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Leucine Zipper-mediated Homodimerization of the p21-activated Kinase-interacting Factor, beta Pix

IMPLICATION FOR A ROLE IN CYTOSKELETAL REORGANIZATION*

Seyun Kim, Seung-Hye Lee, and Dongeun ParkDagger

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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Pix, a p21-activated kinase-interacting exchange factor, is known to be involved in the regulation of Cdc42/Rac GTPases. The 85-kDa beta 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, beta 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 beta Pix-a homodimers. Using in vitro and in vivo methodologies, we show that deletion of the leucine zipper domain is sufficient to abolish beta Pix-a homodimerization. In NIH3T3 fibroblast cells, expression of wild type beta 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 beta Pix-a to homodimerize, and homodimerization is essential for beta Pix-a signaling functions leading to the cytoskeletal reorganization.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 beta Pix or p85Cool-1) and suggested as a putative GEF for Cdc42/Rac1 (12, 13). Two alternative spliced forms of beta Pix, named beta Pix-b and beta Pix-c, that are predominantly expressed in nervous tissue were also reported (14). beta Pix-a, which corresponds to beta 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, beta 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 beta Pix-a include a proline-rich region and a putative leucine zipper domain at the C terminus (11). Recently, beta 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 beta Pix-a (16-18).

Earlier studies have focused on the interactions of beta 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 beta Pix homodimerizes through its leucine zipper domain in vitro and in vivo. This ability of beta Pix to homodimerize was found to be necessary for the beta Pix-mediated membrane ruffle formation in NIH3T3 fibroblast. Our results suggest that beta Pix-a homodimerization plays an essential role in beta Pix-a signaling leading to the cytoskeletal reorganization.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Plasmid Construction-- Schematic diagrams of the various beta Pix-a expression constructs used in this study are shown in Fig. 1A. Expression plasmids for hemagglutinin (HA)-tagged beta Pix-a, FLAG-tagged beta Pix-a, and Myc-tagged beta Pix-a were constructed by subcloning the polymerase chain reaction-amplified cDNA fragments from beta Pix-a.

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 beta Pix-a. The recombinant vector was transformed into Escherichia coli DH5alpha , and the fusion proteins were induced with isopropyl-1-thio-beta -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.

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 beta 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

To address whether beta Pix-a forms dimers (or oligomers), we first tested the ability of full-length beta Pix-a to form oligomers by a co-immunoprecipitation approach. For this study, two different epitope-tagged (FLAG and Myc) beta Pix-a expression vectors were transfected either alone or together into COS7 cells. Anti-Myc antibody, 9E10, was used to immunoprecipitate the putative beta Pix-a complexes. As shown in Fig. 1B, the FLAG-tagged form of beta Pix-a was detected only when co-expressed with Myc-tagged beta Pix-a, indicating that these different epitope-tagged forms of beta Pix-a do, in fact, form oligomers in vivo when overexpressed in COS7 cells. We next examined whether the domain of beta Pix-a that is responsible for the oligomerization resides in the N or C terminus of the beta Pix-a. For this purpose, the GST-SH3 domain and the GST-C-terminal half of beta Pix-a were used for GST pulldown assay using Myc-tagged beta Pix-a overexpressed in COS7 cell lysates. As shown in Fig. 1C, the GST-C-terminal half could form a complex with Myc-tagged beta Pix-a, whereas the GST-SH3 domain failed to associate with the beta Pix-a. These results indicate that the possible intermolecular interaction of the beta Pix-a SH3 domain with its own proline-rich region is not involved in beta Pix-a oligomerization. Thus, we focused on the involvement of the C-terminal leucine zipper domain in the oligomerization of beta Pix-a.



