From the
Division of Cellular Biochemistry and
Centre for Biomedical Genetics, and the ¶Division
of Cell Biology, Plesmanlaan 121, The Netherlands Cancer Institute, 1066 CX
Amsterdam, The Netherlands
Received for publication, March 7, 2003 , and in revised form, April 22, 2003.
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ABSTRACT |
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INTRODUCTION |
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In our ongoing studies to delineate Rho signaling by the lipid growth factor lysophosphatidic acid (LPA) (11, 12), we previously identified a ubiquitously expressed protein of 116 kDa, provisionally named p116Rip, which binds relatively weakly to activated RhoA in a yeast two-hybrid assay (13). The p116Rip sequence predicts several protein interaction domains, including at least one PH domain, two proline-rich stretches, and a C-terminal region predicted to form a coiled-coil domain. This suggests that p116Rip may have a scaffolding role recruiting different proteins into a RhoA-regulated macromolecular complex. When overexpressed in N1E-115 neuroblastoma cells, p116Rip promotes cell flattening and process extension and inhibits cytoskeletal contraction in response to LPA (13). The p116Rip phenotype was reminiscent of what is observed after RhoA inactivation (using dominant-negative RhoA or C3 toxin), which led to the suggestion that p116Rip may negatively regulate RhoA signaling (13). However, the function of p116Rip remains unknown; importantly, no evidence that p116Rip binds directly to RhoA in mammalian cells has been discovered (13).
In the present study, we set out to characterize p116Rip in further detail. We find that p116Rip, rather than directly binding to RhoA, interacts with F-actin via its N-terminal region and colocalizes with dynamic F-actin structures such as stress fibers, cortical microfilaments, filopodia, and lamellipodial ruffles. Furthermore, we show that p116Rip induces bundling of F-actin in vitro, with the bundling activity residing in the N-terminal region. Yet overexpression of p116Rip or its N-terminal actin-binding domain disrupts the actin cytoskeleton and thereby interferes with growth factor-induced contractility and lamellipodia formation. Our studies specify p116Rip as a novel F-actin-binding protein and demonstrate that p116Rip can affect, either directly or indirectly, the integrity of the actomyosin-based cytoskeleton.
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EXPERIMENTAL PROCEDURES |
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Expression ConstructsGeneration of full-length (FL)
pcDNA3-p116Rip (aa 11024),
pcDNA3-HA-CTp116Rip (aa 5451024),
pcDNA3-HA-RBDp116Rip (aa 545823), and
prp261-RBDp116Rip (aa 545823) has been described
previously (13).
pcDNA3-HA-FLp116Rip was engineered by use of a polylinker
created by annealing primers
5'-ggatggcttacccatacgatgttccagattacgcgtgc-3' and
5'-acgcgtaatctggaacatcgtatgggtaagccatccgc-3' encoding the HA-tag
sequence and a SacII restriction site. The polylinker was ligated
into pcDNA3-FLp116Rip via the SacII site.
pcDNA3-HA-NTp116Rip (aa 1382) and GST-NT were
generated by PCR using primers 5'-cggggtaccacatgtcggcggccaaggaa-3'
(forward) and 5'-cggaattccggcgtcatggaggattctgt-3' (reverse) for
NT. GST-CT was generated from pcDNA3-HA-CTp116Rip.
GST-5 (aa 1152), GST-
6 (aa 1212), GST-
7 (aa
43152), GST-
8 (aa 43212); GST-
9 (aa 212390)
were generated similarly by PCR using specific primers. The PCR products were
digested with KpNI-EcoRI and ligated into pcDNA3-HA and
pRP265, a derivative of pGEX-1N. FLp116Rip-peGFPN1-was
constructed by ligation of a HindIII-ScaI fragment out of
pcDNA3-FLp116Rip and a PCR fragment using primers
5'-cagagcagtactcccaaaagtgcctgg-3' (forward) and
5'-cgcggtaccagtcgacagaattcgttatcccatgagac-3' (reverse), encoding
ScaI and Asp718 restriction sites, into peGFPN1 (Clontech).
pMT2sm-FLp116Rip-GST was generated by ligation of a
NotI-ScaI fragment of pcDNA3-FLp116Rip
and a PCR fragment using primers 5'-cagagcagtactcccaaaagtgcctgg-3'
(forward) and 5'-cggggtaccggaattcgttatcccatgagacctg-3' (reverse),
encoding ScaI and Asp718 restriction sites, into pMT2sm-GST.
pMT2sm-FLp116Rip-myc was generated by cloning
FLp116Rip-myc into pMT2sm. Sequence of all constructs was
verified by automated sequencing.
