From the School of Biological Sciences, Seoul National University, Seoul 151-742, Republic of Korea
Received for publication, November 14, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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Pix, a p21-activated kinase-interacting exchange
factor, is known to be involved in the regulation of Cdc42/Rac GTPases.
The 85-kDa The Rho family GTPases, which include Rho, Rac, and Cdc42,
function as molecular switches in a variety of cellular signaling pathways, many of which regulate the cell cytoskeletal organization and
affect on physiological properties of cells such as cell motility (1-3). In fibroblasts, RhoA induces stress fibers associated with
focal adhesions, Rac1 produces lamellipodia or membrane ruffles, and
Cdc42 induces filopodia on the plasma membrane (4-6). Similar to all
members of the Ras superfamily GTPase, the activity of Rho family
proteins cycles between active GTP-bound and inactive GDP-bound
conformational states, regulated by three kinds of proteins: GTPase
activating proteins, guanine nucleotide dissociation inhibitors, and
guanine nucleotide exchange factors
(GEFs1) (7).
All members of the Dbl family possess a Dbl homology (DH) domain in
tandem with a Pleckstrin homology (PH) domain. The DH domain typically
represents the motif for binding the Rho family GTPases and stimulating
GDP/GTP exchange, whereas the PH domain appears to be essential for
mediating the appropriate cellular localization of the protein (8-10).
Additionally, most members of the GEFs contain a number of other
structural motifs that indicate a role in signal transduction. These
domains presumably function to mediate protein/protein or protein/lipid
interactions and serve to link GEFs to upstream regulators and
downstream effectors (8).
Previously, we reported p85SPR (an SH3 domain-containing proline-rich
protein) as a widely expressed focal protein (11). The same protein was
identified as a p21-activated kinase (Pak)-interacting exchange factor
(named Earlier studies have focused on the interactions of Plasmid Construction--
Schematic diagrams of the various
In Vitro Transcription and Translation--
In vitro
transcription and translation were performed with the TNT
reticulocyte lysate system (Promega). 1 µg of supercoiled plasmid was
used according to the manufacturer's protocol. The 50-µl reaction
mixture contained 25 µl of TNT rabbit reticulocyte lysate, TNT
reaction buffer, T7 RNA polymerase, 20 units of RNasin, and amino acid
mixture without methionine, supplemented with
[35S]methionine as the radioactive precursor (1000 Ci/mmol; PerkinElmer Life Sciences). After a 90-min incubation
at 30 °C, the reaction mixture was used for co-immunoprecipitation assay.
GST Fusion Proteins and Pulldown Assay--
GST-SH3 domain and
GST-C-terminal fusion proteins were prepared for this study. Primers
were taken directly from the published cDNA sequence of Cell Lysis and Immunoprecipitation--
Transfected cells were
lysed on ice with 1 ml per 100-mm dish of ice-cold lysis buffer (50 mM HEPES, pH 7.3, 150 mM NaCl, 15 mM NaF, 2 mM EDTA, 1% Nonidet P-40, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl
fluoride, 1 mg/ml leupeptin, 10 µg/ml aprotinin). After 20 min,
lysates were precleared by centrifugation at 20,000 × g for 20 min at 4 °C and immunoprecipitated with a
primary antibody for 2 h followed by incubation with protein
A-Sepharose for 1 h. Immunoprecipitates were washed three times
with the same lysis buffer, and samples were resolved by SDS-PAGE.
Proteins were transferred to Immobilon P membranes (Millipore) and
subjected to immunoblotting analysis. All immunoblots were developed
using enhanced chemiluminescence.
