Báo cáo khoa học: Dematin interacts with the Ras-guanine nucleotide exchange factor Ras-GRF2 and modulates mitogen-activated protein kinase pathways

Eur. J. Biochem. 269, 638–649 (2002) (cid:211) 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

1Section 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

and

co-immunoprecipitation

co-transfection, 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 indepen- dent pathway. Because much of the present evidence has centered on the identification 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.

Keywords: dematin, erythrocyte, limatin, Ras-GRF2, head- piece domain.

Erythroid dematin is a major component of red blood cell junctional complexes that link the spectrin–actin cytoskel- eton 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 filaments in a phosphorylation-dependent manner. The primary structure of dematin consists of a C-terminal domain homologous to the (cid:212)headpiece(cid:213) domain of villin, an actin-binding protein of the brush border cyto- skeleton. Except filamentous actin, no other binding part- ners of dematin have been identified. 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 confirmed by blot overlay, surface plasmon resonance,

Dematin is a cytoskeletal protein that binds and bundles actin filaments in vitro [1,2]. It was originally identified 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 dephosphoryla- tion [2]. Dematin is part of a junctional complex, together with protein 4.1, adducin, tropomyosin, and tropomodulin, that links spectrin tetramers and actin protofilaments 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 character- ized the dematin gene and have identified 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 significance of this insertion is not known, we have postulated that the 52-kDa subunit provides a molecular framework for the formation of disulfide-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 (cid:25) 75-residue domain of dematin is homologous to the (cid:212)headpiece(cid:213) 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 microfilament assembly of microvilli in absorptive tissues is compensated for by dematin and/or other (cid:212)headpiece(cid:213)-containing proteins [11,12]. The N-terminal core domain of dematin is homo- logous to only one other known protein, a (cid:212)LIM(cid:213) protein termed limatin (abLIM) [13]. Limatin contains four double zinc finger LIM domains at its N-terminus with the C-terminus sharing (cid:25) 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: + 1 617 789 3111, Tel.: + 1 617 789 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-ethyl- N¢-[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 sub- mitted to the GenBank with the accession numbers AF181250 and AF186017. (Received 25 September 2001, accepted 20 November 2001)

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[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 8p21- linked prostate tumors [14].

adult human brain library with the C-terminal half of dematin as the bait probe. The identification 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 significance 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.

E X P E R I M E N T A L P R O C E D U R E S

Yeast two-hybrid screen

The vectors, yeast strains, and library employed in two- hybrid 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-1,2,4- triazole (5 mM). Colonies that grew on selective media were then scored for b-galactosidase activity by the filter 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

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 GTP- bound 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-ter- minal 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].

Yeast mating experiments were utilized to test the specificity 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 exper- iments. The segment of limatin (amino acids 597–778) corresponding to the dematin (cid:212)bait(cid:213) 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. Pair- wise matings between all pAS2-1 transformants and all pGAD10 transformants were plated on minimal media and scored for b-galactosidase activity.

the

Cloning of Ras-GRF2cDNA and expression constructs

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 Ras- GRFs 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 exchange activity of Ras-GRF1 (or stimulate CDC25Mm) in a phosphorylation-dependent manner [23]. Calcium influx 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 ilimaquinone- dependent 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].

pcDNA3.1/myc-His

vector

Primer pair 7/8 (7 : 5¢-ATGCAGAAGAGCGTGCGC TAC-3¢; 8 : 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 amplified and subcloned into the vector pCR2.1 (Invitrogen, CA, USA) for sequence analysis. The full-length Ras-GRF2 cDNA was PCR-amplified with BamHI adaptors and subcloned into the mammalian expression (Invitrogen). Immunodetection of Ras-GRF2 protein was carried out

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

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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-amplified with BamHI/EcoRI adaptors for in-frame subcloning into the bacterial expression vector pGEX-2T (Pharmacia Bio- tech): Ras-GRF2 (amino acids 176–474), Ras-GRF2 (ami- no 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–lima- tin(597–778), respectively. Recombinant proteins were expressed and purified accordig to the manufacturer’s instructions (Pharmacia Biotech).

Expression analysis

dematin(224–383) fusion protein was affinity-purified 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 ((cid:25) 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- fied 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. Purified recombinant GST was used as a control sample. Association and dissociation rates were measured at 25 (cid:176)C at a flow rate of 10 lLÆmin)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 significant denaturation of the immobilized ligand during the repeated cycles of regener- ation 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 1 : 1 mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N¢-[3-(diethyla- mino)propyl]carbodiimide (EDC) and blocked with 1 M ethanolamine hydrochloride (pH 8.5). The data were ana- lyzed using the BIAEVALUATION 3.0 (Pharmacia Biosensor) software.

