From the Laboratory of Cell Structure and Signal Integration, Van Andel Research Institute, Grand Rapids, Michigan 49503
Received for publication, July 13, 2000, and in revised form, September 9, 2000
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ABSTRACT |
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Mammalian and fungal
Diaphanous-related formin homology
(DRF) proteins contain several regions of conserved sequence
homology. These include an amino-terminal GTPase
binding domain (GBD) that interacts with
activated Rho family members and formin homology domains that mediate
targeting or interactions with signaling kinases and actin-binding
proteins. DRFs also contain a conserved Dia-autoregulatory domain (DAD) in
their carboxyl termini that binds the GBD. The GBD is a bifunctional
autoinhibitory domain that is regulated by activated Rho. Expression of
the isolated DAD in cells causes actin fiber formation and stimulates
serum response factor-regulated gene expression. Inhibitor experiments show that the effects of exogenous DAD expression are dependent upon
cellular Dia proteins. Alanine substitution of DAD consensus residues
that disrupt GBD binding also eliminate DAD biological activity. Thus,
DAD expression activates nuclear signaling and actin remodeling by
mimicking activated Rho and unlatching the autoinhibited state of the
cellular complement of Dia proteins.
Formin homology
(FH)1 proteins and Rho small
GTPases modulate cytoskeletal remodeling during cytokinesis, polarized
cell growth, and development (1-4). The Diaphanous or Dia-related FH
proteins (DRFs) constitute a subclass of FH proteins that bind
activated Rho family small GTP-binding proteins (5). The DRFs include in Saccharomyces cerevisiae, Bni1p and Bnr1p (6-8),
Aspergillus nidulans, SepA (9), and in
Drosophila, Diaphanous (10). Three mammalian DRF
genes have been identified in mice/humans, respectively, mDia1/DFNA1 (11, 12),
mDia2/Dia2, and
mDia32/DIA
(14). Both mDia1 and mDia2 bind to activated RhoA-C, and mDia2 also
interacts with Cdc42 (11, 15). Based on primary amino acid sequence
homology, the DRF family contains several conserved domains; the
GTPase-binding domain (GBD) in the amino termini (15, 16),2
three formin homology domains that include the highly
conserved proline-rich FH1 and FH2 domains, and a loosely conserved FH3 domain (10, 17). This study identifies a new homology domain unique to
the Diaphanous-related FH family members termed the Dia-autoregulatory
domain or DAD.
The DRFs bridge signaling and cell remodeling pathways by binding
to several signaling kinases and scaffolding proteins via SH3 domain
interactions with the proline-rich FH1 domain. These include the Src
nonreceptor-tyrosine kinase family (18, 19), Hof1p (20), and
IRSp53/BAIAP2 (21). The actin-binding protein profilin also interacts
with FH1 domains (6, 15, 22, 23). Other actin-binding factors EF1 Bni1p, mDia1, and mDia2 have been shown to be activated or
deregulated by removal of their GTPase binding domains (11, 18, 26).
Expression of Many signaling molecules contain autoregulatory domains. For
example, p21-activated kinase (PAK1) (30, 31) and Src family kinases
bear domains that modulate their activity through intramolecular associations (32). The PAK1 autoinhibitory domain is adjacent to the
CRIB domain (31), and this association is regulated by binding
to activated Cdc42. A similar observation has been made for the
Cdc42-binding Wiskott-Aldrich
syndrome protein (WASP) (34). Kim et
al. demonstrated that the amino-terminal CRIB domain of
WASP is a bifunctional autoinhibitory and GTPase binding domain (33).
It binds to the carboxyl-terminal verprolin,
cofilin acidic (VCA) region located in the
carboxyl terminus. Upon binding to Cdc42, the CRIB domain releases the
carboxyl terminus, and the exposed VCA region of WASP functions as an
effector by direct binding to the Arp2/3 actin-nucleating complex
(35).
A similar mechanism has been proposed for the Dia-related proteins
(11). The amino terminus of mDia1 has been shown to interact with the
carboxyl terminus in a manner that is disrupted by activated Rho
binding to the GBD. Here, a conserved carboxyl-terminal autoregulatory domain, termed DAD, that facilitates intramolecular binding is identified. When exogenously expressed in cells, DAD is a potent activator of the cellular complement of DRFs and causes the induction of both cytoskeletal remodeling and the SRF signaling.
