Eur J Biochem 269, 638±649 (2002) Ó FEBS 2002 Dematin interacts with the Ras-guanine nucleotide exchange factor Ras-GRF2 and modulates mitogen-activated protein kinase pathways Mohini Lutchman1, Anthony C Kim1, Li Cheng2, Ian P Whitehead2, S Steven Oh1, Manjit Hanspal1, Andrey A Boukharov1, Toshihiko Hanada1 and Athar H Chishti1 Section of Hematology-Oncology Research, Departments of Medicine, Anatomy, and Cellular Biology, St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA, USA; 2Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ, USA Erythroid dematin is a major component of red blood cell junctional complexes that link the spectrin±actin cytoskeleton to the overlying plasma membrane Transcripts of dematin are widely distributed including human brain, heart, lung, skeletal muscle, and kidney In vitro, dematin binds and bundles actin ®laments in a phosphorylation-dependent manner The primary structure of dematin consists of a C-terminal domain homologous to the ÔheadpieceÕ domain of villin, an actin-binding protein of the brush border cytoskeleton Except ®lamentous actin, no other binding partners of dematin have been identi®ed To investigate the physiological function of dematin, we employed the yeast two-hybrid assay to identify dematin-interacting proteins in the adult human brain Here, we show that dematin interacts with the guanine nucleotide exchange factor Ras-GRF2 by yeast two-hybrid assay, and this interaction is further con®rmed by blot overlay, surface plasmon resonance, co-transfection, and co-immunoprecipitation assays Human Ras-GRF2 is expressed in a variety of tissues and, similar to other guanine nucleotide exchange factors (GEFs), displays anchorage independent growth in soft agar Co-transfection and immunoblotting experiments revealed that dematin blocks transcriptional activation of Jun by Ras-GRF2 and activates ERK1 via a Ras-GRF2 independent pathway Because much of the present evidence has centered on the identi®cation of the Rho family of GTPases as key regulators of the actin cytoskeleton, the direct association between dematin and Ras-GRF2 may provide an alternate mechanism for regulating the activation of Rac and Ras GTPases via the actin cytoskeleton Dematin is a cytoskeletal protein that binds and bundles actin ®laments in vitro [1,2] It was originally identi®ed as a component of human erythrocyte membrane skeleton, and migrates in the zone of polypeptides collectively designated as band 4.9 on polyacrylamide gels [1,2] Phosphorylation by the cAMP-dependent protein kinase abolishes dematin's actin-bundling activity that is restored by dephosphorylation [2] Dematin is part of a junctional complex, together with protein 4.1, adducin, tropomyosin, and tropomodulin, that links spectrin tetramers and actin proto®laments to the erythrocyte plasma membrane [3] Erythroid dematin exists as a trimer consisting of one polypeptide of 52-kDa and two polypeptides of 48-kDa [1,4] Recently, we have characterized the dematin gene and have identi®ed exon 13 as an alternatively spliced exon present in the 52-kDa polypeptide but absent in the 48-kDa subunit [5,6] Exon 13 encodes a 22-amino-acid insertion that includes a motif homologous to protein 4.2 and a motif that binds to ATP in vitro [7] Although the functional signi®cance of this insertion is not known, we have postulated that the 52-kDa subunit provides a molecular framework for the formation of disul®de-linked trimeric dematin [4] Dematin was originally isolated from red blood cells However, dematin transcripts have been detected in a wide variety of tissues including brain, heart, kidney, skeletal muscle, and lung [5,6,8] The C-terminal  75-residue domain of dematin is homologous to the ÔheadpieceÕ domain of villin, an actin-binding protein of the brush border cytoskeleton [5,9] Previously, it was believed that this module played a crucial role in the morphogenesis of microvilli [10] However, the recent generation of villin null mice strongly suggests that villin's role in the micro®lament assembly of microvilli in absorptive tissues is compensated for by dematin and/or other ÔheadpieceÕ-containing proteins [11,12] The N-terminal core domain of dematin is homologous to only one other known protein, a ÔLIMÕ protein termed limatin (abLIM) [13] Limatin contains four double zinc ®nger LIM domains at its N-terminus with the C-terminus sharing  50% identity to full-length dematin Correspondence to A Chishti, Biomedical Research, ACH-404, St Elizabeth's Medical Center, 736 Cambridge Street, Boston, MA 02135, USA Fax: + 617 789 3111, Tel.: + 617 789 3118, E-mail: Athar.Chishti@Tufts.edu Abbreviations: GRF, guanine nucleotide releasing factor; GEF, guanine-nucleotide exchange factor; DH, Dbl homology domain; PH, pleckstrin homology domain; AbLIM, actin-binding LIM protein; IQ, Ilimaquinone; NHS, N-hydroxysuccinimide; EDC, N-ethylN¢-[3-(diethylamino)propyl]carbodiimide; Sos, Son of Sevenless; SAPK, stress-activated protein kinase; JNK, Jun N-terminal kinase Note: M Lutchman, A C Kim, and L Cheng contributed equally to this work Note: the nucleotide sequences reported in this paper have been submitted to the GenBank with the accession numbers AF181250 and AF186017 (Received 25 September 2001, accepted 20 November 2001) Keywords: dematin, erythrocyte, limatin, Ras-GRF2, headpiece domain Ó FEBS 2002 Dematin binds to Ras-GRF2 nucleotide exchange factor (Eur J Biochem 269) 639 [13] The dematin and limatin genes are located on human chromosomes 8p21.1 and 10q25, respectively, regions frequently deleted in prostate and other epithelial cancers [4,14] Interestingly, we have recently demonstrated the loss of heterozygosity of the dematin gene in a majority of 8p21linked prostate tumors [14] The Ras superfamily of GTPases plays critical roles in the regulation of signaling pathways from the cell surface to the nucleus [15] Approximately 40% of human cancers are caused by activated ras alleles [16] In addition, Ras proteins are also involved in synaptic transmission and long-term potentiation [17] These observations generated a great deal of interest in proteins that are involved in the regulation of Ras proteins Ras GTPases cycle between an active GTPbound state and an inactive GDP-bound state GTPase activating proteins (GAPs) catalyze the intrinsic GTPase activity of Ras proteins, thereby down-regulating Ras signaling molecules [17±19] In contrast, the Ras-guanine nucleotide exchange factor (GEF) proteins are factors that catalyze the exchange of GDP for GTP, thus activating Ras GTPases Two of the better-known GEFs are Son of Sevenless (Sos) and the Ras guanine nucleotide release factor (Ras-GRF) [20±24] Both proteins contain a C-terminal domain homologous to the Saccharomyces cerevisiae Cdc25 protein, a Ras-GEF, and regions homologous to the Dbl oncogene product (DH domain) in tandem with a pleckstrin homology (PH) domain [21±23] The Sos protein contains C-terminal proline-rich domain not found in the other related GEFs It is via this proline-rich domain that Sos is constitutively associated with the SH3 domain of the adaptor protein Grb2 [20] Grb2 protein also contains an SH2 domain that interacts with a phosphorylated tyrosine residue of activated EGF receptor [20] The formation of this complex recruits the Sos exchange factor within proximity of membrane-bound Ras, thus providing a coupling mechanism between receptor tyrosine kinases and Ras signaling [20±24] While the upstream events that lead to Sos activation and the subsequent activation of the Ras-MAP kinase cascade are well known, the signals involved in the Ras-GRF activation are not yet fully characterized Ras-GRFs are of two types, the neuronally expressed Ras-GRF1, and the more widely expressed Ras-GRF2 [19,21,22,24] Both RasGRFs are exchange factors for Ras-GTPases via their Cdc25-like catalytic domains Recent in vitro evidence suggests that the Ras-GRFs are activated by G-protein coupled receptors [23] Stimulation of muscarinic receptors or the expression of the G-protein bc subunits is known to stimulate the exchange activity of Ras-GRF1 (or CDC25Mm) in a phosphorylation-dependent manner [23] Calcium in¯ux is also shown to activate Ras-GRF1 [24] The DH domain of Ras-GRF1 catalyzes nucleotide exchange of Rac1 in response to a signal triggered by the Gbc25 Moreover, the co-expression of Ras-GRF1 and Gbc subunits leads to the activation of the MAP kinases JNK1 and ERK2 in heterologous cells [25] Ras-GRF2 stimulates the ERK1 MAP kinase in a Ras- and ilimaquinonedependent manner [22] More recent evidence has shown that the DH domain of Ras-GRF2 also activates the JNK pathway in a Rac-dependent manner [26] To further understand the role of dematin in normal cells, we proceeded to identify binding partners that interact with dematin The yeast two-hybrid assay was used to screen an adult human brain library with the C-terminal half of dematin as the bait probe The identi®cation of Ras-GRF2 as a binding partner for the dematin provides evidence for a direct association between Ras-GRF2 and dematin and therefore suggests a novel mechanism for linking the Ras signaling complex to the actin cytoskeleton The functional signi®cance of the dematin interaction with Ras-GRF2 was further explored by examining the modulatory effects of dematin on the pathways of ERK and JNK activation EXPERIMENTAL PROCEDURES Yeast two-hybrid screen The vectors, yeast strains, and library employed in twohybrid screen were purchased from Clontech The C-terminal half of human 48 kDa dematin (amino acids 224±383) was subcloned in-frame into the EcoRI/BamHI site of the GAL4 DNA binding domain plasmid pAS2-1 and used to screen a human brain Matchmaker cDNA library constructed in the GAL4 activation domain plasmid pGAD10 The dematin bait and the library was transformed into CG-1945 and plated on media lacking the amino acids tryptophan, leucine, and histidine in the presence of 3-amino-1,2,4triazole (5 mM) Colonies that grew on selective media were then scored for b-galactosidase activity by the ®lter assay according to the manufacturer's instructions (Clontech) Plasmid DNA from the positive clone, as shown by a blue color, was recovered from yeast and transformed into bacteria for DNA isolation Yeast mating Yeast mating experiments were utilized to test the speci®city of interaction between dematin and Ras-GRF2 Limatin and Ras-GRF1, the closest known homologues of dematin and Ras-GRF2, respectively, were included in these experiments The segment of limatin (amino acids 597±778) corresponding to the dematin ÔbaitÕ sequence was subcloned into pAS2-1, while the segment of Ras-GRF1 (amino acids 172±471), corresponding to the isolated fragment of Ras-GRF2, was subcloned into pGAD10 The pAS2-1 constructs (including pAS2-1 only) were transformed into the yeast strain Y187 while pGAD10 constructs (including pGAD10 only) were subcloned into strain CG1945 Pairwise matings between all pAS2-1 transformants and all pGAD10 transformants were plated on minimal media and scored for b-galactosidase activity Cloning of Ras-GRF2 cDNA and expression constructs Primer pair 7/8 (7 : 5¢-ATGCAGAAGAGCGTGCGC TAC-3¢; : 5¢-TCAAGCAGGGAGTCGAGGTTC-3¢) was used to amplify the full-length Ras-GRF2 from a human fetal brain cDNA pool (Invitrogen, CA) These primers were designed from the murine Ras-GRF2 cDNA sequence due to the high nucleotide identity A single band of 3.7 kb was ampli®ed and subcloned into the vector pCR2.1 (Invitrogen, CA, USA) for sequence analysis The full-length Ras-GRF2 cDNA was PCR-ampli®ed with BamHI adaptors and subcloned into the mammalian expression vector pcDNA3.1/myc-His (Invitrogen) Immunodetection of Ras-GRF2 protein was carried out 640 M Lutchman et al (Eur J Biochem 269) using a monoclonal antibody directed against the myc-epitope (9E10 clone, Upstate Biotechnology, Lake Placid, NY, USA) The full-length 48-kDa subunit of dematin cDNA (1.15 kb) was subcloned into the BamHI site of pcDNA3.0GFPmyc vector in sense and antisense orientations The following cDNAs were PCR-ampli®ed with BamHI/EcoRI adaptors for in-frame subcloning into the bacterial expression vector pGEX-2T (Pharmacia Biotech): Ras-GRF2 (amino acids 176±474), Ras-GRF2 (amino acids 909±1237), Ras-GRF1 (amino acids 172±471), dematin (amino acids 224±383), and limatin (amino acids 597±778) These constructs will be referred to in this manuscript as GST±GRF2-DH, GST±GRF2-Cdc25, GST±GRF1-DH, GST±dematin(224±383) and GST±limatin(597±778), respectively Recombinant proteins were expressed and puri®ed accordig to the manufacturer's instructions (Pharmacia Biotech) Expression analysis The primer pair 31/21 (31 : 5¢-AGCGCCTCTTGGAAC GACTGA-3¢; 21 : 5¢-GCGGCGGCTTTCCTTTCTT-3¢) was used to amplify a 961-bp Ras-GRF2 fragment to probe the Human Multiple Tissue Northern Blot (Clontech) The probe was 32P-labeled with the DECAprime DNA labeling kit (Ambion) and hybridized to the Northern blot in Rapid-Hyb buffer according to the manufacturer's instructions (Pharmacia Biotech) The primer pair 33/21 (33 : 5¢-CCGCTGCGTCTCCACCACCACAC-3¢) was used to amplify the Multiple Tissue cDNA Panel #2 (Clontech) These primers amplify a 577-bp product from the Ras-GRF2 cDNA Primers speci®c for glyceraldehyde3-phosphate dehydrogenase (G3PDH) were also used to ensure equal cDNA loading Blot overlay assay Equal amounts ( lg) of GST and GST±GRF2-DH fusion proteins were separated by SDS/PAGE and either Coomassie-stained or transferred to a nitrocellulose membrane The nitrocellulose blot was blocked overnight at °C in 5% (w/v) nonfat dry milk/NaCl/Tris (25 mM Tris, 137 mM NaCl, 2.5 mM KCl, pH 8)/0.1% Tween-20 (blocking solution) The blot was then incubated in the blocking solution containing 10 lg of puri®ed dematin Dematin, which is a trimeric protein of two 48-kDa polypeptides and one 52-kDa polypeptide, was puri®ed from human erythrocyte membranes [27] After an overnight incubation in the cold room, the blot was washed twice for 10 at room temperature in NaCl/Tris/0.1% Tween-20 and incubated for h in a : 3000 dilution of af®nity-puri®ed polyclonal antidematin Ig Following two 10-min washes, the blot was then incubated in an horseradish peroxidase-conjugated secondary antibody (1 : 3000 dilution) for h at room temperature After two ®nal washes, bound dematin was immunodetected using the ECL system (Pharmacia Biotech) Surface plasmon resonance analysis A BIAcore 1000 (Pharmacia Biosensor, NJ, USA) was used to measure the speci®c interaction and to determine the binding af®nity between the C-terminal domain of dematin [dematin(224±383)] and GST±Ras-GRF2 The GST± Ó FEBS 2002 dematin(224±383) fusion protein was af®nity-puri®ed using GSH-Sepharose 4B beads, and treated with thrombin (Pharmacia Biotech) to proteolytically cleave the dematin(224±383) domain from the GST fusion protein A homogeneous sample of the dematin(224±383) (free of the GST domain) was immobilized ( 1.0 ng of protein per mm2 of surface) to the Dextran matrix of a CM5 sensor chip (Pharmacia Biosensor) using an amine coupling kit (Pharmacia Biosensor), as previously described [28] Puri®ed GST±Ras-GRF2-DH fusion protein (66 kDa) was extensively dialyzed against HBS buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3.0 mM EDTA, 0.005% v/v Surfactant P20) and diluted to desired concentrations using the same buffer Puri®ed recombinant GST was used as a control sample Association and dissociation rates were measured at 25 °C at a ¯ow rate of 10 lLámin)1 The binding surface was successfully regenerated with a short pulse (5.0 lL) of 20 mM HCl followed by a short pulse (5.0 lL) of 0.01% SDS After the last injection of analyte samples, the analyte at an initial concentration was re-injected to check for signi®cant denaturation of the immobilized ligand during the repeated cycles of regeneration process The contribution of bulk solution in the surface plasmon resonance (SPR) signal