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 (Clon- tech). 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 specific for glyceraldehyde- 3-phosphate dehydrogenase (G3PDH) were also used to ensure equal cDNA loading.

Transfection of Ras-GRF2 and dematin into NIH 3T3 cells

Blot overlay assay

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 modified 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 lgÆmL)1 of G418 over a period of 2 weeks. Stable clones were expanded for further analysis. After 2 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

Equal amounts ((cid:25) 2 lg) of GST and GST–GRF2-DH fusion proteins were separated by SDS/PAGE and either Coomassie-stained or transferred to a nitrocellulose mem- brane. The nitrocellulose blot was blocked overnight at 4 (cid:176)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 (block- ing solution). The blot was then incubated in the blocking solution containing 10 lg of purified dematin. Dematin, which is a trimeric protein of two 48-kDa polypeptides and one 52-kDa polypeptide, was purified from human erythro- cyte membranes [27]. After an overnight incubation in the cold room, the blot was washed twice for 10 min at room temperature in NaCl/Tris/0.1% Tween-20 and incubated for 1 h in a 1 : 3000 dilution of affinity-purified polyclonal anti- dematin Ig. Following two 10-min washes, the blot was then incubated in an horseradish peroxidase-conjugated second- ary antibody (1 : 3000 dilution) for 1 h at room tempera- ture. After two final washes, bound dematin was immunodetected using the ECL system (Pharmacia Biotech).

Surface plasmon resonance analysis

Stable NIH 3T3 clones expressing both Ras-GRF2 and dematin were plated at 40% confluency for use in immuno- localization studies. Stable clones were washed in NaCl/Pi (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) and fixed with formaldehyde (Sigma). After washing in NaCl/Pi, cells were permeabilized in NaCl/Tris/ 1% Triton X-100 for 5 min. Cells were washed in NaCl/Pi and incubated in a 1 : 100 dilution of monoclonal anti-myc Ig for 1 h. Stable clones were washed in NaCl/Pi and incu- bated with a fluorescein isothiocyanate (FITC)-conjugated

A BIAcore 1000 (Pharmacia Biosensor, NJ, USA) was used to measure the specific interaction and to determine the binding affinity between the C-terminal domain of dematin [dematin(224–383)] and GST–Ras-GRF2. The GST–

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goat anti-(mouse IgG) Ig (Pierce; 1 : 64 dilution) (Sigma) for 1 h. After rinsing in NaCl/Pi, cells were incubated for 1 h with polyclonal anti-dematin Ig followed by subsequent washes in NaCl/Pi and incubation with a rhodamine- conjugated goat anti-(rabbit IgG) Ig (Pierce; 1 : 100 dilu- tion; Sigma) for 1 h. After two final washes, cover slips were mounted onto slides using an Antifade reagent (Bio-Rad) and observed under a Zeiss fluorescence microscope linked to a Cooke CCD camera. Photographs were taken using IMAGE-PRO PLUS v. 300 (Mediacybernatics, Silver Spring, MD, USA).

ERK1 activation

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, 2 mM MgCl2, 0.2 mM Na2S2O5, 10% glycerol, 20% sucrose, 2 mM dithiothreitol, 1 lgÆmL)1 leupeptin, 1 lgÆmL)1 pepstatin, and 1 lgÆmL)1 aprotinin. Cell lysates were then cleared by centrifugation at 10 000 g for 10 min at 4 (cid:176)C. The expression of proteins was confirmed by Western blotting prior to affinity purification. Lysates used for affinity purification were normalized for endogenous RacI levels. Affinity purifications were carried out at 4 (cid:176)C for 1 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 Biotech- nology, Santa Cruz, CA, USA).

R E S U L T S

Isolation of human Ras-GRF2 by yeast two-hybrid screening

A293 cells were transiently transfected with Lipofectamine the cells were 2000 (Gibco-BRL). After transfection, allowed to recover for 48 h in DMEM/10% fetal bovine serum. The cells were then starved for 18 h and treated with 5 lM ionomycin (Calbiochem) for 5 min at 37 (cid:176)C. Cells were scraped with cell lysis buffer and used for ERK activation assays. ERK1 assays were as described previ- ously [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 1 : 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 constit- utively 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 five 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

To investigate the function of erythroid dematin in none- rythroid 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 dematin- interacting 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 pre- cluding 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 defined 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 iden- tified in the dematin core domain [5,8]. A total of (cid:25) 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 identified 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 confirmed using controls as specified by the manufacture’s protocol. This indicated that the two proteins interacted in vitro using the yeast two-hybrid assay.