Cell Culture, Microinjection, and Fluorescence
Microscopy--
NIH 3T3 cells grown on glass coverslips were
maintained in Dulbecco's modified essential medium (Life Technologies,
Inc.) containing 10% (v/v) fetal calf serum (Life Technologies, Inc.) until 24 h prior to microinjection when cells were changed to medium containing 0.1% (v/v) FCS. The NIH 3T3 SRE-FosHA reporter cell
line HA13 was used in all SRE gene expression studies (36). Cells were
microinjected with pulled-glass capillaries using an Eppendorf 5171 semi-automated injection system as described previously (18). Purified
plasmid DNA expression vectors were microinjected at a concentration of
10 µg/ml each in a buffer of phosphate-buffered saline/dH20 (1:1), unless indicated otherwise. Empty vector
(pEFm) was included in experiments to normalize injected
DNA concentrations when necessary. For fluorescent detection
experiments, cells were fixed 3 h after microinjection with 3.7%
formaldehyde in phosphate-buffered saline and permeabilized with 0.3%
Triton X-100 (Sigma) prior to staining. SRE-regulated FosHA staining
was detected by indirect immunofluorescence using primary rabbit
anti-HA antisera (Y-11, Santa Cruz Biotechnology) followed by
aminomethyl coumarin acetate (AMCA)-coupled anti-rabbit
(Jackson). Filamentous actin was monitored in cells by staining with
TRITC-labeled phalloidin (Sigma). After staining, coverslips
were mounted in gelvatol. Fluorescent images were captured with a
digital camera (SPOT R100, Diagnostics) mounted on a Nikon E400
epifluorescence microscope using fixed exposure times with either × 40 or × 100 magnification (1.4 NA) where indicated. Images were saved as TIFF files and assembled into figures using Clarisdraw.
Plasmids and GenBankTM/EBI Accession
Numbers--
mDia1, mDia2, and various domain expression constructs
were made in pEFm (courtesy of R. Marais),
pEFHA, pEFmEGFP, pT7-plink, pGEX-KG, and pGAD10
from polymerase chain reaction products using standard methods and
confirmed by direct sequencing; complete details are available upon
request. In vitro translation plasmids were made using
pT7-plink (37). GenBankTM/EBI accession numbers for the
gene products discussed are mDia1, U96963; mDia2, AF094519; DIA156,
NP006720; Diaphanous, AAA67715; Bni1, P41832; and SepA, AAB63335. For
most of the experiments, plasmids encoded the following amino acids for
mDia2: GBD, 101-216; FH1, 521-630; FH2, 801-910; and DAD, 1031-1171
unless otherwise indicated.
In Vitro GST Pull-down and Two-hybrid Assays--
Two-hybrid
assays and in vitro translation/GST pull-down assays were
conducted as described previously (14, 18). In short, the indicated
bait proteins were generated by subcloning the indicated cDNAs into
pGBT9 Gal4 DNA binding domain plasmid; prey were Gal4 activation domain
fusion proteins generated in either pGAD10 or pSE1107. HF7c
(CLONTECH) reporter yeast strain was cotransformed with the indicated plasmids and selected on appropriate plates for bait
and prey auxotrophic markers and then restreaked onto His-plate to
select for Gal4-regulated His reporter expression. Levels of reporter
activity were monitored by replicate streaking onto Trp/Leu/His-plates
with increasing concentrations (0-64 mM) of
3-aminotriazole.
For in vitro translation and pull-down assays, plasmids were
constructed using pT7-plink or pCAN (37) containing the indicated coding sequences for mDia2 (18), GBD, or FH1 and were in
vitro translated using the TNT kit (Promega) using
35S-labeled methionine. 4 µl of labeled in
vitro translation product were incubated with 10 µg of GST,
GST·DAD, or GST·SrcSH3, bound to glutathione beads as indicated.