were minimal as determined by injecting the analyte sample onto a blank CM5 sensor chip surface activated with a : mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N¢-[3-(diethylamino)propyl]carbodiimide (EDC) and blocked with M ethanolamine hydrochloride (pH 8.5) The data were analyzed using the BIAEVALUATION 3.0 (Pharmacia Biosensor) software Transfection of Ras-GRF2 and dematin into NIH 3T3 cells The pcDNA3.1-GRF2-myc-His (full length Ras-GRF2) plasmid was transfected into NIH 3T3 cells using the pFx-6 lipid reagent following the manufacturer's protocol (Invitrogen) Cells were plated in duplicate on plastic and glass discs in six-well Falcon plates After 5±8 h in Opti-Mem (Gibco-BRL) and 24 h in complete media [Dulbecco's modi®ed Eagle's serum (DMEM) plus 10% fetal bovine serum; Hyclone, Logan, UT, USA], Ras-GRF2 expressing colonies were selected by growth in medium containing 400 lgámL)1 of G418 over a period of weeks Stable clones were expanded for further analysis After months of selection, Ras-GRF2 stable clones were cotransfected with pcDNA3-GFPdematin (full length 48-kDa subunit of human dematin) and selected in G418 using the procedures described above Immunocytochemistry Stable NIH 3T3 clones expressing both Ras-GRF2 and dematin were plated at 40% con¯uency for use in immunolocalization studies Stable clones were washed in NaCl/Pi (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) and ®xed with formaldehyde (Sigma) After washing in NaCl/Pi, cells were permeabilized in NaCl/Tris/ 1% Triton X-100 for Cells were washed in NaCl/Pi and incubated in a : 100 dilution of monoclonal anti-myc Ig for h Stable clones were washed in NaCl/Pi and incubated with a ¯uorescein isothiocyanate (FITC)-conjugated Ó FEBS 2002 Dematin binds to Ras-GRF2 nucleotide exchange factor (Eur J Biochem 269) 641 goat anti-(mouse IgG) Ig (Pierce; : 64 dilution) (Sigma) for h After rinsing in NaCl/Pi, cells were incubated for h with polyclonal anti-dematin Ig followed by subsequent washes in NaCl/Pi and incubation with a rhodamineconjugated goat anti-(rabbit IgG) Ig (Pierce; : 100 dilution; Sigma) for h After two ®nal washes, cover slips were mounted onto slides using an Antifade reagent (Bio-Rad) and observed under a Zeiss ¯uorescence microscope linked to a Cooke CCD camera Photographs were taken using IMAGE-PRO PLUS v 300 (Mediacybernatics, Silver Spring, MD, USA) ERK1 activation A293 cells were transiently transfected with Lipofectamine 2000 (Gibco-BRL) After transfection, the cells were allowed to recover for 48 h in DMEM/10% fetal bovine serum The cells were then starved for 18 h and treated with lM ionomycin (Calbiochem) for at 37 °C Cells were scraped with cell lysis buffer and used for ERK activation assays ERK1 assays were as described previously [22] The anti-(phospho-ERK) Ig (sc-94, Santa Cruz) and anti-ERK1 Ig (sc-93, Santa Cruz) were used for the ERK activation assays Antibodies were used at dilutions of : 1000 for Western blots Blots were normalized with the monoclonal anti-(a-tubulin) Ig (CP06, Oncogene Science, Cambridge, MA, USA) Molecular constructs RacI (WT) and RacI (12 V) encode wild-type and constitutively activated derivatives of RacI, respectively, that have been described previously [29] The reporter construct utilized in the luciferase-coupled transcriptional assay has been described previously [30] The 5XGal4-luc contains the luciferase gene under the control of a minimal promoter that contains ®ve Gal4 DNA-binding sites Gal-Jun(1±223) contains the Gal4 DNA-binding domain fused to the transactivation domain of Jun The pCMVnlac encodes the sequences for the b-galactosidase gene under the control of the cytomegalovirus promoter Transient-expression reporter gene assays For transient expression reporter assays, COS-7 cells were transfected by DEAE-dextran, as described previously [31] COS-7 cells were maintained in high glucose DMEM supplemented with 10% fetal bovine serum Cells were allowed to recover for 30 h, and were then starved in DMEM supplemented with 0.5% fetal bovine serum for 14 h before lysate preparation Analysis of luciferase expression was as described previously [30] with enhanced chemiluminescent reagents and a Monolight 3010 luminometer (Analytical Luminescence, San Diego, CA, USA) b-Galactosidase activity was determined using Lumi-Gal substrate (Lumigen, South®eld, MI, USA) according to the manufacturer's instructions All assays were performed in triplicate Rac1 activation assay The p21-binding domain of Pak3 was expressed as a GST fusion in Escherichia coli and immobilized by binding to glutathione-coupled Sepharose 4B beads (Amersham Phar- macia, Piscataway, NJ, USA) The immobilized RacI binding domain was then used to precipitate activated GTP-bound Rac1 from COS-7 cell lysates Cells were washed in cold NaCl/Pi and then lysed in 50 mM Tris/HCl, pH 8.0, mM MgCl2, 0.2 mM Na2S2O5, 10% glycerol, 20% sucrose, mM dithiothreitol, lgámL)1 leupeptin, lgámL)1 pepstatin, and lgámL)1 aprotinin Cell lysates were then cleared by centrifugation at 10 000 g for 10 at °C The expression of proteins was con®rmed by Western blotting prior to af®nity puri®cation Lysates used for af®nity puri®cation were normalized for endogenous RacI levels Af®nity puri®cations were carried out at °C for h, washed three times in an excess of lysis buffer, and then analyzed by Western blot GTP-Rac1 was detected with the monoclonal anti-(C-14) Ig (Santa Cruz Biotechnology, Santa Cruz, CA, USA) RESULTS Isolation of human Ras-GRF2 by yeast two-hybrid screening To investigate the function of erythroid dematin in nonerythroid tissues, we employed the yeast two-hybrid assay to identify the dematin-interacting proteins As the dematin transcript is most abundantly expressed in brain [5,6], we screened a brain cDNA library prepared from adult human brain tissue to isolate cDNAs encoding for the dematininteracting proteins In the initial screen, the full-length coding sequence of human erythroid dematin (48-kDa polypeptide) was used as the bait However, control tests with the bait alone indicated that the full-length dematin cDNA strongly autoactivated transcription thereby precluding its use as a bait in the yeast two-hybrid assay (data not shown) To overcome this limitation, several cDNA constructs were designed that encoded de®ned segments of dematin and tested for the autoactivation of transcription The bait construct containing the C-terminal half of dematin was used to screen a human brain cDNA library This construct, designated as dematin(224±383), includes complete headpiece domain (75 amino acids) and a portion of the dematin core domain (85 amino acids) that precedes the headpiece domain (Fig 1) The dematin(224±383) construct does not include the PEST sequence or the poly(glutamic acid) motif that have been previously identi®ed in the dematin core domain [5,8] A total of  6.0 ´ 105 clones of the brain cDNA library were screened using dematin(224±383) as the bait Five colonies that grew on media lacking histidine were assayed for b-galactosidase activity as described in the Experimental procedures Sequence analysis of the plasmid inserts identi®ed the clones as Ras-GRF2 encoding for the IQ motif, the DH domain, and a small portion of the second PH domain (Fig 1) The interaction between dematin and Ras-GRF2 was con®rmed using controls as speci®ed by the manufacture's protocol This indicated that the two proteins interacted in vitro using the yeast two-hybrid assay Cloning and complete primary structure of human Ras-GRF2 Our initial identi®cation of the human Ras-GRF2 cDNA was based on its sequence alignment with the mouse 642 M Lutchman et al (Eur J Biochem 269) Ras-GRF2 cDNA that was isolated from the mouse brain cDNA library [22] To isolate full-length human Ras-GRF2 cDNA, a PCR-based strategy was used to amplify the required cDNA from human fetal brain cDNA pool The details of the ampli®cation strategy are described in Experimental