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Fig. 1.   Oligomerization of beta Pix-a is mediated by the C terminus of beta Pix-a. A, schematic representation of mouse beta Pix-a constructs (SH3, DH, PH, PXXP (proline-rich region), GBD (GIT1-binding domain), and LZ). B, beta Pix-a forms oligomers when overexpressed in COS7 cells. FLAG- and Myc-tagged beta 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 beta Pix-a requires the GST-C terminus of beta Pix-a in vitro. COS7 cells were transiently transfected with Myc-tagged beta 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 beta Pix-a contains several leucines that are spaced seven residues apart, a feature characteristic of leucine-zippered coiled-coil domains. Thus, we hypothesized that beta Pix-a could dimerize/oligomerize through this putative leucine zipper sequence. To better characterize the leucine zipper domain, we analyzed the sequences of mouse beta 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 beta 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 beta Pix of other species and alpha 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 beta Pix the LZ domain.



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Fig. 2.   The leucine zipper domain of beta Pix-a is predicted to mediate dimer formation. A, beta Pix-a amino acid sequences were analyzed using the Multicoil program, which predicts the presence of coiled-coils and the probability for dimer and trimer formation based on pairwise residue interactions. The probabilities for dimer (blue) and trimer (red), as well as overall score (black), are shown. B, alignment of the predicted dimerization regions of beta Pix-a proteins shows heptad repeats with characteristically placed hydrophobic residues. The critical amino acid residues forming the heptad repeat are shown in bold.

To confirm a direct interaction between the beta Pix-a leucine zipper domain and itself, an HA-tagged C-terminal half of beta Pix-a was transcribed and translated in vitro either alone or in combination with Myc-tagged full-length beta Pix-a (beta Pix-aWT) or leucine zipper domain-deleted beta Pix-a (beta Pix-aDelta LZ). As shown in Fig. 3A, beta Pix-aWT was co-immunoprecipitated with the HA-tagged C-terminal region, but beta Pix-aDelta LZ failed to co-immunoprecipitate. To determine whether the leucine zipper domain is required for beta Pix-a homodimerization in vivo, Myc-tagged beta Pix-aWT and beta Pix-aDelta LZ were transiently transfected in COS7 cells with FLAG-tagged beta Pix-aWT. As shown in Fig. 3B, FLAG-tagged beta Pix-aWT co-immunoprecipitated with Myc-tagged beta Pix-aWT but not with Myc-tagged beta Pix-aDelta LZ. These data demonstrate that beta Pix-a homodimerizes in cells in a leucine zipper domain-dependent manner.



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Fig. 3.   Homodimerization of beta Pix-a is mediated by the leucine zipper domain. A, in vitro dimerization of beta Pix-a. HA-tagged beta Pix C-terminal cDNA was translated in vitro alone or together with beta Pix-aWT or beta Pix-aDelta LZ as indicated in the presence of [35S]methionine (left panel, IVT). Immunoprecipitates of each translated products with anti-HA antibody were shown in the right panel (IP). Each sample was run on a 10% SDS-PAGE and exposed to autoradiography. B, beta Pix-a homodimerization via LZ domain in vivo. FLAG-tagged beta Pix-aWT was transiently expressed in COS7 cells either alone or together with the Myc-tagged beta Pix-aWT or beta Pix-aDelta LZ as indicated. Anti-Myc antibody was used for immunoprecipitation (IP) and anti-FLAG antibody for immunoblotting (IB).

