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• W2037844242 abstract "Neuritogenesis requires active actin cytoskeleton rearrangement in which Rho GTPases play a pivotal role. In a previous study (Shin, E. Y., Woo, K. N., Lee, C. S., Koo, S. H., Kim, Y. G., Kim, W. J., Bae, C. D., Chang, S. I., and Kim, E. G. (2004) J. Biol. Chem. 279, 1994-2004), we demonstrated that βPak-interacting exchange factor (βPIX) guanine nucleotide exchange factor (GEF) mediates basic fibroblast growth factor (bFGF)-stimulated Rac1 activation through phosphorylation of Ser-525 and Thr-526 at the GIT-binding domain (GBD). However, the mechanism by which this phosphorylation event regulates the Rac1-GEF activity remained elusive. We show here that βPIX binds to Rac1 via the GBD and also activates the GTPase via an associated GEF, smgGDS, in a phosphorylation-dependent manner. Notably, the Rac1-GEF activity of βPIX persisted for an extended period of time following bFGF stimulation, unlike other Rho GEFs containing the Dbl homology domain. We demonstrate that C-PIX, containing proline-rich, GBD, and leucine zipper domains can interact with Rac1 via the GBD in vitro and in vivo and also mediated bFGF-stimulated Rac1 activation, as determined by a modified GEF assay and fluorescence resonance energy transfer analysis. However, nonphosphorylatable C-PIX (S525A/T526A) failed to generate Rac1-GTP. Finally, βPIX is shown to form a trimeric complex with smgGDS and Rac1; down-regulation of smgGDS expression by short interfering RNA causing significant inhibition of βPIX-mediated Rac1 activation and neurite outgrowth. These results provide evidence for a new and unexpected mechanism whereby βPIX can regulate Rac1 activity. Neuritogenesis requires active actin cytoskeleton rearrangement in which Rho GTPases play a pivotal role. In a previous study (Shin, E. Y., Woo, K. N., Lee, C. S., Koo, S. H., Kim, Y. G., Kim, W. J., Bae, C. D., Chang, S. I., and Kim, E. G. (2004) J. Biol. Chem. 279, 1994-2004), we demonstrated that βPak-interacting exchange factor (βPIX) guanine nucleotide exchange factor (GEF) mediates basic fibroblast growth factor (bFGF)-stimulated Rac1 activation through phosphorylation of Ser-525 and Thr-526 at the GIT-binding domain (GBD). However, the mechanism by which this phosphorylation event regulates the Rac1-GEF activity remained elusive. We show here that βPIX binds to Rac1 via the GBD and also activates the GTPase via an associated GEF, smgGDS, in a phosphorylation-dependent manner. Notably, the Rac1-GEF activity of βPIX persisted for an extended period of time following bFGF stimulation, unlike other Rho GEFs containing the Dbl homology domain. We demonstrate that C-PIX, containing proline-rich, GBD, and leucine zipper domains can interact with Rac1 via the GBD in vitro and in vivo and also mediated bFGF-stimulated Rac1 activation, as determined by a modified GEF assay and fluorescence resonance energy transfer analysis. However, nonphosphorylatable C-PIX (S525A/T526A) failed to generate Rac1-GTP. Finally, βPIX is shown to form a trimeric complex with smgGDS and Rac1; down-regulation of smgGDS expression by short interfering RNA causing significant inhibition of βPIX-mediated Rac1 activation and neurite outgrowth. These results provide evidence for a new and unexpected mechanism whereby βPIX can regulate Rac1 activity. Rho GTPases regulate cytoskeletal dynamics and thus play a pivotal role in a wide range of biological responses, including cell polarity, cell adhesion and migration, and neuritogenesis (1Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3794) Google Scholar, 2Luo L. Nat. Rev. Neurosci. 