Beads were incubated at 4 °C with rocking for 2 h in 25 mM Tris pH 7.2, 100 mM NaCl, and 10 mM MgCl2. Binding reactions were warmed to
30 °C before addition of recombinant RhoA-V14 (34). Beads were
collected by centrifugation and after washing beads three times in 5 bed volumes binding buffer, beads were suspended in SDS sample buffer,
electrophoresed, and then stained with Coomassie Blue R250 and dried
prior to autoradiography.
Identification of DAD--
Amino acid sequence alignments of the
growing FH protein superfamily have delineated the proline-rich FH1 and
FH2 regions of homology (5, 10). Comparative alignment of only the
Dia-related subfamily yields a conserved domain in the carboxyl termini
that is shown in Fig. 1. The consensus
sequence
(G/A)(V/A)MDXLLEXL(K/R/Q)X(G/A)(S/G/A)(A/P) was designated the Dia-autoregulatory domain or DAD. There is also a
conserved region of basic residues several residues toward the carboxyl
termini. The DAD domain was found in all the three mouse/human
DRFs2 (14, 15), Drosophila Diaphanous (10),
budding yeast S. cerevisiae Bni1p (37), and
Aspergillus nidulans SepA proteins (9); the exception
appeared to be Bnr1p (6).
Self-Association: DAD Interaction with the GBD--
Watanabe
et al. (11) have previously reported that the amino terminus
of mDia1 could bind to its carboxyl terminus. A similar intramolecular
interaction has also been reported for the Cdc42-binding WASP (34). To
determine whether DAD could mediate this interaction in mDia2, the DAD
domain was tested for binding to the GBD in both two hybrid and GST
pull-down assays as shown in Fig. 2,
A and B, respectively. In vitro both
mDia2 and the isolated GBD bound specifically to the DAD domain (data
not shown). The DAD·GBD or DAD interaction with mDia2 was tested for
regulation by activated RhoA-V14. Both in vitro translated
[35S]methionine-labeled GBD and mDia2 were incubated with
increasing concentrations of purified recombinant RhoA-V14
protein.2 Activated Rho disrupted the association of both
the isolated mDia2 GBD and mDia2 itself as shown in Fig. 2B
but did not have any effect on FH1-SrcSH3 binding.
A model based on these observations is shown in Fig. 2C
after Watanabe et al. (11) In cells with low levels of
activated Rho·GTP, Dia protein assumes an inactive state with the
carboxyl-terminal DAD directly interacting with the amino-terminal GBD.
Small GTPase activation and GBD binding induces release of the DAD and
then effectors are recruited through the FH1 and FH2 domains. The
carboxyl-terminal DAD, like the VCA domain of WASP, could be a
bifunctional regulator. In this event, DAD would not only associate
with the GBD but also recruit effectors such as the ARP2/3 complex in a
manner analogous to the WASP-VCA domain (32). If DAD lacks effector
activity, it is possible that expression of this isolated domain in
cells might disrupt the intramolecular GBD-DAD association. In this case, DAD expression might activate endogenous DRFs in cells. These two
possibilities were then examined.
DAD Expression Activates Actin Remodeling and SRF--
The
mDia2 DAD domain was fused to green fluorescent protein (EGFP) in a
mammalian expression plasmid (pEFmEGFP-DAD (18)) and was
microinjected into NIH 3T3 cells previously maintained for 24 h in
0.1% serum, which diminishes the appearance of actin fibers. 3 h
after injection, the effects on actin reorganization and activation of
SRF were assayed. Actin polymerization was observed by fixing and
staining cells with fluorescent TRITC-phalloidin, and SRF-regulated
gene expression was monitored by indirect immunofluorescence by
staining HA13 cells for the induction of a stably transformed SRE-controlled c-fos reporter gene that contains an
HA-epitope tag (34). The effects of EGFP·DAD were compared with
similar EGFP fusion proteins containing other mDia2 domains, including the GBD, FH1, and FH2 sequences, in addition to the previously described activated
The localization of the fusion proteins differed greatly. The
EGFP·FH1 fusion was predominantly nuclear with some diffuse cytoplasmic localization. However, upon stimulation with LPA, the
EGFP·FH1 fusion began to decorate actin stress fibers as shown in the
example in Fig. 3B. The inset shows a region from
the EGFP·FH1-expressing cell in the top row; merged images
clearly show overlapping EGFP·FH1/stress fibers which appear
yellow. EGFP·FH2 localization was consistently diffuse
throughout the cell, whereas EGFP·DAD was excluded from the nucleus.