Cell Culture, Transfection, and Immunofluorescence
Microscopy--
COS7 cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) containing 10% fetal bovine serum and
maintained in 10% CO2 at 37 °C. Mouse NIH3T3 fibroblast
cells were grown in medium consisting of DMEM containing 10% bovine
calf serum, 100 IU/ml penicillin, and 50 µg/ml streptomycin and
maintained at 37 °C in an atmosphere of 5% CO2. The
cells were transfected using LipofectAMINE Plus reagent (Life
Technologies, Inc.) according to the manufacturer's protocol. 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
rhodamine-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. Myc-tagged To address whether Pix-a protein contains an Src homology 3 domain,
the tandem Dbl homology and Pleckstrin homology domains, a proline-rich
region, and a GIT1-binding domain. In addition to those domains,
Pix-a also contains a putative leucine zipper domain at the
C-terminal end. In this study, we demonstrate that the
previously identified putative leucine zipper domain mediates the
formation of
Pix-a homodimers. Using in vitro and
in vivo methodologies, we show that deletion of the leucine
zipper domain is sufficient to abolish
Pix-a homodimerization. In
NIH3T3 fibroblast cells, expression of wild type
Pix-a induces the
formation of membrane ruffles. However, cells expressing the leucine
zipper domain deletion mutant could not form membrane ruffle
structures. Moreover, platelet-derived growth factor-mediated
cytoskeletal changes were completely blocked by the leucine zipper
domain deletion mutant. The results suggest that the leucine
zipper domain enables
Pix-a to homodimerize, and homodimerization is
essential for
Pix-a signaling functions leading to the cytoskeletal reorganization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Pix or p85Cool-1) and suggested as a putative GEF for
Cdc42/Rac1 (12, 13). Two alternative spliced forms of
Pix, named
Pix-b and
Pix-c, that are predominantly expressed in nervous
tissue were also reported (14).
Pix-a, which corresponds to
Pix/p85Cool-1/p85SPR, is a multidomain protein with many potential binding sites that can mediate protein-protein interactions. In addition to the conventional tandem, a DH domain and a PH domain,
Pix-a has an SH3 domain that directly binds to the proline-rich region of Pak, an important downstream effector in cellular signaling governed by Cdc42/Rac GTPases (12, 15). Other domains of
Pix-a include a proline-rich region and a putative leucine zipper domain at
the C terminus (11). Recently,
Pix-a was reported to interact with
ADP ribosylation factor-GTPase activating proteins such as GIT
(G-protein-coupled receptor kinase-interacting
targets), p95PKL (paxillin-kinase
linker), and Cat (cool-associated,
tyrosine-phosphorylated) through the GIT1-binding
domain of the C-terminal end of
Pix-a (16-18).
Pix with other
signaling proteins but have not considered the possibility of its
self-association. The leucine zipper domain is known to mediate the
formation of homo- or heterodimers in various kinds of proteins such as
transcription factor, adaptor, and kinase and regulate the functions of
the proteins (19). In this study, we demonstrated that
Pix
homodimerizes through its leucine zipper domain in vitro and
in vivo. This ability of
Pix to homodimerize was found to
be necessary for the
Pix-mediated membrane ruffle formation in
NIH3T3 fibroblast. Our results suggest that
Pix-a homodimerization
plays an essential role in
Pix-a signaling leading to the
cytoskeletal reorganization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Pix-a expression constructs used in this study are shown in Fig.
1A. Expression plasmids for hemagglutinin (HA)-tagged
Pix-a, FLAG-tagged
Pix-a, and Myc-tagged
Pix-a were
constructed by subcloning the polymerase chain reaction-amplified
cDNA fragments from
Pix-a.
Pix-a.
The recombinant vector was transformed into Escherichia
coli DH5
, and the fusion proteins were induced with
isopropyl-1-thio-
-D-galactopyranoside and
affinity-purified according to the standard protocol of Amersham
Pharmacia Biotech. 2 µg of GST fusion proteins was incubated with 1 mg of cell lysates in a volume of 1 ml. To immobilize the GST protein,
glutathione-agarose beads equilibrated in binding buffer were added to
the reaction mixture and incubated for 1 h at 4 °C under
constant rotation. After washing the resin with the same buffer, the
bound proteins were analyzed by immunoblotting.
