Sequence alignment analysis between human and mouse brain Ras-GRF2 sequences indicated that human Ras-GRF2 protein contains several well-de®ned motifs including: an N-terminal PH pleckst
Trang 1Dematin 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 Hanada1and Athar H Chishti1
1 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; 2 Department 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
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 ®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
cyto-skeleton Except ®lamentous actin, no other binding
part-ners 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 indepen-dent 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
Keywords: dematin, erythrocyte, limatin, Ras-GRF2, head-piece domain
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
dephosphoryla-tion [2] Dematin is part of a juncdephosphoryla-tional 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 character-ized 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 homo-logous 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: + 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)
Trang 2[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]
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-GTP-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]
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 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
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
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]
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
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-hybridscreenwerepurchasedfromClontech.TheC-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 ®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 exper-iments 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 Pair-wise 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
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
Trang 3using 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
Bio-tech): Ras-GRF2 (amino acids 176±474), Ras-GRF2
(ami-no acids 909±1237), Ras-GRF1 (ami(ami-no 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 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
(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
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
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were also used to
ensure equal cDNA loading
Blot overlay assay
Equal amounts ( 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 °C
in 5% (w/v) nonfat dry milk/NaCl/Tris (25 mM Tris,
137 mMNaCl, 2.5 mMKCl, pH 8)/0.1% Tween-20
(block-ing solution) The blot was then incubated in the block(block-ing
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
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 af®nity-puri®ed 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 ®nal washes, bound dematin was
immunodetectedusingtheECLsystem(PharmaciaBiotech)
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±
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 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 1M ethanolamine hydrochloride (pH 8.5) The data were ana-lyzed using theBIAEVALUATION3.0 (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)1of 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 Stable NIH 3T3 clones expressing both Ras-GRF2 and dematin were plated at 40% con¯uency 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 ®xed 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/Piand incu-bated with a ¯uorescein isothiocyanate (FITC)-conjugated
Trang 4goat 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 ®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
5 lM ionomycin (Calbiochem) for 5 min at 37 °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 ®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/Piand then lysed in 50 mMTris/HCl,
pH 8.0, 2 mM MgCl2, 0.2 mM Na2S2O5, 10% glycerol, 20% sucrose, 2 mM dithiothreitol, 1 lgámL)1 leupeptin,
1 lgámL)1pepstatin, and 1 lgámL)1aprotinin Cell lysates were then cleared by centrifugation at 10 000 g for 10 min
at 4 °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 4 °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
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 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 iden-ti®ed in the dematin core domain [5,8] A total of
6.0 ´ 105clones 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
Trang 5Ras-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
well-de®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
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
proteins One notable difference is the presence of an additional 50 amino-acid sequence found in the human Ras-GRF2 The I1insertion 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 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 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 (I1for 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 GRF 1 and Ras-GRF2 (C) Amino-acid sequence of
insertion-1 sequence The ƠextraÕ 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.
Trang 6and 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)
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
two-hybrid 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 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 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 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 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 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 7.64 ´ 103 M)1ás)1 and kd 3.53 ´ 10)3s)1 An apparent dissociation con-stant Kd 462 nMwas 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 anti-dematin Ig The anti-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 7.5 kb is detected in most tissues.
(B) A multiple tissue cDNA panel was screened by PCR using
Ras-GRF2 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.
Trang 7by 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
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 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 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 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 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 proteinámm )2 ) The sensorgrams were generated by injecting dierent concentrations of GST±Ras-GRF2 (2.3 l M , 1.2 l M , 0.46 l M ) at a ¯ow rate of 10 lLámin )1 at 25 °C Puri®ed recombinant GST (6.6 l M ) did not bind under the same conditions Apparent association and dissociation rate constants were estimated from the sensorgrams using BIAEVALUATION 3.0 software: k a 7.64 ´ 10 3 M)1ás )1 and
k d 3.53 ´ 10 )3 s )1 An apparent dissociation constant (K D ) of 462 n M was obtained from the ratio of k d /k a The avidity eect caused by the dimerization of the GST domain has not been discounted from the data in the determination of kinetic constants.
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 buer 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 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.
Trang 8mammalian 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 Ras-GRF2 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 mod-ulate the in vivo function of small GTPases in mammalian cells
Effect of dematin expression on ERK1 and JNK activation
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 over-expression did not result in any measurable modulatory
Fig 6 Eect 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
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).
Fig 5 Immuno¯uorescent 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 Magni®cation 100´.
Trang 9effect 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
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)
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 quanti®ed
by measuring the transcriptional activation of Jun by human
Ras-GRF2 As expected, the expression of Ras-GRF2 and
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, cotrans-fection 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
D I S C U S S I O N
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
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 anity 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.
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 eciency 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.
Trang 10showing 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
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
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 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
multi-domain 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
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 ®broblasts (Fig 5),
suggests that the protein complex may regulate cytoskeletal reorganization in mammalian cells
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 micro®lament 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 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 activa-tion of Rac and cdc42 leads to the formaactiva-tion of lamello-podia and ®lolamello-podia, 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 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 ®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