2000; 1: 173-180Crossref PubMed Scopus (830) Google Scholar, 3da Silva J.S. Dotti C.G. Nat. Rev. Neurosci. 2002; 3: 694-704Crossref PubMed Scopus (357) Google Scholar). Of these GTPases Rac1, Cdc42, and RhoA have been well characterized to induce specific cytoskeletal structures as follows: membrane ruffles and lamellipodia by Rac1; filopodia by Cdc42; stress fibers by RhoA (4Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar). Guanine nucleotide exchange factor (GEF), 4The abbreviations used are: GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; bFGF, basic fibroblast growth factor; GBD, GIT-binding domain; DH, Dbl homology; LZ, leucine zipper; GST, glutathione S-transferase; FBS, fetal bovine serum; PBS, phosphate-buffered saline; siRNA, short interfering RNA; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; RSID, Rac1-binding domain; PBD, p21-binding domain; TRITC, tetramethylrhodamine isothiocyanate; βPIX, βPak-interacting exchange factor; GTPγS, guanosine 5′-3-O-(thio)triphosphate; ERK, extracellular signal-regulated kinase; SH3, Src homology 3; PH, pleckstrin homology; CRD, cysteine-rich domain; FL-PIX, full-length βPIX; C-PIX, C-terminal half of βPIX; N-PIX, N-terminal half of βPIX; Pak, p21-activated kinase. 4The abbreviations used are: GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; bFGF, basic fibroblast growth factor; GBD, GIT-binding domain; DH, Dbl homology; LZ, leucine zipper; GST, glutathione S-transferase; FBS, fetal bovine serum; PBS, phosphate-buffered saline; siRNA, short interfering RNA; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; RSID, Rac1-binding domain; PBD, p21-binding domain; TRITC, tetramethylrhodamine isothiocyanate; βPIX, βPak-interacting exchange factor; GTPγS, guanosine 5′-3-O-(thio)triphosphate; ERK, extracellular signal-regulated kinase; SH3, Src homology 3; PH, pleckstrin homology; CRD, cysteine-rich domain; FL-PIX, full-length βPIX; C-PIX, C-terminal half of βPIX; N-PIX, N-terminal half of βPIX; Pak, p21-activated kinase. GTPase-activating protein, and guanine nucleotide dissociation stimulator coordinate the intracellular activities of these Rho GTPases by regulating their interconversion between the inactive GDP-bound and active GTP-bound forms. βPak-interacting exchange factor (βPIX) is a specific GEF for Rac1/Cdc42 (5Manser E. Loo T.H. Koh C.G. Zhao Z.S. Chen X.Q. Tan L. Tan I. Leung T. Lim L. Mol. Cell. 1998; 1: 183-192Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). As a member of the Dbl family of GEFs, βPIX has a Dbl homology (DH) domain responsible for GDP-GTP exchange on Rho GTPases. Additionally, it has a number of other structural motifs as follows: SH3 domain for interaction with p21-activated kinase (Pak) (5Manser E. Loo T.H. Koh C.G. Zhao Z.S. Chen X.Q. Tan L. Tan I. Leung T. Lim L. Mol. Cell. 1998; 1: 183-192Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar); PH domain for protein/lipid interaction; proline-rich domain as yet uncharacterized; GB domain (GBD) for GIT binding (6Bagrodia S. Bailey D. Lenard Z. Hart M. Guan J.L. Premont R.T. Taylor S.J. Cerione R.A. J. Biol. Chem. 1999; 274: 22393-22400Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar); and leucine zipper (LZ) domain for homo- or heterodimerization of PIX (7Kim S. Lee S.H. Park D. J. Biol. Chem. 2001; 276: 10581-10584Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 8Koh C.G. Manser E. Zhao Z.S. Ng C.P. Lim L. J. Cell Sci. 2001; 114: 4239-4251Crossref PubMed Google Scholar).DH domain-mediated GEF activity is transient in principle, because it has a lower affinity toward the active GTP-bound form of Rho GTPases than inactive GDP-bound or nucleotide-depleted ones (9Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1346) Google Scholar, 10Erickson J.W. Cerione R.A. Biochemistry. 