Pix was detected by incubating with anti-Myc
antibody, 9E10, for 1 h followed by a fluorescein-conjugated
anti-IgG-specific secondary antibody for 1 h. Cells were observed
and photographed under a Zeiss fluorescence microscope.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Pix-a forms dimers (or oligomers), we first
tested the ability of full-length
Pix-a to form oligomers by a
co-immunoprecipitation approach. For this study, two different epitope-tagged (FLAG and Myc)
Pix-a expression vectors were
transfected either alone or together into COS7 cells. Anti-Myc
antibody, 9E10, was used to immunoprecipitate the putative
Pix-a
complexes. As shown in Fig.
1B, the FLAG-tagged form of
Pix-a was detected only when co-expressed with Myc-tagged
Pix-a,
indicating that these different epitope-tagged forms of
Pix-a do, in
fact, form oligomers in vivo when overexpressed in COS7
cells. We next examined whether the domain of
Pix-a that is
responsible for the oligomerization resides in the N or C
terminus of the
Pix-a. For this purpose, the GST-SH3 domain
and the GST-C-terminal half of
Pix-a were used for GST pulldown
assay using Myc-tagged
Pix-a overexpressed in COS7 cell lysates. As
shown in Fig. 1C, the GST-C-terminal half could form a
complex with Myc-tagged
Pix-a, whereas the GST-SH3 domain failed to
associate with the
Pix-a. These results indicate that the possible
intermolecular interaction of the
Pix-a SH3 domain with its own
proline-rich region is not involved in
Pix-a oligomerization. Thus,
we focused on the involvement of the C-terminal leucine zipper domain
in the oligomerization of
Pix-a.
View larger version (30K):
[in a new window]
Fig. 1.
Oligomerization of
Pix-a is mediated by the C terminus of
Pix-a. A, schematic representation
of mouse
Pix-a constructs (SH3, DH,
PH, PXXP (proline-rich region), GBD
(GIT1-binding domain), and LZ). B,
Pix-a forms
oligomers when overexpressed in COS7 cells. FLAG- and Myc-tagged
Pix-a were transiently expressed in COS7 cells either alone or
together as indicated. Anti-Myc antibody was used for
immunoprecipitation (IP), and anti-FLAG antibody was used
for immunoblotting (IB). C, oligomerization of
Pix-a requires the GST-C terminus of
Pix-a in vitro.
COS7 cells were transiently transfected with Myc-tagged
Pix-a, and
the cell lysates were incubated with 2 µg of immobilized GST fusion
proteins for 2 h at 4 °C, and samples were then washed and
subjected to SDS-PAGE and immunoblotting with anti-Myc antibody.
The region between amino acid 587 and 634 of mouse Pix-a contains
several leucines that are spaced seven residues apart, a feature
characteristic of leucine-zippered coiled-coil domains. Thus, we
hypothesized that
Pix-a could dimerize/oligomerize through this
putative leucine zipper sequence. To better characterize the leucine
zipper domain, we analyzed the sequences of mouse
Pix-a by using the
program Multicoil (20). As shown in Fig. 2A, the Multicoil program
predicted the existence of a coiled-coil domain capable of forming a
parallel side by side homodimer in
Pix-a. Although the dimer
probability is very high and considered significant, the trimer peaks
at the shoulders of the dimer are due to artifacts of the algorithm and
are not significant. Although we cannot exclude formation of high order
oligomers, we assume and will refer to this interaction as
dimerization. The alignment of
Pix of other species and
Pix
leucine zipper domains is shown in Fig. 2B. The key features
of the leucine zipper sequence are well conserved in all Pix proteins.
In the search using the BLAST network, a number of other GEFs such as
p115Rho-GEF and GEF-H1 did not show any homology with
Pix the LZ
domain.