procedures Both strands of cDNA were sequenced to con®rm the identity of the human Ras-GRF2 and ensure the ®delity of PCR The predicted sequence of human Ras-GRF2 consists of 1237 amino acids and encodes a protein of 140 763 Da with an isoelectric point of 7.44 (GeneBank accession no AF181250, data reviewed but not shown) Sequence alignment analysis between human and mouse brain Ras-GRF2 sequences indicated that human Ras-GRF2 protein contains several wellde®ned motifs including: an N-terminal PH (pleckstrin homology) domain, an a helical coiled coil (cc) motif, an IQ motif that is known to bind calmodulin, a DH (Dbl homology) domain, a second PH domain, a Ras exchanger motif (REM) that is conserved among the Ras-speci®c exchange factors, a CDB motif similar to the cyclin destruction box, and a Cdc25-like catalytic exchange domain at the C-terminus (Fig 1A) [21] The primary structure of human Ras-GRF2 is 90.5% identical to the mouse Ras-GRF2 [22], 65.2% identical to human Ras-GRF1 (22), and 64.1% identical to the mouse Ras-GRF1 [22] The extent of sequence identity is even greater when individual protein domains are compared, as shown by the 97.7% identity between DH domains of human and mouse Ras-GRF2 Ó FEBS 2002 proteins One notable difference is the presence of an additional 50 amino-acid sequence found in the human Ras-GRF2 The I1 insertion sequence is located between the CDB and Cdc25-like domains of human Ras-GRF2 protein (Fig 1A,C) These results indicate that the overall domain organization of Ras-GRF2 is highly conserved across species thus permitting functional analysis of human and murine Ras-GRF2 proteins by switching their cDNAs in mutagenesis and immunohistochemistry experiments Human Ras-GRF2 is widely distributed but most abundantly expressed in brain Northern blot analysis showed an abundant expression of Ras-GRF2 transcript ( 8.0 kb) in human brain tissue (Fig 2A) The enrichment of Ras-GRF2 in human brain is consistent with the highly abundant expression of dematin in human brain [5,6] In addition, low levels of the RasGRF2 transcript were also detected in human heart, placenta, kidney, and pancreas (Fig 2A) A highly sensitive PCR-based assay was then used to detect Ras-GRF2 in the cDNA pool of human tissues As shown in Fig 2B, a relatively signi®cant amount of Ras-GRF2 was detected in human ovary and spleen tissues In the testis, an additional band was detected that migrated just above the expected size of the PCR product (Fig 2B) The extra band was subcloned and its cDNA was sequenced The additional PCR band encoded a 50-amino acid insert (I1 for insertion 1) Fig Yeast two-hybrid analysis (A) Schematic representation of dematin and Ras± GRF2 interaction The carboxyl-terminal half of dematin (amino acids 224±383) was used as the bait for the yeast two-hybrid screening Yeast transformed with both dematin and Ras-GRF2 grew on media lacking histidine (+) and turned blue (marked with a B) in the presence of X-gal indicative of a binding interaction Absence of growth was designated by (±) while failure to activate the LacZ reporter gene was designated as (W) (B) Yeast mating between dematin and Ras-GRF1 and between limatin and Ras-GRF and RasGRF2 (C) Amino-acid sequence of insertion1 sequence The ÔextraÕ exon is located between the amino acids KHAQ-Insertion1-DFEL of the human Ras-GRF2 sequence The underlined sequence of insertion-1 shows homology with an isoform of Trio nucleotide exchanger as discussed in the Results section Ó FEBS 2002 Dematin binds to Ras-GRF2 nucleotide exchange factor (Eur J Biochem 269) 643 Fig Tissue expression of human Ras-GRF2 (A) Northern blot analysis of Ras-GRF2 Ras-GRF2 expression is most abundant in the brain A single band of  7.5 kb is detected in most tissues (B) A multiple tissue cDNA panel was screened by PCR using RasGRF2 speci®c primers The bottom panel shows equal amount of starting cDNA pool in each tissue as detected by the glyceraldehyde 3-phosphate dehydrogenase-speci®c primers and is located between the candidate-destruction box and Cdc25-like catalytic domains of Ras-GRF2 (Fig 1C) Genebank database analysis revealed that a 16-amino-acid segment of insertion is 75% identical to a sequence found in an isoform of the Trio protein (Fig 1C) Speci®city of the binding interaction between dematin and human Ras-GRF2 Several independent techniques were employed to establish the speci®city of binding interaction between dematin and Ras-GRF2 First, the yeast two-hybrid assay was used to demonstrate the speci®city of binding between members of the dematin and Ras-GRF families As shown in Fig 1B, the C-terminal half of dematin [dematin (224± 383)] binds to the DH domain of human Ras-GRF2 The dematin(224±383) construct was intentionally engineered to delete the poly(glutamic) acid motif found in the N-terminal half of the dematin core domain [5,6] In preliminary control tests, the poly(glutamic) acid motif appeared to contribute in the autoactivation of the full-length dematin construct The design of the dematin(224±383) construct was also in¯uenced by our previous studies showing a stable expression of the headpiece domain in solution whereas the bacterially expressed core domain of dematin was relatively susceptible to proteolysis [4,5] For this reason, the dematin(224±383) construct was selected for the yeast twohybrid and other biochemical assays A second bait construct for the yeast two-hybrid screen contained only the headpiece domain of dematin The dematin(309)383) headpiece construct failed to bind the DH domain of Ras-GRF2 in the yeast two-hybrid assay (data not shown) suggesting that the Ras-GRF2 binding site is likely to be located within the 84-residue [dematin(224± 308)] segment of the core domain of dematin Similarly, the dematin(224±383) construct failed to bind to the DH domain of human Ras-GRF1 that is  88% identical to the DH domain of human Ras-GRF2 This result suggests that the human dematin binds speci®cally to the DH domain of human Ras-GRF2 but not human Ras-GRF1 (Fig 1B) We have recently identi®ed human limatin (abLIM) as the closest homologue of dematin in mammalian tissues [13] A construct of human limatin(597±778) corresponding to dematin(224±383) (40% identity) also did not bind to the DH domain of either Ras-GRF2 or RasGRF1 (Fig 1B) Based on the results of the yeast twohybrid assay, we conclude that the interaction between dematin and Ras-GRF2 is highly speci®c and is mediated by a novel sequence located within the core domain of dematin An in vitro overlay assay was used to demonstrate direct biochemical interaction between dematin and Ras-GRF2 Native dematin was puri®ed from human erythrocyte membranes and tested for binding to the recombinant Ras-GRF2-DH protein immobilized on the nitrocellulose membrane As shown on Fig 3A, native dematin speci®cally bound to the GST fusion protein of Ras-GRF2-DH domain but not GST alone Again, no binding was observed between native dematin and the GST fusion protein of human Ras-GRF1-DH domain (data not shown) Speci®c binding of the GST fusion protein of Ras-GRF2-DH domain to the dematin(224±383) was quanti®ed by surface plasmon resonance technique using a BIAcore biosensor instrument A homogeneous preparation of dematin(224± 383) domain (18 kDa) (free of GST) was immobilized to a CM5 sensor chip by a standard amine coupling protocol [28] The binding interaction of GST±Ras-GRF2-DH domain (66 kDa) to the immobilized dematin(224±383) was concentration dependent (Fig 3B) No such binding was observed when GST samples were injected at increasing concentrations (up to 6.6 lM) onto the same dematin(224± 383)-immobilized ligand surface under the same experimental conditions The binding was reproducible after repeated cycles of the regeneration process These results demonstrate that the DH domain of Ras-GRF2 protein speci®cally binds to a segment of dematin encoded by dematin(224±383) Apparent on/off rate constants for the