2004; 43: 837-842Crossref PubMed Scopus (115) Google Scholar, 11Rossman K.L. Der C.J. Sondek J. Nat. Rev. Mol. Cell Biol. 2005; 6: 167-180Crossref PubMed Scopus (1302) Google Scholar). Once Rho GTPase is activated by a DH domain, their interaction becomes weak and is followed by dissociation. Several laboratories have reported results consistent with the presence of a Rac/Cdc42-directed DH domain of βPIX (12Lee S.H. Eom M. Lee S.J. Kim S. Park H.J. Park D. J. Biol. Chem. 2001; 276: 25066-25072Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 13Park H.S. Lee S.H. Park D. Lee J.S. Ryu S.H. Lee W.J. Rhee S.G. Bae Y.S. Mol. Cell. Biol. 2004; 24: 4384-4394Crossref PubMed Scopus (200) Google Scholar, 14Chahdi A. Miller B. Sorokin A. J. Biol. Chem. 2005; 280: 578-584Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Platelet-derived growth factor-induced membrane ruffle was βPIX-dependent as determined 15 min after stimulation (12Lee S.H. Eom M. Lee S.J. Kim S. Park H.J. Park D. J. Biol. Chem. 2001; 276: 25066-25072Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). βPIX is also involved in epidermal growth factor-induced reactive oxygen species generation as an intermediary step in sequential activation of phosphatidylinositol 3-kinase, PIX, Rac1, and Nox1 (13Park H.S. Lee S.H. Park D. Lee J.S. Ryu S.H. Lee W.J. Rhee S.G. Bae Y.S. Mol. Cell. Biol. 2004; 24: 4384-4394Crossref PubMed Scopus (200) Google Scholar). Rac1 activation occurred in a transient manner, reaching a peak 15 min after epidermal growth factor stimulation. In mesangial cells through the protein kinase A-dependent pathway, endothelin-1 stimulates Cdc42 activation, which peaks at 2 min, decreases at 10 min, and returns to a basal level at 30 min after stimulation (14Chahdi A. Miller B. Sorokin A. J. Biol. Chem. 2005; 280: 578-584Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Taken together, DH-mediated activation of Rac1/Cdc42 appears to reach a peak and proceed to completion within 30 min after agonist stimulation. By contrast, we observed that βPIX stably interacts with Rac1, and more surprisingly, it exhibits prolonged activity toward Rac1 for up to 4 h following growth factor stimulation (15Shin E.Y. Woo K.N. Lee C.S. Koo S.H. Kim Y.G. Kim W.J. Bae C.D. Chang S.I. Kim E.G. J. Biol. Chem. 2004; 279: 1994-2004Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). This kinetic profile does not match the profile reported for other Rho GEFs (16Billadeau D.D. Brumbaugh K.M. Dick C.J. Schoon R.A. Bustelo X.R. Leibson P.J. J. Exp. Med. 1998; 188: 549-559Crossref PubMed Scopus (153) Google Scholar, 17Welch H.C. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Erdjument-Bromage H. Tempst P. Hawkins P.T. Stephens L.R. Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 18Fleming I.N. Batty I.H. Prescott A.R. Gray A. Kular G.S. Stewart H. Downes C.P. Biochem. J. 2004; 382: 857-865Crossref PubMed Scopus (42) Google Scholar, 19Aoki K. Nakamura T. Fujikawa K. Matsuda M. Mol. Biol. Cell. 2005; 16: 2207-2217Crossref PubMed Scopus (117) Google Scholar).Recent evidence indicates that a non-DH domain can participate in Rho GTPase binding and regulation of its activity as well. The Vav family of GEFs has a cysteine-rich domain (CRD), which is located C-terminal to the DH-PH bidomain and allows binding to GTPases (20Heo J. Thapar R. Campbell S.L. Biochemistry. 