Studies in transfected cells have shown that beta 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 beta Pix-a-mediated cytoskeletal reorganization such as membrane ruffle formation, NIH3T3 fibroblast cells were transfected with either Myc-tagged beta Pix-aWT or beta Pix-aDelta LZ cDNAs. Expression of beta Pix-aWT caused formation of membrane ruffle at cell periphery, and beta Pix-aWT is concentrated at F-actin-rich membrane ruffle structures (Fig. 4, B and C). However, in cells expressing beta Pix-aDelta LZ, morphological changes were not exhibited, and beta Pix-aDelta 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 beta Pix-aWT exhibited polarized membrane ruffles in response to PDGF (Fig. 4, G and H). The extent of membrane ruffling in cells expressing beta 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 beta Pix-aDelta LZ indicates that beta Pix-aDelta LZ could function as a dominant inhibitory mutant. Diffuse cytoplasmic localization of beta Pix-aDelta LZ suggests that it might sequester the downstream effector molecules, such as Pak and/or other beta Pix-a interacting proteins, essential for membrane ruffling at the cytoplasm. To examine the importance of the C terminus in beta Pix-a function, the effect of co-expression of beta Pix-aWT with beta Pix-a C-ter was investigated. Cells expressing beta Pix-aWT alone exhibited membrane ruffles as expected (Fig. 5A). However, beta Pix-aWT-induced membrane ruffling was inhibited in cells co-expressing beta Pix-aWT with beta Pix-a C-ter (Fig. 5B). Quantitation of membrane ruffle-bearing cells showed that co-expression of beta Pix-a C-ter inhibits membrane ruffling in 60-70% of cells expressing beta Pix-aWT (Fig. 5C).



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Fig. 4.   Overexpression of a beta Pix-aDelta LZ mutant in NIH3T3 fibroblast cells inhibits membrane ruffle formation in response to PDGF. NIH3T3 fibroblast cells untransfected or transfected with Myc-tagged beta Pix-aWT or beta Pix-aDelta LZ were serum-starved for 16 h and fixed before (A-E) or after (F-J) stimulation with 20 ng/ml PDGF for 10 min. beta Pix distribution was visualized using an anti-Myc antibody, 9E10, and fluorescine-conjugated secondary antibody. Actin distribution was visualized with rhodamine-conjugated phalloidin.



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Fig. 5.   Co-expression of beta Pix-a C-ter blocked beta Pix-a-induced membrane ruffling in NIH3T3 fibroblast cells. Cells were transfected with Myc-tagged beta Pix-aWT alone (A) or together with HA-tagged beta Pix-a C-ter (B), serum-starved for 16 h, and fixed. Double immunofluorescence staining for Myc-beta Pix-a and HA-C-ter were carried out using anti-beta Pix-a antiserum (30,000:1 diluted) or anti-HA antibody (data not shown), respectively. Quantitation of a typical result from three independent experiments is shown in C. Values are expressed as the percentage of membrane ruffle-bearing cells over all transfected cells. Data represent means ± S.E. from three different fields.

In this study, we demonstrated that the leucine zipper domain at the C-terminal end of beta Pix-a mediates the formation of beta Pix-a homodimers in vitro and in vivo. The leucine zipper domain is an alpha -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 beta Pix-a-mediated cytoskeletal reorganization, we found that the deletion of the leucine zipper domain and the resulting loss of homodimerization made beta Pix-a fail to induce the formation of membrane ruffles in NIH3T3 fibroblasts. These results suggest that the leucine zipper domain of beta Pix-a plays an important role in the regulation of the beta 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, beta Pix-a homodimerization is required for the PDGF receptor-mediated signaling cascade leading to the morphological changes.

beta 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 beta Pix proteins could function as molecular bridges to recruit and juxtapose other beta Pix-binding proteins such as Pak and Nck within the signaling complex. Dimer formation could also mask or expose functional domains or residues of beta 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 beta Pix heterodimerizes with other leucine zipper domain-containing proteins or other beta Pix isoforms such as beta Pix-b and beta Pix-c (14). In our preliminary data, beta Pix-a could interact with beta Pix-b and beta Pix-c.2 Because three beta Pix isoforms differ in the primary structure at the C terminus, these differences may affect the abilities of beta Pix isoforms to interact with each other or form distinct signaling complexes, and such heterodimerization could be crucial to understand the function of beta Pix isoforms.

In conclusion, we have shown that the putative leucine zipper domain at the C terminus of beta Pix-a mediates the formation of homodimers and that the homodimerization of beta Pix is required for the cytoskeletal changes by beta Pix-a overexpression. Moreover, beta 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 beta Pix-a homodimerization will provide additional important insights in the regulation of beta Pix activities and functions.


    FOOTNOTES

* 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.

Dagger 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.


    ABBREVIATIONS

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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