observed binding interaction between dematin and Ras-GRF2 protein was determined from the association and dissociation phases of the sensorgram using a nonlinear regression algorithm in the BIAEVALUATION 3.0 software package Estimated kinetic constants for the immobilized dematin(224±383) and GST± Ras-GRF2±DH interaction were ka ˆ 7.64 ´ 103 M)1ás)1 and kd ˆ 3.53 ´ 10)3 s)1 An apparent dissociation constant Kd ˆ 462 nM was obtained from the ratio of kd/ka It is noteworthy here that the GST domain of ligand-bound and free GST±Ras-GRF2-DH domain could in principal, undergo dimerization causing an avidity effect in both association and dissociation phases of the interaction Dematin and Ras-GRF2 associate in mouse brain lysate and in transfected epithelial cells To test whether Ras-GRF2 and dematin associate in vivo, we examined their association in mouse brain lysate and mammalian cells Dematin was immunoprecipitated from mouse brain lysate using an af®nity-puri®ed polyclonal antidematin Ig The dematin immunoprecipitate was analyzed 644 M Lutchman et al (Eur J Biochem 269) Ó FEBS 2002 Fig Interaction of dematin with the DH domain of human Ras-GRF2 (A) Blot overlay assay Approximately lg of GST and GST-RasGRF2-DH fusion protein was immobilized on the nitrocellulose The immunoblot was incubated with puri®ed native dematin, and the binding of dematin was detected by immunoblot analysis The details of the blot overlay are described in the Experimental procedures A similar analysis was carried out using GST-Ras-GRF1-DH fusion protein No binding was observed between dematin and Ras-GRF1 (data not shown) (B) An overlay plot of sensorgrams showing the binding interaction of GST±Ras-GRF2 and the C-terminal domain of dematin [dematin(224±383)] A homogeneous sample of the dematin(224±383) protein was immobilized to the dextran matrix of a CM sensor chip by a standard amine coupling procedure (1.0 ng proteinámm)2) The sensorgrams were generated by injecting di€erent concentrations of GST±Ras-GRF2 (2.3 lM, 1.2 lM, 0.46 lM) at a ¯ow rate of 10 lLámin)1 at 25 °C Puri®ed recombinant GST (6.6 lM) did not bind under the same conditions Apparent association and dissociation rate constants were estimated from the sensorgrams using BIAEVALUATION 3.0 software: ka ˆ 7.64 ´ 103 M)1ás)1 and kd ˆ 3.53 ´ 10)3 s)1 An apparent dissociation constant (KD) of 462 nM was obtained from the ratio of kd/ka The avidity e€ect caused by the dimerization of the GST domain has not been discounted from the data in the determination of kinetic constants by SDS/PAGE and Western blotted with the Ras-GRF2 monoclonal antibody generated against the PH domain of Ras-GRF2 (Transduction Laboratories, Lexington, KY, USA) A control without the addition of anti-dematin Ig did not show any Ras-GRF2 band (Fig 4A, lane 1) A speci®c 140-kDa band consistent with the mobility of mouse RasGRF2 was detected in total lysate (Fig 4A, lane 2) and in lysate immunoprecipitated with the polyclonal anti-dematin Ig (Fig 4A, lane 3) These results demonstrate that endogenous dematin and Ras-GRF2 associate within the same protein complex in mouse brain lysate To examine this interaction further, we transfected human embryonic kidney epithelial cells (A293) with either dematin or Ras-GRF2 or both The expression of Ras-GRF2 and dematin in the transfected cells was con®rmed using an anti-myc Ig (data not shown) Dematin, Ras-GRF2, and dematin/Ras-GRF2 lysates were immunoprecipitated with the anti-dematin Ig and immunoprecipitates were blotted with the monoclonal anti-(Ras-GRF2) Ig (Fig 4B) Total Ras-GRF2 lysate was used as the control indicating the position of 140-kDa band (Fig 4B) The Ras-GRF2 band was detected only in the cotransfected A293 cells (Fig 4B) Together, these results indicate that dematin and Ras-GRF2 associate with each other in vivo under the conditions described above Ras-GRF2 and dematin colocalize in the transfected ®broblasts Direct binding of dematin to Ras-GRF2 suggested that the two proteins might colocalize when over-expressed in the Fig In vivo interaction of dematin with Ras-GRF2 (A) Co-immunoprecipitation of dematin and Ras-GRF2 from mouse brain lysate Mouse brain was homogenized in NP-40 lysis bu€er and the homogenate was centrifuged at 14 000 g The supernatant was precleared with protein G beads and incubated with anti-dematin Ig The immune complexes were recovered by protein G beads that were extensively washed Lane 1, protein G beads were added in samples that were not incubated with anti-dematin Ig (negative control) Lane 2, total brain lysate (positive control) Lane 3, dematin immune complexes that were immunoblotted with Ras-GRF2 antibody The140 kDa band corresponds to Ras-GRF2 (B) Co-transfection and coimmunoprecpitation of dematin and Ras-GRF2 complex from A293 epithelial cells A293 cells were transiently transfected with either dematin or Ras-GRF2 or both for immunoprecipitation experiments Lane 1, total lysate of the dematin/Ras-GRF2 cotransfected cells Lane 2, anti-dematin immunoprecipitate of dematin transfected cells Lane 3, anti-dematin immunoprecipate of Ras-GRF2 transfected cells Lane shows anti-dematin immunoprecipitate of dematin/Ras-GRF2 cotransfected cells Note that the 140 kDa Ras-GRF2 was detected only in the cotransfected cells Ó FEBS 2002 Dematin binds to Ras-GRF2 nucleotide exchange factor (Eur J Biochem 269) 645 Fig Immuno¯uorescent colocalization of dematin and Ras-GRF2 (A) Phase contrast picture of stably cotransfected dematin/RasGRF2 NIH 3T3 cells (B) Rhodamine-labeled dematin antibody showing localization of dematin in the perinuclear and cytoplasmic compartments of the transfected cells (C) FITC-labeled anti-myc in the stably transfected cells showing perinuclear and cytoplasmic localization of human Ras-GRF2 (D) An overlay of B/C panels indicating that dematin and Ras-GRF2 localize to the same compartments of these overexpressing cells Magni®cation 100´ mammalian cells Full-length cDNA constructs of dematin and Ras-GRF2 were transfected into NIH 3T3 ®broblasts to generate stable cell lines The expression of Ras-GRF2 protein in the stable clones was con®rmed by the detection of a 140-kDa polypeptide by Western blot analysis using an anti-myc Ig (data not shown) The overexpression of dematin was detected using a speci®c anti-dematin Ig By indirect immuno¯uorescence analysis, dematin and RasGRF2 were colocalized in the perinuclear and cytoplasmic compartments of the transfected ®broblasts (Fig 5) Nuclear staining of neither dematin nor Ras-GRF2 was not detectable under these conditions These results suggest that the two proteins may interact with each other in the cytoplasmic compartment, and directly or indirectly modulate the in vivo function of small GTPases in mammalian cells Effect of dematin expression on ERK1 and JNK activation Fig E€ect of dematin on ERK1 activation (A) A293 cells were transfected with either vector, or constitutively active Ras, or dematin, or Ras-GRF2 Cells were stimulated with ionomycin, as described in the Experimental procedures, and lysates were immunoblotted with respective antibodies Anti-tubulin Ig was used to normalize the protein content of each lysate ERK1 activation was detected with an antibody against phospho-ERK1 This antibody detects a doublet of activated ERK1 Note that dematin overexpression alone induced signi®cant increase in the activation of ERK1 (B) Dematin does not modulate the Ras-GRF2 induced activation of ERK1 Anti-tubulin Ig normalized lysates were then tested for the presence of total ERK protein using an anti-ERK2 Ig Activated ERK1 was detected as described in (A) Recent studies have shown that the Cdc25-like domain of Ras-GRF2 stimulates the activation of the MAP kinase ERK1 and Ras upon in¯ux of intracellular calcium in A293 cells [22,26] First, we wanted to test whether the binding of dematin to the DH domain of human Ras-GRF2 had any downstream regulatory effects on the activation of