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, Southfield, MI, USA) according to the manufacturer’s instructions. All assays were performed in triplicate.

Rac1 activation assay

Cloning and complete primary structure of human Ras-GRF2

Our initial identification of the human Ras-GRF2 cDNA was based on its sequence alignment with the mouse

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-

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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

the PCR product

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 amplification strategy are described in Experimental procedures. Both strands of cDNA were sequenced to confirm the identity of the human Ras-GRF2 and ensure the fidelity 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 well- defined 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-specific exchange factors, a CDB motif similar to the cyclin destruc- tion 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

Northern blot analysis showed an abundant expression of Ras-GRF2 transcript ((cid:25) 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 Ras- GRF2 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 significant 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 (Fig. 2B). The extra band was of subcloned and its cDNA was sequenced. The additional PCR band encoded a 50-amino acid insert (I1 for insertion 1)

Fig. 1. Yeast two-hybrid analysis. (A) Sche- matic 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 1 and Ras- GRF2. (C) Amino-acid sequence of insertion- 1 sequence. The (cid:212)extra(cid:213) exon is located between the amino acids KHAQ-Insertion1-DFEL of the human Ras-GRF2 sequence. The under- lined sequence of insertion-1 shows homology with an isoform of Trio nucleotide exchanger as discussed in the Results section.

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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 1 is 75% identical to a sequence found in an isoform of the Trio protein (Fig. 1C).

Specificity of the binding interaction between dematin and human Ras-GRF2

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 (cid:25) 88% identical to the DH domain of human Ras-GRF2. This result suggests that the human dematin binds specifically to the DH domain of human Ras-GRF2 but not human Ras-GRF1 (Fig. 1B). We have recently identified human limatin (abLIM) as the closest homologue of dematin in mamma- lian 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 Ras- GRF1 (Fig. 1B). Based on the results of the yeast two- hybrid assay, we conclude that the interaction between dematin and Ras-GRF2 is highly specific 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 purified 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 specifi- 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). Specific binding of the GST fusion protein of Ras-GRF2-DH domain to the dematin(224–383) was quantified 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 experimen- tal conditions. The binding was reproducible after repeated cycles of the regeneration process. These results demonstrate that the DH domain of Ras-GRF2 protein specifically binds to a segment of dematin encoded by dematin(224–383).

M

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 (cid:136) 7.64 · 103 )1Æs)1 and kd (cid:136) 3.53 · 10)3 s)1. An apparent dissociation con- stant Kd (cid:136) 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.

Several independent techniques were employed to establish the specificity of binding interaction between dematin and Ras-GRF2. First, the yeast two-hybrid assay was used to demonstrate the specificity 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 influenced 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 two- hybrid and other biochemical assays.

Dematin and Ras-GRF2 associate in mouse brain lysate and in transfected epithelial cells

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–

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 affinity-purified polyclonal anti- dematin Ig. The dematin immunoprecipitate was analyzed

Fig. 2. 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 (cid:25) 7.5 kb is detected in most tissues. (B) A multiple tissue cDNA panel was screened by PCR using Ras- GRF2 specific primers. The bottom panel shows equal amount of starting cDNA pool in each tissue as detected by the glyceraldehyde 3-phosphate dehydrogenase-specific primers.

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M

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 fibroblasts

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 specific 140-kDa band consistent with the mobility of mouse Ras- GRF2 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 endog- enous 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 confirmed using an anti-myc Ig (data

Direct binding of dematin to Ras-GRF2 suggested that the two proteins might colocalize when over-expressed in the

Fig. 3. Interaction of dematin with the DH domain of human Ras-GRF2. (A) Blot overlay assay. Approximately 2 lg of GST and GST-Ras- GRF2-DH fusion protein was immobilized on the nitrocellulose. The immunoblot was incubated with purified 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 homo- geneous sample of the dematin(224–383) protein was immobilized to the dextran matrix of a CM 5 sensor chip by a standard amine coupling procedure (1.0 ng proteinÆmm)2). The sensorgrams were generated by injecting di(cid:128)erent concentrations of GST–Ras-GRF2 (2.3 lM, 1.2 lM, 0.46 lM) at a flow rate of 10 lLÆmin)1 at 25 (cid:176)C. Purified 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 (cid:136) 7.64 · 103 )1Æs)1 and kd (cid:136) 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(cid:128)ect caused by the dimerization of the GST domain has not been discounted from the data in the determination of kinetic constants.

that were immunoblotted with Ras-GRF2 antibody. The140 kDa band corresponds

Fig. 4. 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(cid:128)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, to Ras-GRF2. dematin immune complexes (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 4 shows anti-dematin immunoprecipitate of dematin/Ras-GRF2 cotransfected cells. Note that the 140 kDa Ras-GRF2 was detected only in the cotransfected cells.

