimulated activation of JNK. Given that RhoA(T19N)
was less effective than Rac1(S17N) in blocking JNK activation suggests
that Lfc may show a preference for binding Rac1 compared with RhoA in
COS-7 cells. The ability of Lfc to stimulate a Rac1-mediated JNK
pathway may be favored by a specific cellular localization. It is
possible that the demonstrated localization of Lfc to microtubules
favors interactions with Rac1, which has also been reported to localize
to microtubules (27). A recent report has also demonstrated that both
MLK and JNK are localized to microtubules (28), again supporting the
concept of a specific localization and proximity of a full Rac1
signaling pathway. The biochemical data presented here indicate that
the localization of Lfc to microtubules may be mediated by the binding
of its PH domain to tubulin.
An obvious question is how does Lfc promote Rac1-signaling? Typically,
a GEF binds with the highest affinity to the nucleotide-depleted state
of a GTP-binding protein (31). Such a preference is consistent with the
fact that GEFs, by stabilizing the nucleotide-depleted state, stimulate
GDP dissociation from the G protein and enable GDP-GTP exchange to
occur. As we previously reported, this is the case for the interaction
of Lfc with RhoA, but not with Rac1 (22). The in vitro
interaction of Lfc with Rac1 shows no nucleotide specificity. Thus far,
we have not been able to detect an Lfc-stimulated increase in the
amount of GTP-bound Rac1 in cells, as read-out by the ability of
GTP-bound Rac1 to form a complex with its binding domain on Pak3 (Fig.
6). We also have not been able to directly detect Lfc-mediated Rac1
activation when co-expressing Lfc with potential upstream activators,
such as an activated Src mutant (Src(Y527F)), GTPase-defective Ras
(Ras(G12V)), or a constitutively activate form of the
PI3-kinase (data not shown). In addition, we have been
unable to detect Lfc-stimulated nucleotide exchange activity on Rac1
in vitro using an insect cell-expressed GST fusion protein
of Lfc that contains the DH and PH domains (22), nor have we been able
to detect nucleotide exchange activity using immunoprecipitated Lfc
from lysates of COS-7 cells overexpressing the protein (data not
shown). Thus, our data raise the possibilities that Lfc regulates
Rac1-mediated signaling and stimulates JNK activation and/or actin
cytoskeletal changes by an alternative mechanism, for example by
recruiting activated Rac-1 to microtubules. This could bring GTP-bound
Rac1 to a site proximal to MLK or another appropriate Rac1 target(s)
that initiates a pathway leading to JNK activation and/or changes in
the actin cytoskeleton and cell morphology. In this model, dominant
negative Rac, by either directly preventing the activation of
endogenous Rac molecules or by binding Lfc and competitively inhibiting
the binding of activated (GTP-bound) Rac, would prevent the
Lfc-mediated activation of Rac-signaling. Such a mechanism would also
explain why we cannot detect an Lfc-stimulated increase in the cellular
levels of activated Rac1 and would be consistent with the reported
GTP-dependent association of Rac1 with microtubules (27).
Thus, rather than acting as a GEF, Lfc may serve to recruit Rac to the
appropriate cellular location such that it can engage the necessary
signaling partners for efficiently activating JNK and/or for triggering
the necessary actin cytoskeletal changes that lead to lamellipodia formation.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
grant GM45478 (to R. A. C.).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.
The abbreviations used are:
GEF, guanine
nucleotide exchange factor; DH, Dbl homology; PH, Pleckstrin homology; Pak, p21-activated kinase; MLK, mixed lineage kinase; JNK, c-Jun
kinase; MBP, myelin basic protein; PBD, p21 (Cdc42/Rac1) binding
domain; PCR, polymerase chain reaction; PAGE, polyacrylamide gel
electrophoresis; GST, glutathione S- transferase.
 |
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