2005; 44: 6573-6585Crossref PubMed Scopus (42) Google Scholar). Isolated CRD associates with Rac1 and RhoA, but Vav-1 appears to be Rac1-specific. This interaction between Rac1/Rho (involving Ser-83 and Lys-116 of Rac1) may facilitate conformational changes and enhancement of Vav DH-mediated GEF activity toward Rac1. Interestingly, Vav-3 has a zinc finger domain, which can interact with RhoA in vitro and stimulate this GTPase (21Movilla N. Bustelo X.R. Mol. Cell. Biol. 1999; 19: 7870-7885Crossref PubMed Scopus (225) Google Scholar). αPIX also has an extra Rac1-binding domain (RSID), which makes its dimeric form function as a unique Rac-specific GEF (22Feng Q. Baird D. Cerione R.A. EMBO J. 2004; 23: 3492-3504Crossref PubMed Scopus (74) Google Scholar). We therefore hypothesized that βPIX might interact and activate Rac1 via a non-DH domain interaction and consequently promote Rac1 activation in a novel DH-independent mechanism. In this study, we identified GBD as a new stable Rac1-binding site. More importantly, this domain mediates Rac1 activation in collaboration with an associated GEF, smgGDS, in a phosphorylation (Ser-525 and Thr-526)-dependent manner.EXPERIMENTAL PROCEDURESMaterials—Human recombinant bFGF, Lipofectamine 2000, G418, and hygromycin B were purchased from Invitrogen. Anti-Rac1 and anti-GFP antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-smgGDS antibody and smgGDS cDNA (FLJ30470) were purchased from BD Biosciences and the National Institute of Technology and Evaluation (Chiba, Japan), respectively. pEGFP-C2 and pCMV-myc were purchased from Clontech. Nonspecific siRNAs and specific siRNAs for smgGDS were purchased from Invitrogen. Raichu-Rac1 probe for fluorescence resonance energy transfer (FRET) analysis was kindly provided by Dr. Matsuda Michiyuki (Osaka University, Osaka, Japan).Cell Culture and Differentiation—PC12 cells overexpressing FGF receptor-1 in a tetracycline-dependent manner were cultured as described previously (23Shin E.Y. Shin K.S. Lee C.S. Woo K.N. Quan S.H. Soung N.K. Kim Y.G. Cha C.I. Kim S.R. Park D. Bokoch G.M. Kim E.G. J. Biol. Chem. 2002; 277: 44417-44430Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Briefly, cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% Tet system approved fetal bovine serum (FBS), 2 mm glutamine, 1× antibiotics (Invitrogen), 50 μg/ml hygromycin B, and 100 μg/ml G418 at 37 °C in 10% CO2. Prior to differentiation, PC12 cells were induced to overexpress FGF receptor-1 in serum-free Dulbecco's modified Eagle's medium containing 1.5 μg/ml doxycycline for 24 h and replaced with Dulbecco's modified Eagle's medium supplemented with 20 ng/ml bFGF, 2% FBS and 1.5 μg/ml doxycycline for 24-48 h.Transient Transfection and siRNA Treatment—PC12 cells were seeded on 60-mm culture dishes or 20 μg/ml poly-l-lysine-coated coverslips. A mixture of 5 μg of DNA and 5 μl of Lipofectamine 2000 was added to culture dishes according to the manufacturer's instructions. For siRNA treatment, nonspecific siRNA and specific siRNA for smgGDS (5′-GAAGATGAATCCATGCAGAAAATTT-3′) at the indicated concentrations were transfected into cells with Lipofectamine 2000. After 3 days, expression of smgGDS was assessed by immunoblotting with anti-smgGDS antibody. The band intensity after exposure and development was digitized and analyzed by Quantity One software version 4.2.1 (Bio-Rad).Immunoprecipitation and Immunoblotting—Cells were washed twice with PBS and lysed in lysis buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 100 mm NaF, 10% glycerol, 1% Triton X-100, 200 μm orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin) for 1 h at 4°C. Proteins were immunoprecipitated with an appropriate antibody for 3 h at 4 °C. Immunoprecipitates were collected by adding protein G-Sepharose and washed five times with lysis buffer and twice with PBS. Samples were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane in a