Solubility AssayCells were lysed in ice-cold lysis buffer (0.1% Triton X-100, 50 mM Tris, pH 7.4, 150 mM NaCl, and 1 mM EDTA, supplemented with protease inhibitors) and were left on ice for 15 min. Lysates were centrifuged for 30 min (13,000 rpm; Eppendorf table centrifuge, 4 °C). Pellet and supernatant fractions were collected, dissolved in sample buffer, and subjected to SDS-PAGE. Proteins were detected by Western blotting using the polyclonal anti-p116Rip antibody (1:1000 dilution).
Expression and Purification of Recombinant Fusion
ProteinsThe E. coli strains BL21 or DH5 were
transformed with plasmids encoding GST-NT, GST-CT, GST-
5, GST-
6,
GST
7, GST
8, GST
9, or GST, respectively. Colonies were
obtained and used to inoculate Luria broth/ampicillin. Cultures were grown and
isopropyl
-D-thiogalactoside was added overnight to induce
expression of the fusion proteins when the cultures reached an OD between 0.4
and 0.6. Bacteria were harvested by centrifugation at 4000 x g,
resuspended in cold lysis buffer (50 mM Tris-HCl, pH 7.6, 50
mM NaCl, 5 mM MgCl2, 1 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride), and lysed
by sonification followed by the addition of 0.5% Nonidet P-40. Lysates were
cleared by centrifugation at 4000 x g, and resulting
supernatants were incubated with glutathione-Sepharose 4B beads (Amersham
Biosciences). Beads with affinity-bound proteins were washed five times with
lysis buffer, and bound proteins were eluted with 50 mM Tris-HCl,
pH 8.0, containing 10 mM reduced glutathione. In some cases, GST
was cleaved off by incubating beads with affinity-bound proteins with thrombin
(Amersham Biosciences) according to manufacturer's protocol.
Purified full-length p116Rip fused to GST or purified full-length p116Rip fused to Myc were obtained by transfection of COS-7 cells with the constructs pMT2sm-FLp116Rip-GST and pMT2sm-FLp116Rip-myc, respectively. Cells were lysed in ice-cold lysis buffer (1% Nonidet P-40, 50 mM Tris, pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, and 10% glycerol, supplemented with protease inhibitors). Lysates were cleared by centrifugation (13,000 rpm; 10 min). Further purification of the full-length GST fusion proteins occurred in the same manner as the purification of GST fusion proteins produced in bacteria. FLp116Rip-myc fusion protein was purified using a column of monoclonal myc antibodies (9E10) chemically cross-linked to protein G-Sepharose beads (Amersham Biosciences). The protein was eluted off with 0.1 M glycine, pH 2.5, and fractions were collected in tubes containing 0.1 volume of 1 M Tris-HCl, pH 8.0, to make an end pH of 7.0. Eluted proteins were subjected to SDS-PAGE, followed by protein staining with Coomassie Blue to estimate the purity and concentration of the proteins in the fractions. Some of the proteins were concentrated using Centricon 10-kDa cutoff devices (Millipore). Protein concentration was also determined by the Bradford method using BSA as a standard. Purified proteins were stored in aliquots at -80 °C in 10% glycerol.
F-actin Cosedimentation AssayCOS-7 cells were transfected with the indicated expression vectors and lysed for 48 h after transfection in ice-cold lysis buffer (1% Nonidet P-40, 50 mM Tris, pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, and 10% glycerol, supplemented with protease inhibitors). Lysates were cleared by centrifugation (13,000 rpm; 10 min) and supernatant aliquots were run out on SDS-PAGE gels to check for expression (data not shown). 10-µl aliquots were used in the in vitro actin-binding assay.
F-actin cosedimentation assays were performed according to the
manufacturer's protocol (Cytoskeleton, Denver, CO). Briefly, prespun aliquots
of COS-7 cell lysates (100,000 x g, 30 min) or purified
proteins GST-NTp116Rip, GST,
FLp116Rip-GST, BSA, -actinin (Cytoskeleton), or
GST-
5p116Rip;
GST-
6p116Rip;
GST-
7p116Rip;
GST-
8p116Rip;
GST-
9p116Rip were incubated for 1 h at room
temperature with 40 µg of pure actin filaments. The final concentration of
F-actin was 18 µM. Filaments were subsequently pelleted by
centrifugation 100,000 x g (Beckman airfuge). As a control for
actin-independent sedimentation, the various proteins were also centrifuged
under conditions in which filamentous actin was omitted from the mix.