DAD did appear to concentrate at the ends of a subset of actin fibers
in cells expressing higher levels of DAD but not to the extent seen
with EGFP·FH1 (data not shown).
EGFP·DAD strongly induced SRF as summarized in Fig. 3C,
where bars represent the number of FosHA-positive GFP
fusion-expressing cells. This result is consistent with previous
results showing a role for the DRFs in an SRF-signaling pathway (18,
40). The model (Fig. 2C) predicts that overexpression of the
GBD would squelch the effects of DAD domain expression through direct
binding. To test this, plasmids encoding both GBD
(pEFm-GBD) and DAD (pEFmEGFP-DAD) were
microinjected into cells, and actin and SRF activity were monitored as
before. As shown in Fig. 4A,
GBD expression blocked DAD-induced actin remodeling. Coinjection of
either FH1 or FH2 domains were without effect. Similar results were
obtained when SRF activity was assayed (Fig. 4B). These
results showed that the GBD expression inhibited the DAD domain in
trans and was, in effect, squelching the DAD domain.
The GBD could also block SRF and actin remodeling if DAD was activating
Rho. Expression of other Rho GTPase binding domains have been shown to
block Rho signaling (36). DAD activation of SRF via Rho was tested
despite the presumption that the DRFs were downstream effectors of Rho
signaling. DAD was coexpressed with either the amino terminus of PKN
(PKN.N) or C3 transferase, which also inhibits Rho signaling but not
the activated DRFs (18, 36, 40). Neither C3 or PKN.N inhibited DAD
activation of SRF (Fig. 4B) though each blocked serum
activation of SRF as previously reported (data not shown). Because of
this specific blockade of DAD, it was concluded that DAD was triggering
signals downstream of the Rho GTPases to activate SRF. These
experiments also show that DAD is biologically active and is capable of
specifically binding the GBD in cells. The molecular nature of DAD
activity in cells was then examined.
DAD Expression Unlatches the GBD·DAD Autoregulatory
Mechanism--
If DAD was an effector domain analogous to the WASP-VCA
domain (34), GBD squelching of DAD activity would be predicted. The GBD
would also be expected to block DAD if the alternative occurred; DAD
inhibits a negative intramolecular DAD-GBD association. In this case,
DAD domain expression would be activating endogenous Dia proteins by
unlatching the GBD·DAD autoregulatory mechanism. This was tested by
expressing DAD and simultaneously inhibiting endogenous mDia1 by
coinjection of affinity-purified anti-mDia1 with the EGFP·DAD
expression vector (18). NIH 3T3 cells express mDia1 but not mDia2;
anti-mDia1 antibody recognizes mDia1 amino acids 66-77 (YGDDPTAQSLQD).
Anti-mDia1 effectively blocked DAD activity toward actin remodeling
(data not shown) and SRF (Fig. 5,
A and B). Anti-mDia1 did not inhibit
To correlate DAD-DRF binding with DAD biological activity, a series of
alanine substitutions were introduced into the DAD consensus region and
were tested for binding to mDia2 by two-hybrid analysis. After
determining the effects on DAD-GBD association, the resultant DAD
domain mutants were then expressed in cells. The results of the binding
experiments performed by two-hybrid analysis are summarized in Fig.
6A. Mutations that disrupted
the mDia2-DAD interaction are indicated by the filled
arrowheads; mutations that had no effect on mDia2 interaction are
shown by the open arrowheads. M1041A, L1044A, L1048A (data
not shown) (amino acid positions are from the mDia2 peptide sequence;
conserved residues are indicated by black boxes, similar
residues by grey boxes) substitutions of conserved
DAD residues disrupted binding and were inactive toward the activation
of both SRF (data not shown) and actin fiber formation as shown in Fig.