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the transfected fibroblasts

mammalian cells. Full-length cDNA constructs of dematin and Ras-GRF2 were transfected into NIH 3T3 fibroblasts to generate stable cell lines. The expression of Ras-GRF2 protein in the stable clones was confirmed 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 specific anti-dematin Ig. By indirect immunofluorescence analysis, dematin and Ras- GRF2 were colocalized in the perinuclear and cytoplasmic compartments of (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 mod- ulate the in vivo function of small GTPases in mammalian cells.

Effect of dematin expression on ERK1 and JNK activation

Fig. 5. Immunofluorescent colocalization of dematin and Ras-GRF2. (A) Phase contrast picture of stably cotransfected dematin/Ras- GRF2 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. Magnification 100·.

Recent studies have shown that the Cdc25-like domain of Ras-GRF2 stimulates the activation of the MAP kinase ERK1 and Ras upon influx 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 significant enhancement of ionomycin-induced activation of ERK1 (Fig. 6A). However, dematin over- expression did not result in any measurable modulatory

Fig. 6. E(cid:128)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 pro- tein 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 significant 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).

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Fig. 7. Dematin does not regulate Ras-GRF2 encoded Rac-GRF activ- ity. 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(cid:129)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 Ras- GRF2.

constitutively active Rac(12V) resulted in the transcriptional activation of Jun (Fig. 8). Interestingly, the coexpression of dematin caused a significant inhibition of Jun activation by Ras-GRF2 as well as Rac(12V) (Fig. 8). Similarly, cotrans- fection of dematin and Ras-GRF2 in A293 cells suppressed JNK activation by (cid:25) fivefold (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.

Fig. 8. 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(cid:129)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 per- formed on duplicate plates. The error bars indicate standard devi- ations.

D I S C U S S I O N

The identification 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

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 first 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). More- over, 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 quantified by measuring the transcriptional activation of Jun by human Ras-GRF2. As expected, the expression of Ras-GRF2 and

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suggests that the protein complex may regulate cytoskeletal reorganization in mammalian cells.

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 recom- binant 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-amino- acid 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 mutagen- esis will be required to precisely map the Ras–GRF2 binding interface and its stability within the core domain of dematin.

Direct binding of dematin to the DH domain of Ras- GRF2 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 microfilament reorganization. It has recently been demon- strated that Ras-GRF1 and Ras-GRF2 can form homo- and 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 resi- dues [35]. The adjacent PH domain structure is so oriented that its interaction with inositol(1,4,5)-triphosphate is likely to influence 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 specific 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.

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 specificity of the binding interaction between dematin and Ras-GRF2. The primary structure of human brain Ras-GRF2 encodes a highly conserved multidomain pro- tein 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 identified during PCR amplification of human testis cDNA pool, and likely to represent an alternatively spliced exon. Interestingly, a segment of the I1 insertion sequence shows significant homology with another nucleotide exchanger termed Trio [32]. Trio is a multi- domain protein consisting of Rac- and Rho-specific 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).

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 fiber and focal adhesion formation while the activa- tion of Rac and cdc42 leads to the formation of lamello- podia and filopodia, respectively [36]. The induction of membrane ruffles by microinjection of activated mutant Ras into fibroblasts 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 Ras- GRF2 (Fig. 6), the activation of ERK1 by dematin alone suggests a potential modulatory role of the actin cytoskel- eton in the Ras signaling pathways. More importantly, the data shown in Figs 7 and 8 provide the first 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

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 Ras- GRF2 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 fibroblasts (Fig. 5),

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is transmitted via Ras-GRF2 or Rac1, hightlights the functional importance of the dematin-mediated reorganiza- tion 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 man- ifest functionally upon post-translational modification of either protein in vivo under specific 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 fibroblasts. For instance, Tiam-1 is an exchange factor for Rac and was identified by virtue of its contribu- tion in tumor invasion and metastasis pathways [39,40]. Similarly, the APC colon tumor suppressor binds to a Rac- specific guanine nucleotide exchange factor (Asef) and regulates membrane ruffling and lamellipodia formation in epithelial cells [41]. The mechanism by which these nucle- otide 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 neurofibromatosis type II (NF2) tumor suppressor protein in the inhibition of Rac- induced signaling as a possible mechanism of tumor initiation and progression [42].

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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.

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