Tris-glycine/methanol buffer (25 mm Tris base, 200 mm glycine, 20% methanol). Membranes were blocked with 3% skimmed milk in phosphate-buffered saline (PBS) for 1 h, incubated with primary antibodies for 1 h at room temperature, and washed three times (10 min each) with PBS containing 0.1% Tween 20. Membranes were blotted with secondary horseradish peroxidase-conjugated antibodies for 1 h at room temperature. After five washes with PBS and 0.1% Tween 20, signals were detected using enhanced chemiluminescence reagent (Amersham Biosciences). In some cases, membranes were stripped and reprobed with different antibodies.In Vitro Binding Assay—GST, GST-Rac1, and GST-smgGDS proteins were expressed in Escherichia coli (DH5α) and purified by glutathione-Sepharose affinity chromatography. C-PIXHis, GBD-His, and p21-binding domain (PBD)-His proteins were expressed in M15 and purified by Ni2+ affinity chromatography. Equal amounts of GST (GST-Rac1) glutathione-Sepharose beads were incubated with His-tagged proteins or lysates from cells expressing various domains of GFP-βPIX. Beads were washed five times with lysis buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 100 mm NaF, 10% glycerol, 1% Triton X-100, 5 mm MgCl2, 1 mm dithiothreitol, 200 μm orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin), resolved on 9% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Membranes were immunoblotted with anti-His, GFP, or GST antibody.Modified GST-PBD Binding Assay—GEF activities of βPIX/truncated βPIX were measured as described previously (15Shin E.Y. Woo K.N. Lee C.S. Koo S.H. Kim Y.G. Kim W.J. Bae C.D. Chang S.I. Kim E.G. J. Biol. Chem. 2004; 279: 1994-2004Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Briefly, GST-PBD was expressed in E. coli (DH5α) and purified with glutathione-Sepharose affinity chromatography. Cells were stimulated with or without 30 ng/ml bFGF for 1 h, lysed, and immunoprecipitated with anti-βPIX or anti-GFP antibody. Immunoprecipitates were further incubated with purified soluble GST-PBD (1 μg) at 4 °C for 2 h in binding buffer (25 mm Tris-HCl, pH 7.5, 1 mm dithiothreitol, 30 mm MgCl2, 40 mm NaCl, 0.5% Nonidet P-40) and washed five times with lysis buffer. Beads were boiled for 5 min, resolved by 12% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. Membranes were immunoblotted with anti-GST antibody and then reprobed with anti-GFP, βPIX, Pak2, or Rac1 as described under “Transient Transfection and siRNA Treatment.”FRET Analysis—PC12 cells were plated on 40-mm dishes containing poly-l-lysine-coated 18-mm glass coverslips. One day after plating, Myc-tagged PIX constructs were co-transfected with Raichu-Rac1 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Thirty six hours after transfection, cells were starved for 16 h and then treated with 30 ng/ml bFGF for 1 h. For Myc-tagged PIX staining, cells were fixed in 3.7% paraformaldehyde/PBS for 10 min at room temperature, permeabilized with ice-cold methanol for 15 min, and then blocked with 10% FBS for 1 h. Cells were incubated with anti-Myc antibody in 2% FBS/PBS for1hat37 °C, followed by incubation with Alexa Fluor 633-conjugated goat anti-mouse IgG antibody (Molecular Probes, OR) for 1 h at 37°C. After staining, coverslips were mounted onto a glass slide with mounting medium (DakoCytomation). FRET was analyzed using a Leica TCS SP2 confocal microscope system with HCX PL APO ×63 objective (Leica, Wetzlar, Germany). Excitation was provided by 20-milliwatt multimode argon ion laser lines. Donor (CFP) was excited at 458 nm, and fluorescence was detected in a bandwidth of 470-500 nm (CFP channel), whereas for acceptor (YFP), excitation was at 514 nm and emission at