6B. The L1044A localization was also significantly altered.
Instead of the diffuse membrane localization seen with wild-type DAD,
L1044A had a punctate localization in the membrane and appeared at the
cell edge. The L1044A substitution allowed the fusion protein to appear
in the nucleus and also caused the formation of lamellipod-like
extensions. Alanine substitutions of the nonconserved residues S1043A
and Q1049A had no effect on binding, and both were able to induce fiber
formation and SRF (data not shown). Interestingly, the E1046A substitution of the conserved glutamate residue was also inactive, suggesting that DAD·mDia2 binding is largely mediated through hydrophobic interactions. The interaction is also dependent upon the
stretch of basic residues adjacent to the core DAD sequence. The
introduction of a stop codon at position 1050 weakened but did not
eliminate the interaction as determined by two-hybrid assay and also
reduced the number and density of apparent fibers in cells (data not
shown). The laboratory is in the process of examining the importance of
these residues and the variable distance from the DAD core in its
biological activity. Taken together, the results of these DAD
mutational experiments show that complete DAD activity is dependent
upon its ability to bind to a full-length DRF.
The current study defines a regulatory domain that accounts for
Rho GTPase activation of the Diaphanous-related FH protein subfamily.
The DAD·GBD autoregulatory mechanism is analogous to the interaction
of the WASP GTPase binding and the VCA domains (34); the DRFs are
regulated by intramolecular GBD·DAD binding where Rho-GTP activates
the DRFs by disrupting GBD-DAD interaction. DAD is highly conserved,
and its identification further explains the nature of several prior
observations where truncation of Bni1, mDia1, and mDia2 GTPase binding
domains resulted in their activation (11, 16, 18). Here, it is shown
that ectopic DAD expression mimics Rho·GTP binding by interfering
with normal autoregulation of cellular DRFs. Based on this model, it
may be possible that overexpression of the GTPase binding domain would
also unlatch the autoinhibited Dia proteins. This is not the case as
DAD clearly activates both actin fiber production and SRF, whereas the
GBD was inactive and inhibits DAD in coexpression experiments. These results suggest the GBD is truly a bifunctional autoinhibitory domain
in addition to the GTPase binding domain. Even if GBD binding to the
carboxyl terminus of the endogenous Dia protein in trans unlatched intramolecular autoinhibition, it would likely interfere with
endogenous Dia function by masking exposed effector domains contained in the carboxyl terminus or by inducing the inactive state of
the Dia proteins through direct binding. This may be reflected in
results obtained by Nakano et al. (29) who have shown
that expression of a portion the mDia1 amino terminus interfered with
the integrity of the actin fiber network in MDCK cells. It is critical,
however, to determine whether these types of interfering proteins block
by binding to functional domains of endogenous Dia proteins or simply
squelch activated Rho GTPases by binding to a cryptic GTPase
binding domain found within these Dia regions. Unfortunately, there is
currently little information regarding the specific structural
requirements of the DRF GTPase binding domains. This interaction is
likely complex, and Rho effector loop mutations suggest that the
interaction of Rho with mDia2 is unique (36). Also, several of the
Rho-binding proteins, including PKN and Kinectin, bear multiple binding
sites for activated GTPases and cannot be restricted to a limited
peptide domain (36, 43, 44).2 This may also be true for
the DRFs.
The carboxyl terminus of the DRFs, including regions between DAD
and the FH2 domain, may still serve in a signaling or actin remodeling
capacity like the WASP carboxyl terminus. DAD itself may also contain
intrinsic effector activity. The L1044A-substituted mDia2 DAD, for
example, was unable to bind the mDia2 GBD and therefore unlatch the
autoinhibited cellular Dia proteins. Expression of this mutated DAD was
still able to effect the actin cytoskeleton by causing the disruption
of pre-existing actin fibers and the formation of lamella-like
extensions. This suggests that either this mutant directly targets
cellular components that participate in actin remodeling or it
interferes with endogenous DRFs. Both possibilities are being explored
as the complete induction of stress fibers by DAD will likely be shown
to be dependent upon multiple activities being recruited to activated
Rho- or DAD-bound Dia proteins. The current results also raise the
possibility that expression of dual function autoregulatory domains
from proteins like WASP may have multiple effects in cells; those that
are dependent upon the recruitment of cellular factors to the domain of
interest, but also those caused by the relief of self-inhibition.