ERK1 via its Cdc25 domain The recombinant Cdc25-like domain of human Ras-GRF2 stimulated guanine nucleotide exchange on Ha-Ras protein (data reviewed but not shown) We then transfected the A293 cells with various constructs and measured the extracellular-signal-regulated kinase (ERK) activity as described in the Experimental procedures Interestingly, the transfection of dematin alone in A293 cells caused a signi®cant enhancement of ionomycin-induced activation of ERK1 (Fig 6A) However, dematin overexpression did not result in any measurable modulatory 646 M Lutchman et al (Eur J Biochem 269) Fig Dematin does not regulate Ras-GRF2 encoded Rac-GRF activity COS-7 cells were transiently transfected with pAX142-RacI (WT) and with pCDNA3 that contained the indicated cDNAs Lysates were collected at 48 h and examined by Western blot for expression of RacI (B), Ras-GRF2 (C), and Dematin (D) Lysates were then normalized for RacI expression and subjected to anity precipitation using immobilized GST-Pak GTP-bound RacI that was precipitated with GST-Pak was visualized by Western blot (A) using an anti-RacI Ig (C14, Santa Cruz Biotechnology) Dematin was immunoblotted using a monoclonal antibody from Transduction Laboratories effect on the ionomycin-induced activation of ERK1 through Ras-GRF2 (Fig 6B) These results suggest that dematin does not directly modulate the Ras signaling pathway mediated by the Cdc25 domain of human RasGRF2 The DH domain of several exchange proteins has been shown to exhibit guanine nucleotide exchange activity [22,23,25,26] To investigate the nucleotide exchange activity of the DH domain of human Ras-GRF2, we ®rst tested whether the recombinant DH domain could catalyze the nucleotide exchange of RhoA GTPase In vitro exchange assays did not show any stimulation of the nucleotide exchange on RhoA irrespective of whether dematin was bound to the DH domain of Ras-GRF2 (data reviewed but not shown) Recently, the DH domain of mouse Ras-GRF2 has been reported to enhance the nucleotide exchange activity of Rac1 and stimulates stress-activated protein kinase (SAPK), also known as Jun N-terminal kinase (JNK), in transfected 293 cells [26] Indeed, the human Ras-GRF2 activated Rac1 in transfected COS-7 cells as demonstrated by a GST-pulldown assay (Fig 7) Moreover, the coexpression of dematin did not modulate the Rac activation (Fig 7) Although it appears that the dematin overexperssion may slightly inhibit the Rac exchange activity (Fig 7), it is probably accounted for by the slightly lower expression of Ras-GRF2 in that particular condition We then proceeded to examine the effect of dematin overexpression on JNK activation via Ras-GRF2 in the transfected COS-7 cells The JNK activation was quanti®ed by measuring the transcriptional activation of Jun by human Ras-GRF2 As expected, the expression of Ras-GRF2 and Ó FEBS 2002 Fig Dematin blocks transcriptional activation of Jun by Ras-GRF2 COS-7 cells were transfected with plasmids encoding the indicated proteins (3 lg each), along with an expression vector for the Gal4 DNA binding domain fused to transactivation domain of Jun [0.25 lg Gal-Jun (1±223)] and a Gal4 luciferase reporter (2.5 lg 5XGal4-luc) For each condition, pCMVnlac (0.25 lg) was also included in the transfection as an internal control for transfection eciency and/or growth inhibition All values were normalized against b-galactosidase activity Fold activation was determined by the number of luciferase units relative to the number of units seen with the vector control Data shown are representative of at least three independent assays performed on duplicate plates The error bars indicate standard deviations constitutively active Rac(12V) resulted in the transcriptional activation of Jun (Fig 8) Interestingly, the coexpression of dematin caused a signi®cant inhibition of Jun activation by Ras-GRF2 as well as Rac(12V) (Fig 8) Similarly, cotransfection of dematin and Ras-GRF2 in A293 cells suppressed JNK activation by  ®vefold (data reviewed but not shown) Together, these results indicate that dematin functions downstream of the signaling cascade mediated by Rac1 and Ras-GRF2 in the mammalian epithelial cells DISCUSSION The identi®cation of dematin as a component of erythrocyte cytoskeleton revealed many aspects of its actin binding/ bundling properties [1,2,27] However, the function of dematin in nonerythroid cells remains to be elucidated The primary structure of dematin suggested that its modular sequence might encode distinct cellular functions [4,5] The C-terminal headpiece domain of dematin is specialized for its actin binding function, and is likely to modulate dematin's actin bundling activity [2,27] In contrast, the core domain of dematin may serve as a docking site for the binding of unknown proteins With this modular structure, dematin could be ideally suited as a molecular adaptor linking the cytoplasmic or membrane-associated proteins to the actin cytoskeleton Due to the abundant expression of dematin in the brain, we searched for dematin-interacting proteins by screening a human brain cDNA library using the yeast two-hybrid system Guided by our previous studies Ó FEBS 2002 Dematin binds to Ras-GRF2 nucleotide exchange factor (Eur J Biochem 269) 647 showing poor expression of the core domain, most likely due to the presence of a PEST sequence that marks proteins for proteolysis, we designed a dematin bait construct expressing only 84 amino acids of the core domain fused to the headpiece domain The headpiece domain is a protease-resistant module that expresses as a stable recombinant protein in vitro [4] This bait construct of dematin containing 84 amino acids of the core domain and complete headpiece domain mediated binding with the DH domain of human Ras-GRF2 (Fig 1) In contrast, a bait construct containing only the headpiece domain of dematin failed to bind to the DH domain of human Ras-GRF2 (data not shown) This observation suggests that a novel 84-aminoacid sequence originating from the core domain mediates dematin binding to the DH domain of human Ras-GRF2 protein Clearly, a detailed evaluation by in vitro mutagenesis will be required to precisely map the Ras±GRF2 binding interface and its stability within the core domain of dematin The inability of dematin to bind to the DH domain of human Ras-GRF1, as well as lack of binding between limatin (abLIM) and Ras-GRF2/Ras-GRF1 underscores the speci®city of the binding interaction between dematin and Ras-GRF2 The primary structure of human brain Ras-GRF2 encodes a highly conserved multidomain protein consisting of an N-terminal PH domain, followed by the coiled coil (cc) and IQ motifs, a single DH domain that is closely linked to another PH domain, REM and CDB motifs, and a C-terminal Cdc25 exchanger domain (Fig 1) The overall domain organization of human Ras-GRF2 is similar to its mouse homologue except for the presence of an additional sequence of 50 amino acids located just upstream of the Cdc25 exchanger domain (Fig 1) [22] The I1 insertion sequence was identi®ed during PCR ampli®cation of human testis cDNA pool, and likely to represent an alternatively spliced exon Interestingly, a segment of the I1 insertion sequence shows signi®cant homology with another nucleotide exchanger termed Trio [32] Trio is a multidomain protein consisting of Rac- and Rho-speci®c guanine nucleotide exchanger domains, and binds to the leukocyte antigen-related transmembrane tyrosine phosphatase [32] Whether the Ras-GRF2 isoform bearing the I1 insertion sequence binds to a similar transmembrane protein remains to be determined While our manuscript was under review, the primary structure of human Ras-GRF2 was published [33] Our results are consistent with the reported primary structure of human Ras-GRF2 [33] The presence of I1 insertion upstream of the Cdc25-like domain of Ras-GRF2 remains unique in our sequence (Fig 1) The widespread tissue distribution of Ras-GRF2 (Fig 2), in