a bandwidth of 535-565 nm (YFP channel). For FRET, the excitation was at 458 nm and emission at a bandwidth 535-565 nm (FRET channel). Donor and FRET images were acquired from the respective CFP and FRET channels under the similar conditions. After background subtraction, FRET: CFP ratio images were generated by dividing the FRET image by the CFP image using MetaMorph software version 6.01 (Universal Imaging), and these ratios were used to represent FRET efficiency.Statistical Analysis—Paired t test was applied for statistical analysis of neurite outgrowth assay and FRET using SPSS version 10.0 for windows and the statistical significance was set at p < 0.05.RESULTSPIX Binds to Rac1 via GIT-binding Domain—To assess which domain of βPIX might be involved in a non-DH domain interaction, we generated two truncated βPIX constructs as follows: the first contains the N-terminal half of βPIX (N-PIX), including the SH3-DH-PH domains in sequence, and the second contains the C-terminal half of βPIX (C-PIX) (as illustrated in Fig. 1A). These constructs were introduced into PC12 cells and assessed for GST-Rac1 binding. As expected, both DH-containing full-length βPIX (FL-PIX) and N-PIX interacted with GST-Rac1 but not with GST or SH3 domain of βPIX (Fig. 1B, left). Interestingly, GST-Rac1 also bound C-PIX to the same extent as the N-terminal domain, although there did not appear to be any cooperativity of binding in the case FL-PIX. These results, however, did not exclude the possibility that the C-PIX: Rac1 association occurs indirectly through heterodimerization of C-PIX with endogenous FL-PIX, via the LZ dimerization (7Kim S. Lee S.H. Park D. J. Biol. Chem. 2001; 276: 10581-10584Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 8Koh C.G. Manser E. Zhao Z.S. Ng C.P. Lim L. J. Cell Sci. 2001; 114: 4239-4251Crossref PubMed Google Scholar). We therefore tested the ability of bacterially expressed His-tagged C-PIX to bind GST-Rac1 in vitro. C-PIX interacted specifically with GST-Rac1 (Fig. 1B, right, lane 2), but not with GST alone (lane 1). These results indicate that βPIX binds Rac1 through a region independent of the DH domain. To further determine which part of C-PIX is involved in this binding, C-PIX was divided into three parts, namely a proline-rich region (PXXP), the GBD, and the LZ domains (Fig. 1A). Each domain was expressed in PC12 cells as GFP fusion proteins, and lysates were incubated with GST (control) or GST-Rac1 immobilized on glutathione-Sepharose beads (Fig. 1C, left). C-PIX consistently showed a specific interaction with GST-Rac1, and thus was employed as a positive control (Fig. 1C, left, lanes 1 and 5). Only GBD exhibited a strong interaction comparable with that of the parental C-PIX (Fig. 1C, left, lanes 3 and 7). Further binding study with bacterially expressed His-tagged GBD revealed that direct interaction occurs between Rac1 and GBD (Fig. 1C, right). These results indicate that GBD plays a role in binding Rac1, independent of classical DH interactions. The related region (amino acid 546-779) derived from αPIX, which has 56% homology to βC-PIX (amino acids 401-646), also bound to GST-Rac1 (but not GST) (Fig. 1D, lanes 2 and 5). We thus determined whether the GBD of αPIX plays a similar role in binding Rac1. It also interacted with wild-type Rac1 and Cdc42 (Fig. 1E, lanes 1-6). Finally, we tested whether interaction between the C-PIX (GBD) and Rac1 is altered depending on the activation status of Rac1. It is well known that the p21-binding domain (PBD) of p21-activated kinase (Pak) specifically binds to Rac1-GTP but not Rac1-GDP. This interaction was therefore presented as a positive control (Fig. 1F, lanes 1 and 