Once the DRFs are activated by Rho binding, what is the mechanism of
signal transduction to effectors? The current model suggests that once
intramolecular inhibition because of GBD-DAD association is relieved by
binding to activated Rho, the DRFs then recruit downstream effectors
that mediate signals via the proline-rich FH1, the FH2, or other
uncharacterized Dia domains. Whereas the integrity of the FH1 domain is
required for the ability of The DAD peptide represents a useful biological tool to study Rho
signaling and cytoskeletal regulation pathways. Because DAD effects are
largely dependent upon the endogenous complement of cellular DRFs, the
results generated from further experiments using DAD will likely be
more useful in the dissection of Dia-dependent signaling
events. For example, in comparison to the activated
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and Bud6p/Aip3p associate with Bni1p through other regions (16, 24).
The significance of profilin binding to the mammalian DRF family
members has yet to be elucidated, although it does not appear to be an
important factor in Rho-regulated actin remodeling (25).
GBD-mDia1 and -mDia2 in fibroblasts activates a
Rho-regulated signaling pathway that leads to the activation of the
serum response factor (SRF)
transcriptional regulator in the nucleus (18, 40). The activated Dia
proteins also cooperate with another small GTPase effector, Rho-kinase
or ROCK, to induce stress fibers (11, 18, 29). These truncation
experiments suggest that the GTPase binding domain of the DRFs contains
a negative regulatory activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A conserved carboxyl-terminal domain in the
Diaphanous-related FH protein subfamily. Comparative alignments of
mouse DRFs mDia1, mDia3, and mDia2 with Drosophila
melanogastar Diaphanous, S. cerevisiae Bni1p, and
A. nidulans SepA. Numbers in the second column
correspond to the first residue shown from each respective sequence
(accession nos. provided in "Experimental Procedures." Identical
residues black and similar amino acids are gray.
A consensus sequence can be described by
(G/A)(V/A)MDXLLEXL(K/R/Q)X(G/A)(S/G/A)(A/P)
where X represents any residue.
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Fig. 2.
Interaction of DAD with the amino-terminal
GBD. A, two-hybrid analysis. Yeast reporter strain HF7c
was transformed with the indicated bait (Trp ) and prey
(Leu
) plasmids before restreaking on selective plates
(His
) with increasing concentrations of
3-aminotriazole (0, 2, 4, 8, 16, 32, and 64 mM) that
selects for correlating expression of the His reporter gene. The
indicated numbers correspond to the highest concentration of
3-aminotriazole on which there was growth after 3 days. Both activated
RhoA-V14S190 (GTPase-deficient and CAAX mutation to prevent
lipid modification), and DAD interacted with both mDia2 and the GBD but
not other FH domains. FH1 binding to the SrcSH3 domain was used as a
positive control for binding. The mDia2 plasmid encodes amino acids
47-800. B, GST pull-down experiments to analyze the effects
of activated RhoA on the GBD-DAD interaction. pT7-plink plasmids were
used to generate [35S]methionine-labeled GBD, FH1, and
mDia2 (18) by in vitro translation (IVT). This
input IVT material was incubated with either GST·DAD or GST·FH1
fusion proteins bound to agarose beads for 2 h at 4 °C and was
warmed to 30 °C for 5 min before addition of purified
recombinant-activated Rho-V14 at 2, 10, and/or 20-fold molar excess
compared with GST fusion proteins (34). Beads were washed with cold
binding buffer three times before resuspension in SDS sample buffer.
Samples were separated by gel electrophoresis and autoradiographed.
C, model describing an autoregulatory mechanism for the DRFs
based on an intramolecular interaction between the GBD and DAD. The GBD
is a bifunctional autoinhibitory domain that binds DAD when the Dia
proteins are inactive; autoinhibition is relieved by activated
GTP-bound Rho. Thus, coexpression of the GBD would block DAD
effects. If DAD expression activates endogenous Dia proteins by binding
to the GBD and unlatching their autoinhibited state, then interfering
Src or anti-mDial should inhibit DAD activity. If DAD were an
"effector" domain sufficient to trigger downstream signaling, then
these inhibitors should not have an effect.