contrast to restricted neuronal expression of Ras-GRF1, is consistent with the tissue expression of dematin [5,6] Both dematin and Ras-GRF2 are enriched in human brain suggesting a functional interdependence of their interaction in vivo The co-immunoprecipitation of dematin and RasGRF2 from brain lysate (Fig 4A) and transfected A293 epithelial cells (Fig 4B) suggest that the two proteins are found in the same protein complex in vivo Biochemical analysis of cellular fractionation assays revealed that the two proteins are predominantly associated with the particulate fraction of transfected cells (data not shown) This result, together with the cytosolic and perinuclear localization of dematin and Ras-GRF2 in transfected ®broblasts (Fig 5), suggests that the protein complex may regulate cytoskeletal reorganization in mammalian cells Direct binding of dematin to the DH domain of RasGRF2 raises important issues regarding the function of these domains in Ras signaling and actin reorganization Nucleotide exchange factor proteins carrying deletions and targeted mutations within the DH domains lose their transformation potential and catalytic exchange activity [34] A physical link between the DH domains, cellular transformation, and cytoskeletal association is likely to be afforded by the activation of Rho and Rac family GTPases [34] These observations imply that an alternate mechanism must exist that can couple Ras-GRF exchangers to micro®lament reorganization It has recently been demonstrated that Ras-GRF1 and Ras-GRF2 can form homoand hetero-oligomers via their DH domains [33] This observation suggests that DH domains, in addition to their nucleotide exchange function, may be involved in protein± protein interactions While our results indicate that dematin does not directly interact with Ras-GRF1, dematin may indirectly recruit GRF1 to the actin cytoskeleton via its association with Ras-GRF2 It is therefore plausible that the direct binding of dematin to the DH domain of Ras-GRF2 may provide a functional link between Ras signaling and the actin cytoskeleton Elucidation of the crystal structure of tandem DH and PH domains of human Sos1 protein highlights the dramatic complexity of the DH domain±mediated interactions [35] The crystal structure revealed that the DH domain is composed of three helical segments, two of which provide a highly conserved surface bearing functionally critical residues [35] The adjacent PH domain structure is so oriented that its interaction with inositol(1,4,5)-triphosphate is likely to in¯uence the binding of DH domain with potential GTPases This pivotal insight into the structure of the DH± PH domains opens a case for precise mapping of dematin binding to a speci®c helical segment(s) of Ras-GRF2 protein The reported interaction of dematin with the DH domain of Ras-GRF2 may therefore provide a rationale for the modulation of cytoskeletal integrity by phosphorylation, phospholipid binding, and GTPase activation Much of the current evidence implicates the Rho family of GTPases as key regulators of the actin cytoskeleton [36] For instance, the activation of the Rho GTPase leads to stress ®ber and focal adhesion formation while the activation of Rac and cdc42 leads to the formation of lamellopodia and ®lopodia, respectively [36] The induction of membrane ruf¯es by microinjection of activated mutant Ras into ®broblasts strongly suggested a role of Ras in the remodeling of actin cytoskeleton [37] The association of Ras-GRF2 with dematin, an actin binding and bundling protein, provides a potential coupling mechanism between Ras signaling and the actin cytoskeleton without Rho protein intermediaries Although our data indicate that the direct binding of dematin to the DH domain does not affect the activation of ERK1 via the Cdc25-like domain of RasGRF2 (Fig 6), the activation of ERK1 by dematin alone suggests a potential modulatory role of the actin cytoskeleton in the Ras signaling pathways More importantly, the data shown in Figs and provide the ®rst evidence for a functional role of dematin in the regulation of Rac1-JNK signaling pathway Suppression of JNK activation by the overexpression of dematin, irrespective of whether the signal 648 M Lutchman et al (Eur J Biochem 269) is transmitted via Ras-GRF2 or Rac1, hightlights the functional importance of the dematin-mediated reorganization of the actin cytoskeleton in intracellular signaling pathways It is noteworthy here that Vav, a proto-oncogene that plays a major role in cell proliferation and cytoskeletal organization, activates Rac1 and JNK pathway only upon phosphorylation of its tyrosine residues [38] As dematin's actin bundling activity is completely dependent upon its state of phosphorylation, a possibility remains that a physical link between dematin and Ras-GRF2 may manifest functionally upon post-translational modi®cation of either protein in vivo under speci®c stimulatory conditions DH domain-containing proteins, of which there are greater than 20 members, constitute the largest family of oncogenes [34] In fact, many DH domain proteins were discovered by virtue of their transforming ability when expressed in ®broblasts For instance, Tiam-1 is an exchange factor for Rac and was identi®ed by virtue of its contribution in tumor invasion and metastasis pathways [39,40] Similarly, the APC colon tumor suppressor binds to a Racspeci®c guanine nucleotide exchange factor (Asef) and regulates membrane ruf¯ing and lamellipodia formation in epithelial cells [41] The mechanism by which these nucleotide exchangers modulate cell signaling and cytoskeletal reorganization is poorly understood It is of interest to note that we have recently reported loss of heterozygozity of the dematin gene in a majority of 8p21-linked prostate tumors [14] Based on these observations, we postulate that dematin may play a role in the regulation of cell shape with implications in understanding the mechanism of cellular transformation and tumor progression in malignant cells This proposed function of dematin would be analogous to the recently discovered role of the neuro®bromatosis type II (NF2) tumor suppressor protein in the inhibition of Racinduced signaling as a possible mechanism of tumor initiation and progression [42] Ó FEBS 2002 10 11 12 13 14 15 ACKNOWLEDGEMENTS The National Institutes of Health Grants HL51445 (AHC) and CA77493 (IPW) supported this work We are grateful to Dr Larry Feig of Tufts University Biochemistry Department for sharing the cDNA constructs and giving us valuable advice during the course of these studies We thank Dr J Samulski for providing the pCMVnlac construct We are also thankful to Donna Marie-Mironchuk for help with the artwork and Dr Richie Khanna of St Elizabeth's Medical Center for critically reading the manuscript REFERENCES Siegel, D.L & Branton, D (1985) Partial puri®cation and characterization of an actin-bundling protein, band 4.9, from human erythrocytes J Cell Biol 100, 775±785 Chishti, A., Levin, A & Branton, D (1988) Abolition of actinbundling by phosphorylation of human erythrocyte protein 4.9 Nature 334, 718±721 Gilligan, D.M & Bennett, V (1993) The junctional complex of the membrane skeleton Seminars Hematol 30, 74±83 Azim, A.C., Knoll, J.H., Beggs, A.H & Chishti, A.H (1995) Isoform cloning, actin binding, and chromosomal localization of human erythroid dematin, a member of the villin superfamily J Biol Chem 270, 17407±17413 Rana, A.P., Ru€, P., Maalouf, G.J., Speicher, D.W & Chishti, A.H (1993) Cloning of human erythroid dematin reveals another 16 17 18 19 20 21 22 member of the villin family Proc Natl Acad Sci USA 90, 6651± 6655 Kim, A.C., Azim, A.C & Chishti, A.H (1998) Alternative splicing and structure of the human erythroid dematin gene Biochim Biophys Acta 1398, 382±386 Azim, A.C., Marfatia, S.M., Korsgren, C., Dotimas, E., Cohen, C.M & Chishti, A.H (1996) Human erythrocyte dematin and protein 4.2 (pallidin) are ATP binding