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To confirm a direct interaction between the Pix-a leucine zipper
domain and itself, an HA-tagged C-terminal half of
Pix-a was
transcribed and translated in vitro either alone or in
combination with Myc-tagged full-length
Pix-a (
Pix-aWT) or
leucine zipper domain-deleted
Pix-a (
Pix-a
LZ). As shown in
Fig. 3A,
Pix-aWT was
co-immunoprecipitated with the HA-tagged C-terminal region, but
Pix-a
LZ failed to co-immunoprecipitate. To determine whether the
leucine zipper domain is required for
Pix-a homodimerization in vivo, Myc-tagged
Pix-aWT and
Pix-a
LZ were
transiently transfected in COS7 cells with FLAG-tagged
Pix-aWT. As
shown in Fig. 3B, FLAG-tagged
Pix-aWT
co-immunoprecipitated with Myc-tagged
Pix-aWT but not with
Myc-tagged
Pix-a
LZ. These data demonstrate that
Pix-a
homodimerizes in cells in a leucine zipper domain-dependent manner.
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Studies in transfected cells have shown that Pix-a has the
potential to localize to focal adhesions and can promote the formation of membrane ruffles that co-localize with focal adhesions (12). To
examine a role for the leucine zipper domain in
Pix-a-mediated cytoskeletal reorganization such as membrane ruffle formation, NIH3T3
fibroblast cells were transfected with either Myc-tagged
Pix-aWT or
Pix-a
LZ cDNAs. Expression of
Pix-aWT caused formation of
membrane ruffle at cell periphery, and
Pix-aWT is concentrated at
F-actin-rich membrane ruffle structures (Fig.
4, B and C). However, in cells expressing
Pix-a
LZ, morphological changes were
not exhibited, and
Pix-a
LZ showed a diffuse cytoplasmic localization (Fig. 4, D and E). Next,
serum-starved cells were stimulated with platelet-derived growth factor
(PDGF) for 10 min to induce membrane ruffles. Cells expressing
Pix-aWT exhibited polarized membrane ruffles in response to PDGF
(Fig. 4, G and H). The extent of membrane
ruffling in cells expressing
Pix-aWT was comparable with that in
PDGF-stimulated nontransfected cells (Fig. 4, A and
F). However, surprisingly, cells expressing the leucine
zipper domain deletion mutant did not show any apparent ruffle-like
structures (Fig. 4, I and J). Blocking of
PDGF-induced membrane ruffling in cells expressing
Pix-a
LZ
indicates that
Pix-a
LZ could function as a dominant inhibitory
mutant. Diffuse cytoplasmic localization of
Pix-a
LZ suggests that
it might sequester the downstream effector molecules, such as Pak
and/or other
Pix-a interacting proteins, essential for membrane
ruffling at the cytoplasm. To examine the importance of the C terminus
in
Pix-a function, the effect of co-expression of
Pix-aWT with
Pix-a C-ter was investigated. Cells expressing
Pix-aWT
alone exhibited membrane ruffles as expected (Fig.
5A). However,
Pix-aWT-induced membrane ruffling was inhibited in cells
co-expressing
Pix-aWT with
Pix-a C-ter (Fig. 5B).
Quantitation of membrane ruffle-bearing cells showed that co-expression
of
Pix-a C-ter inhibits membrane ruffling in 60-70% of cells
expressing
Pix-aWT (Fig. 5C).
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In this study, we demonstrated that the leucine zipper domain at
the C-terminal end of Pix-a mediates the formation of
Pix-a homodimers in vitro and in vivo. The leucine
zipper domain is an
-helical structure formed by several heptad
repeats of hydrophobic residues, usually leucine and isoleucine, that
are commonly found in nuclear transcription factors, and its role in
promoting the homo- and heterodimerization of these proteins has been
well characterized (21, 22). Leucine zipper domains have also been
identified in many other proteins such as protein kinases, adaptors,
and cytoskeletal proteins, but their function in these proteins has been less extensively studied. Recently, it has been reported that the
leucine zipper domain-dependent homodimerization of a ZIP kinase, a serine/threonine kinase, is necessary for their activity (23).