GBD variants (18). Whereas none of the other
homology domains had an effect on actin or SRF activity, EGFP·DAD
expression strongly induced the formation of actin filaments in cells
as shown in Fig. 3A.
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Fig. 3.
Expression of the isolated DAD activates
actin remodeling and SRF. A, actin cytoskeletal changes
following expression of EGFP·DAD fusion protein. Top
panels display EGFP fusion proteins and bottom panels
the corresponding TRITC-phalloidin staining. Plasmids were injected at
10 ng/µl each (pEFmEGFP·FH2 (mDia2-(801-910)) and
pEFmEGFP·DAD (mDia2-(1031-1171))) and were fixed 3 h later. Prior to injection, cells were maintained in 0.1% fetal calf
serum for 24 h to reduce the number of pre-existing stress fibers.
B, FH1 domain targets EGFP to stress fibers.
pEFmEGFP·FH1 (10 ng/µl, mDia2-(521-630)) was injected
into cells as described in A. 3 h after injection,
cells were treated with 50 µM LPA. Top
row shows a single LPA-treated cell with predominantly nuclear
EGFP. The right panels show merged EGFP and
TRITC-phalloidin. The bottom row shows images taken from the
inset (white box) in the top row.
C, DAD expression activates SRF-regulated gene expression.
HA13 SRE-FosHA reporter cells were microinjected with the indicated
expression plasmids. 3 h later, they were fixed and stained
for FosHA reporter by indirect immunofluorescence. Bars
represent the mean percentage of EGFP-expressing cells staining
positive for FosHA; error bars represent the S.D. from 2-3
experiments with 40-100 EGFP-positive cells counted.
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Fig. 4.
Disruption of DAD effects by GBD
squelching. A, DAD-induced actin rearrangements are
blocked by GBD expression and intefering Src K298M/Y530F.
pEFmEGFP-DAD was injected with empty vector
(pEFmEGFP) or pEFm-GBD, -FH2, or pSGT-Src
K298M/Y530F, each 10 ng/µl as indicated. Cells were fixed
and stained with TRITC-phalloidin (bottom panels; EGFP
fusion proteins shown in top panels). Both GBD and
interfering Src block longitudinal induction of actin fibers.
B, GBD coexpression specifically blocks DAD activation of
SRF. pEFmEGFP-DAD was coinjected with pEFm-GBD, FH1, FH2, PKN.N, or C3
as indicated. Cells were fixed 3 h later for FosHA expression;
bars represent the mean percentage of FosHA-positive
EGFP-expressing cells. Error bars represent the S.D. from
2-3 experiments.
GBD-mDia2
or
GBD-mDia1 SRF induction because the deletion constructs lack the
peptide sequence necessary for recognition by anti-mDia1 (18). Src
binds to and colocalizes with endogenous Dia proteins (18, 19). To test
if Src has a role in DAD activity, DAD was expressed with either
interfering Src (Src K298M/Y530F) or coinjected with
affinity-purified anti-Src (13). Both reagents effectively blocked
DAD-induced stress fiber formation and SRF (Fig. 5B).
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Fig. 5.
DAD effects are dependent upon endogenous
DRFs. A, DAD inhibition by anti-mDia1, interfering Src,
and anti-Src. Anti-mDia1, Cst.1 anti-Src (38), or nonspecific rabbit
IgG (1 mg/ml each) were microinjected along with either
pEFmEGFP- GBD-mDia2 or pEFmEGFP-DAD. 3 h later, cells were fixed
and stained for FosHA expression (Y-11 anti-HA/AMCA donkey anti-rabbit
(blue)). In these experiments, each cell was injected twice
to ensure delivery of expression vector to the nucleus and antibody to
the cytoplasm. Similar results were obtained as the effects on actin
remodeling (data not shown). B, summary of squelching
experiments. Bars represent the number of SRE-FosHA-positive
cells from two experiments where 40-100 cells were injected in each;
bars represent the S.D.