2). Unlike PBD-Rac1 interaction, GDP or GTP loading did not affect interaction of both C-PIX-Rac1 and GBD-Rac1 (Fig. 1F, lanes 3-6). This unexpected interaction between C-PIX (GBD) and Rac1 would make it clear how the modified GSTPBD assay as described under “Experimental Procedures” works (15Shin E.Y. Woo K.N. Lee C.S. Koo S.H. Kim Y.G. Kim W.J. Bae C.D. Chang S.I. Kim E.G. J. Biol. Chem. 2004; 279: 1994-2004Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). More importantly, these results suggest that active Rac1-GTP can remain stably bound to βPIX via the GBD, providing a rationale to persistent βPIX-mediated Rac1 activity.C-PIX Mediates Activation of Rac1 but Not Cdc42 in Response to bFGF—Given that C-PIX stably interacts with Rac1, we wished to test whether this binding influences the regulation of Rac1. We conducted two different assays to measure Rac1 activation. One uses the ability of active GTP-bound, but not inactive GDP-bound Rac1/Cdc42, to bind GST-PBD (the modified GST-PBD binding assay) (15Shin E.Y. Woo K.N. Lee C.S. Koo S.H. Kim Y.G. Kim W.J. Bae C.D. Chang S.I. Kim E.G. J. Biol. Chem. 2004; 279: 1994-2004Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The conventional GST-PBD binding assay has been used to detect active forms of Rac1/Cdc42 bound, but this method can not identify the GEF responsible for their activation. To overcome this handicap, the modified protocol we have been using involves immunoprecipitation of PIX constructs followed by the addition of soluble GST-PBD to bind βPIX-associated GTP-Rac1 or Cdc42. Active Rac1/Cdc42 in the immunoprecipitates of βPIX is able to bind added GST-PBD as detected by anti-GST. An underlying principle of this assay is that βPIX can stably hold the active GTP-bound form of Rac1/Cdc42 via a non-DH domain, GBD interaction (Fig. 1F). The other assay analyzes fluorescence resonance energy transfer (FRET) images using a Raichu-probe for Rac1 in PC12 cells (24Itoh R.E. Kurokawa K. Ohba Y. Yoshizaki H. Mochizuki N. Matsuda M. Mol. Cell. Biol. 2002; 22: 6582-6591Crossref PubMed Scopus (453) Google Scholar, 25Aoki K. Nakamura T. Matsuda M. J. Biol. Chem. 2004; 279: 713-719Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar).As shown in Fig. 2A, left, in both FL-PIX- and C-PIX-expressing cells, bFGF increased levels of associated active Rac1/Cdc42, as determined by the appearance of a GST-PBD band (lanes 4 and 8). Only Rac1 (not Cdc42) was detectable in the immunoprecipitates retrieved by anti-GFP. Note C-PIX but not N-PIX had a stimulatory effect on associated Rac1-GTP levels that is comparable with that of FL-PIX (Fig. 2A, left, lanes 7 and 8). Surprisingly, neither GFP nor N-PIX activated Rac1 by these criteria, indicating that the DH domain in N-PIX by itself is not sufficient to mediate GEF activity (Fig. 2A, left, lanes 1, 2, 5, and 6); the N-PIX is likely compromised by a failure to undergo dimerization. Shin et al. (23Shin E.Y. Shin K.S. Lee C.S. Woo K.N. Quan S.H. Soung N.K. Kim Y.G. Cha C.I. Kim S.R. Park D. Bokoch G.M. Kim E.G. J. Biol. Chem. 2002; 277: 44417-44430Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) previously showed that bFGF treatment resulted in phosphorylation of Ser-525 and Thr-526 within the GBD of βPIX, and this phosphorylation is critical for βPIX-mediated Rac1 activation (15Shin E.Y. Woo K.N. Lee C.S. Koo S.H. Kim Y.G. Kim W.J. Bae C.D. Chang S.I. Kim E.G. J. Biol. Chem. 2004; 279: 1994-2004Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Interestingly, the new Rac1-binding domain falls within the same GBD region; such phosphorylation may therefore affect Rac1 binding/activation. To test this, cells were transfected with plasmids