In the functional study of the leucine zipper domain in
Pix-a-mediated cytoskeletal reorganization, we found that the
deletion of the leucine zipper domain and the resulting loss of
homodimerization made
Pix-a fail to induce the formation of membrane
ruffles in NIH3T3 fibroblasts. These results suggest that the leucine
zipper domain of
Pix-a plays an important role in the regulation of the
Pix-a function. Recently, Yoshii et al. (24) reported
that Pix could form a complex with PDGF receptor and mediate Cdc42/Rac signaling by PDGF stimulation. Therefore, considering our results that
the leucine zipper domain deletion mutant blocked completely the
morphological changes of NIH3T3 cells in response to PDGF,
Pix-a
homodimerization is required for the PDGF receptor-mediated signaling
cascade leading to the morphological changes.
Pix is, to our knowledge, the first GEF protein that is
demonstrated to have the ability to homodimerize through a leucine zipper domain-dependent mechanism. The roles of the
coiled-coil domain of other GEFs have been reported previously. The
leucine zipper domain-mediated interaction between human
immunodeficiency virus (HIV)-1 transmembrane protein gp41 and
p115-RhoGEF is essential for the HIV replication cycle and
p115-RhoGEF-mediated stress fiber formation (25). In the case of
GEF-H1, the coiled-coil domain is essential for the GEF-H1 microtubule
co-localization (26). Dimeric
Pix proteins could function as
molecular bridges to recruit and juxtapose other
Pix-binding
proteins such as Pak and Nck within the signaling complex. Dimer
formation could also mask or expose functional domains or residues of
Pix-a as a result of conformational changes that might be induced by
dimerization. For example, the deletion of the leucine zipper domain of
c-Cbl, a multidomain adaptor protein, caused a decrease in both the
tyrosine phosphorylation of Cbl and its association with the epidermal growth factor receptor (27). Finally, it is also possible that
Pix
heterodimerizes with other leucine zipper domain-containing proteins or
other
Pix isoforms such as
Pix-b and
Pix-c (14). In our
preliminary data,
Pix-a could interact with
Pix-b and
Pix-c.2 Because
three
Pix isoforms differ in the primary structure at the C
terminus, these differences may affect the abilities of
Pix isoforms
to interact with each other or form distinct signaling complexes, and
such heterodimerization could be crucial to understand the function of
Pix isoforms.
In conclusion, we have shown that the putative leucine zipper
domain at the C terminus of Pix-a mediates the formation of homodimers and that the homodimerization of
Pix is required for the
cytoskeletal changes by
Pix-a overexpression. Moreover,
Pix-a homodimerization plays essential roles in PDGF-mediated signaling pathway leading to morphological changes. Further studies on the role
of the leucine zipper domain and
Pix-a homodimerization will provide
additional important insights in the regulation of
Pix activities
and functions.
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FOOTNOTES |
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* This work was supported in part by Korea Science Foundation (KOSEF) Grant 1998G0202 through Center for Cell Signaling Research and by KOSEF Grant 97-0401-07-01-5. S. K. and S.-H. L. were supported by Fellowship BK21 from the Korean Ministry of Education.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: School of Biological
Sciences, Seoul National University, Kwanak-gu, Shilim-dong, Seoul
151-742, Republic of Korea. Tel.: 82-2-880-5753; Fax: 82-2-872-1993; E-mail: depark@snu.ac.kr.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.C000806200
2 Unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: GEF(s), guanine nucleotide exchange factor(s); DH, Dbl homology domain; PH, Pleckstrin homology domain; SH3, Src homology 3 domain; Pak, p21-activated kinase; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; LZ, leucine zipper; PDGF, platelet-derived growth factor; HIV, human immunodeficiency virus; C-ter, C terminus.
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