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Fig. 6.
Mutations of DAD that disrupt DRF binding
inhibit biological activity. A, mutational analysis of
DAD-mDia2 interaction by two-hybrid assay. Alanine substitutions or
stop codons were introduced for several of the conserved residues in
the DAD core region and after the mDia2 basic stretch (RRKR). Mutations
that affected binding are indicated by the black arrowheads,
those that did not have an effect are indicated by the open
arrowheads. B, expression of DAD mutants in cells. NIH
3T3 cells maintained on glass coverslips for 24 h in 0.1% fetal
calf serum were microinjected with the indicated pEGFP-DAD or DAD
variants (10 ng/µl). 3 h later, cells were fixed and stained
with TRITC-phalloidin (shown in right panels). Mutation of
conserved residues disrupted biological effects except for DAD-E1046A,
which was still active.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
GBD-versions of both mDia1 and mDia2 to
signal to actin and DRF (18), it is clear that both FH1 and FH2 domains
are involved in downstream signaling (11, 26). It is also possible that
FH1 is functionally an input domain that receives signals from upstream
signaling modules or mediates cross-talk via cellular factors such as
Src (18, 19) or IRSp53/BAIAP2 (22). Src for example, may be targeting
the DRFs upon activation by Rho, even though prior work has shown that
mDia1 membrane localization is dependent upon Rho (16).
Src-dependent targeting may therefore be a permissive event. This is consistant with the conclusion of this study; ectopic DAD expression triggers endogenous Dia protein in a manner similar to
activated Rho. In this situation, activated cellular Dia proteins are
then directed to the membrane by factors in a FH1
domain-dependent manner. v-Src has been shown to be
directed to focal adhesions in a Rho- and actin-dependent
manner (45). Given our previous findings with interfering Src and
anti-Src antibodies (13) that block activated Dia function, Src and Dia
targeting may be mutually dependent events that are controlled by the
rate-limiting Rho-activation step.
GBD-mDia
variants, DAD expression induces the formation of stable microtubules
whose orientation better reflects those observed after growth factor
treatment.3 DAD and mutated
DAD proteins are now being tested for effects on downstream signaling
pathways. Specifically, it will be interesting to examine the effects
of the DFNA1 mutation (12) on DAD activity in cells and on
its ability to interact with the GBD. The autosomal dominant
DFNA1 mutation in the human Dia1 gene occurs at a
donor splice site at the carboxyl terminus. The mutation causes a
frameshift 15 amino acids away from the core DAD sequence, which
introduces an anomalous 19 amino acids onto the protein. Thus far in
our laboratory, the effects of truncations near this region of DAD have
been equivocal, but this may suggest that it destabilizes the
autoinhibitory GBD-DAD association. Expression of truncated versions of
mDia1 by Watanabe et al. (11) appears to cause disruption of
the actin cytoskeleton in some cells and resembles the results seen
with DAD mutants shown here. However, the effects of the DFNA1 mutation
on Dia1 protein function will not be clear until these Dia1 mutants are
expressed in cells at in vivo levels.
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ACKNOWLEDGEMENTS |
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The author thanks Gregg Gundersen, Andrew Thorburn, Michael Weinreich, and Nick Duesbery for discussions and/or comments on the manuscript and Martin Broome and Sara Courtneidge (Sugen, San Francisco, CA) for providing the anti-Src reagents.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: 333 Bostwick Ave. NE,
Grand Rapids, MI 49503. Tel.: 616-234-5316; Fax: 616-234-5317; E-mail:
art.alberts@vai.org.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M006205200
2 A. S. Alberts, M. Wernick, and C. C. Collins, unpublished observations.
3 G. Gundersen, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: FH, formin homology; DRF, Diaphanous-related formin homology; GBD, GTPase binding domain; WASP, Wiskott-Aldrich syndrome protein; HA, hemagglutinin; VCA, verprolin cofilin acidic; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; DAD, Dia-autoregulatory domain; SRF, serum response factor. CRIB, Cdc42-Rac interactive binding region; TRITC, tetramethylrhodamine; LPA, lysophosphatidic acid.
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