encoding C-PIX or mutant C-PIX (S525A/T526A), and their activity was measured by the modified GST-PBD binding assay (Fig. 2A, right). Wild-type C-PIX mediated the expected bFGF-stimulated Rac1 activation (Fig. 2A, right, lanes 1 and 2). However, cells expressing the nonphosphorylatable C-PIX did not generate Rac1-GTP (Fig. 2A, right lanes 3 and 4). Inability of mutant C-PIX to activate Rac1 was not because of disruption of its interaction with Rac1. Similarly, FL-PIX- and C-PIX-mediated Rac1 activation were dependent on phosphorylation of Ser-525/Thr-526 (15Shin E.Y. Woo K.N. Lee C.S. Koo S.H. Kim Y.G. Kim W.J. Bae C.D. Chang S.I. Kim E.G. J. Biol. Chem. 2004; 279: 1994-2004Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The C-PIX GBD-mediated Rac1 activation in response to bFGF suggested that this domain might associate with another GEF.FIGURE 2C-PIX activates Rac1 in response to bFGF stimulation. A, modified GST-PBD binding assay for Rac1/Cdc42 activation. Left panel, PC12 cells were transfected with plasmids encoding GFP-tagged FL-PIX, N-PIX, or C-PIX and maintained for 24 h. They were then stimulated with 30 ng/ml bFGF for 1 h. Equal amounts of protein from each lysate were immunoprecipitated (IP) with anti-GFP and incubated with soluble GST-PBD to trace active GTP-bound Rac1/Cdc42 in the immunoprecipitate. Bound GST-PBD was probed by immunoblotting with anti-GST antibody (top panel). Expression of each construct as GFP fusion proteins was evaluated by probing with anti-GFP (middle panel) and anti-Cdc42 and Rac1 (bottom panel), respectively. In the right panels, cells were transfected with pEGFP plasmids encoding wild-type C-PIX (lanes 1 and 2) or mutant C-PIX (S525A/T526A) (lanes 3 and 4), and then the above procedure was applied. B, FRET analysis was performed to investigate Rac1 activation using a Raichu-Rac1 probe. Various Myc-tagged βPIX constructs were co-transfected with a Raichu-Rac1 probe into PC12 cells. Cells were starved for 16 h and then treated with bFGF or left untreated. Confocal images were obtained 1 h following bFGF stimulation as described under “Experimental Procedures.” Representative ratio images of FRET:CFP after bFGF stimulation are shown in the intensity-modulated display mode. In the intensity-modulated display mode, eight colors from red to blue were used to represent the FRET:CFP ratio, with the intensity of each color indicating the mean intensity of FRET and CFP image. The upper and lower limits of the ratio image are shown on the right. Bar graphs represent the relative emission ratio (FRET:CFP) of the whole cell area. The number of cells examined for each sample was as follows: Myc (n = 10), FL-PIX (n = 20), N-PIX (n = 15), C-PIX (n = 20). *, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To assess the effect of C-PIX on Rac1 in vivo, we used an established FRET probe designated Raichu-Rac1 (24Itoh R.E. Kurokawa K. Ohba Y. Yoshizaki H. Mochizuki N. Matsuda M. Mol. Cell. Biol. 2002; 22: 6582-6591Crossref PubMed Scopus (453) Google Scholar). PC12 cells were co-transfected with Raichu-Rac1 plasmid and various βPIX constructs, starved for 24 h, and then stimulated with bFGF for 1 h prior to fixation. Representative YFP:CFP ratio images and their emission ratio for each construct were show" @default.
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• W2037844242 date "2006-11-01" @default.
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• W2037844242 title "βPak-interacting Exchange Factor-mediated Rac1 Activation Requires smgGDS Guanine Nucleotide Exchange Factor in Basic Fibroblast Growth Factor-induced Neurite Outgrowth" @default.
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