proteins Biochemistry 35, 3001±3006 Azim, A.C., Kim, A.C., Lutchman, M., Andrabi, S., Peters, L.L & Chishti, A.H (1999) cDNA sequence, genomic structure, and expression of the mouse dematin gene Mamm Gen 10, 1026± 1029 Arpin, M., Pringault, E., Finidori, J., Garcia, A., Jeltsch, J.M., Vandekerckhove, J & Louvard, D (1988) Sequence of human villin: a large duplicated domain homologous with other actinsevering proteins and a unique small carboxy-terminal domain related to villin speci®city J Cell Biol 107, 1759±1766 Friederich, E., Vancompernolle, K., Huet, C., Goethals, M., Finidori, J., Vandekerckhove, J & Louvard, D (1992) An actinbinding site containing a conserved motif of charged amino acid residues is essential for the morphogenic e€ect of villin Cell 70, 81±92 Pinson, K.I., Dunbar, L., Samuelson, L & Gumucio, D.L (1998) Targeted disruption of the mouse villin gene does not impair the morphogenesis of microvilli Dev Dynam 211, 109±121 Ferrary, E., Cohen-Tannoudji, M., Pehau-Arnaudet, G., Lapillonne, A., Athman, R., Ruiz, T., Boulouha, L., El Marjou, F., Doye, A., Fontaine, J.J., Antony, C., Babinet, C., Louvard, D., Jaisser, F & Robine, S (1999) In vivo, villin is required for Ca(2+)-dependent F-actin disruption in intestinal brush borders J Cell Biol 146, 819±830 Roof, D.J., Hayes, A., Adamian, M., Chishti, A.H & Li, T (1997) Molecular characterization of abLIM, a novel actin-binding and double zinc ®nger protein J Cell Biol 138, 575±588 Lutchman, M., Pack, S., Kim, A.C., Azim, A., Emmert-Buck, M., Hu€el, C.V., Zhuang, Z & Chishti, A.H (1999) Loss of heterozygosity on 8p in prostate cancer implicates a role for dematin in tumor progression Cancer Genet Cytogen 115, 65±69 Boguski, M.S & McCormick, F (1993) Proteins regulating Ras and its relatives Nature 366, 643±654 Bos, J.L (1989) Ras oncogenes in human cancer Cancer Res 49, 4682±4689 Brambilla, R., Gnesutta, N., Minichiello, L., White, G., Roylance, A.J., Herron, C.E., Ramsey, M., Wolfer, D.P., Cestari, V., RossiArnaud, C., Grant, S.G., Chapman, P.F., Lipp, H.P., Sturani, E & Klein, R (1997) A role for the Ras signalling pathway in synaptic transmission and long-term memory Nature 390, 281±286 Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T & Bowtell, D (1993) The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1 Nature 363, 83±85 Shou, C., Farnsworth, C.L., Neel, B.G & Feig, L.A (1992) Molecular cloning of cDNAs encoding a guanine-nucleotidereleasing factor for Ras p21 Nature 358, 351±354 Lowenstein, E.J., Daly, R.J., Batzer, A.G., Li, W., Margolis, B., Lammers, R., Ullrich, A., Skolnik, E.Y., Bar-Sagi, D & Schlessinger, J (1992) The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling Cell 70, 431±442 Wei, W., Mosteller, R.D., Sanyal, P., Gonzales, E., McKinney, D., Dasgupta, C., Li, P., Liu, B.X & Broek, D (1992) Identi®cation of a mammalian gene structurally and functionally related to the CDC25 gene of Saccharomyces cerevisiae Proc Natl Acad Sci USA 89, 7100±7104 Fam, N.P., Fan, W.T., Wang, Z., Zhang, L.J., Chen, H & Moran, M.F (1997) Cloning and characterization of Ras-GRF2, a novel Ó FEBS 2002 23 24 25 26 27 28 29 30 31 32 Dematin binds to Ras-GRF2 nucleotide exchange factor (Eur J Biochem 269) 649 guanine nucleotide exchange factor for Ras Mol Cell Biol 17, 1396±1406 Mattingly, R.R & Macara, I.G (1996) Phosphorylation-dependent activation of the Ras-GRF/CDC25Mm exchange factor by muscarinic receptors and G-protein beta gamma subunits Nature 382, 268±272 Farnsworth, C.L., Freshney, N.W., Rosen, L.B., Ghosh, A., Greenberg, M.E & Feig, L.A (1995) Calcium activation of Ras mediated by neuronal exchange factor Ras-GRF Nature 376, 524±527 Kiyono, M., Satoh, T & Kaziro, Y (1999) G protein beta gamma subunit-dependent Rac-guanine nucleotide exchange activity of Ras-GRF1/CDC25 (Mm) Proc Natl Acad Sci USA 96, 4826± 4831 Fan, W.T., Koch, C.A., de Hoog, C.L., Fam, N.P & Moran, M.F (1998) The exchange factor Ras-GRF2 activates Rasdependent and Rac-dependent mitogen-activated protein kinase pathways Curr Biol 8, 935±938 Chishti, A., Faquin, W., Wu, C.C & Branton, D (1989) Puri®cation of erythrocyte dematin (protein 4.9) reveals an endogenous protein kinase that modulates actin-bundling activity J Biol Chem 264, 8985±8991 Johnsson, B., Lofas, S & Lindquist, G (1991) Immobilization of proteins to a carboxymethyldextran-modi®ed gold surface for biospeci®c interaction analysis in surface plasmon resonance sensors Anal Biochem 198, 268±277 Whitehead, I.P., Khosravi-Far, R., Kirk, H., Trigo-Gonzalez, G., Der, C.J & Kay, R (1996) Expression cloning of lsc, a novel oncogene with structural similarities to the Dbl family of guanine nucleotide exchange factors J Biol Chem 271, 18643±18650 Whitehead, I.P., Lambert, Q.T., Glaven, J.A., Abe, K., Rossman, K.L., Mahon, G.M., Trzaskos, J.M., Kay, R., Campbell, S.L & Der, C.J (1999) Dependence of Dbl and Dbs transformation on MEK and NF-kappaB activation Mol Cell Biol 19, 7759± 7770 Whitehead, I., Kirk, H., Tognon, C., Trigo-Gonzalez, G & Kay, R (1995) Expression cloning of lfc, a novel oncogene with structural similarities to guanine nucleotide exchange factors and to the regulatory region of protein kinase C J Biol Chem 270, 18388± 18395 Debant, A., Serra-Pages, C., Seipel, K., O'Brien, S., Tang, M., Park, S.H & Streuli, M (1996) The multidomain protein Trio 33 34 35 36 37 38 39 40 41 42 binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-speci®c and rhospeci®c guanine nucleotide exchange factor domains Proc Natl Acad Sci USA 93, 5466±5471 Anborgh, P.H., Qian, X., Papageorge, A.G., Vass, W.C., DeClue, J.E & Lowy, D.R (1999) Ras-speci®c exchange factor GRF: oligomerization through its Dbl homology domain and calcium-dependent activation of Raf Mol Cell Biol 19, 4611±4622 Whitehead, I.P., Campbell, S., Rossman, K.L & Der, C.J (1997) Dbl family proteins Biochim Biophys Acta 1332, 1±23 Soisson, S.M., Nimnual, A.S., Uy, M., Bar-Sagi, D & Kuriyan, J (1998) Crystal structure of the Dbl and pleckstrin homology domains from the human Son of sevenless protein Cell 95, 259±268 Hall, A (1998) Rho GTPases and the actin cytoskeleton Science 279, 509±514 Bar-Sagi, D & Feramisco, J.R (1986) Induction of membrane ru‚ing and ¯uid-phase pinocytosis in quiescent ®broblasts by ras proteins Science 233, 1061±1068 Crespo, P., Schuebel, K.E., Ostrom, A.A., Gutkind, J.S & Bustelo, X.R (1997) Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product Nature 385, 169±172 Habets, G.G., Scholtes, E.H., Zuydgeest, D., van der Kammen, R.A., Stam, J.C., Berns, A & Collard, J.G (1994) Identi®cation of an invasion-inducing gene, Tiam-1, that encodes a protein with homology to GDP-GTP exchangers for Rho-like proteins Cell 77, 537±549 Hordijk, P.L., ten Klooster, J.P., van der Kammen, R.A., Michiels, F., Oomen, L.C & Collard, J.G (1997) Inhibition of invasion of epithelial cells by Tiam1-Rac signaling Science 278, 1464±1466 Kawasaki, Y., Senda, T., Ishidate, T., Koyama, R., Morishita, T., Iwayama, Y., Higuchi, O & Akiyama, T (2000) Asef, a link between the tumor suppressor APC and G-protein signaling Science 289, 1194±1197 Shaw, R.J., Paez, J.G., Curto, M., Yaktine, A., Pruitt, W.M., Saotome, I., O'Bryan, J.P., Gupta, V., Der Ratner, N.C.J., Jacks, T & McClatchey, A.I (2001) The Nf2 tumor suppressor, Merlin, functions in Rac-dependent signaling Dev Cell 1, 63±72  ... with either dematin or Ras-GRF2 or both The expression of Ras-GRF2 and dematin in the transfected cells was con®rmed using an anti-myc Ig (data not shown) Dematin, Ras-GRF2, and dematin/ Ras-GRF2. .. band (Fig 4B) The Ras-GRF2 band was detected only in the cotransfected A293 cells (Fig 4B) Together, these results indicate that dematin and Ras-GRF2 associate with each other in vivo under the. .. investigate the nucleotide exchange activity of the DH domain of human Ras-GRF2, we ®rst tested whether the recombinant DH domain could catalyze the nucleotide exchange of RhoA GTPase In vitro exchange