Cosedimenting proteins were resolved by SDS-PAGE and detected by either
Coomassie Blue staining or by Western blot analysis using
anti-p116Rip antibodies, anti-GFP rabbit polyclonal
antibodies (16), or an
anti-actin mouse monoclonal antibody (Mab 1501R; Chemicon).
For quantitative analysis, a fixed concentration of FLp116Rip-GST (1 µM) was mixed with increasing amounts of F-actin (03.5 µM) in polymerization buffer and incubated at room temperature for 30 min. Proteins were centrifuged as above and total pellets and supernatants were loaded separately on SDS-polyacrylamide gels. Protein bands were detected by Coomassie Blue staining and were scanned and quantified using the software program TINA. The amount of p116Rip bound to different concentrations of F-actin was fit to a single rectangular hyperbola using Prism (ver. 3; GraphPad Software, San Diego, CA). In all cases, entire pellet and supernatant fractions were loaded separately on SDS-polyacrylamide gels and detected by either Coomassie Blue staining or by Western blot (above).
Electron MicroscopyTo test for bundling activity, actin
filaments (5 µM) were incubated for 30 min with purified
proteins GST, -actinin (both 2 µM),
FL-p116Rip (0.5 µM),
NT-p116Rip (0.5 µM), and
CT-p116Rip (0.5 µM) at room temperature.
Samples were absorbed on to glow-discharged carbon-coated formvar film on a
copper grid and negatively stained with 1% uranyl acetate and examined with a
Philips CM10 electron microscope.
Metabolic LabelingN1E-115 cells were incubated in methionine/cysteine-free media for 30 min and labeled for 4 h with medium containing [35S]methionine/cysteine (200 µCi/ml; Amersham Biosciences). Labeled medium was aspirated and cells were washed once in ice-cold PBS. Cells were scraped in ice-cold lysis buffer (1% Nonidet P-40, 50 mM Tris pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, and 10% glycerol, supplemented with protease inhibitors). Lysates were cleared by centrifugation (13,000 rpm; 10 min) and supernatants were tumbled with protein A-Sepharose beads precoupled to either preimmune rabbit serum, anti-p116Rip antibodies (13), or anti-myosin IIA antibodies (BTI, Oklahoma City, OK) for1hat4 °C. GST pull-down assays were performed with 20 µl of GSH-Sepharose beads loaded with 20 µg of either GST alone or the GST-NT-fusion protein containing the isolated actin-binding domain. Beads were washed five times in ice-cold lysis buffer and resolved by SDS-PAGE. Proteins were detected by autoradiography. In some cases the gels were blotted and further analyzed by Western blotting to assess the identity of labeled proteins using the polyclonal anti-p116Rip antibody (1:1000 dilution) and anti-myosin II antibodies (1:500 dilution).
Antibodies and Confocal MicroscopyThe FRA58 antibody directed against GST-RBD (amino acids 545823) has been described previously (13). N1E-115 cells and NIH3T3 cells were grown on gelatin-coated glass coverslips in six-well plates. N1E-115 cells were serum-starved overnight and NIH3T3 cells for 7 h. Cells were fixed 24 h after transfection in 3.7% formaldehyde in PBS for 10 min, permeabilized (0.1% Triton X-100/PBS; 2 min), blocked (1% BSA/PBS; 30 min), and incubated with primary antibodies (FRA58 preimmune serum, polyclonal FRA58 anti-p116Rip, 3F10 anti-HA rat monoclonal antibodies (Roche; 1 h). Subsequently, cells were washed and incubated with secondary antibodies (Goat-anti-rabbit-fluorescein isothiocyanate (DAKO) and Goat-anti-rat-fluorescein isothiocyanate (Rockland); 30 min)) together with rhodamine-conjugated phalloidin (Molecular Probes). Coverslips were mounted in Vectashield and analyzed using a Leica TCS-NT confocal microscope.
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RESULTS |
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p116Rip Localizes to Dynamic Actin-rich Structures and the
NucleusAs a first step in elucidating the function of
p116Rip, we examined its subcellular localization in
N1E-115 and NIH3T3 cells using a polyclonal anti-p116Rip
antibody raised against the putative "RhoA-binding domain" (RBD;
Ref. 13). Cells were
simultaneously double-stained with rhodamine-phalloidin to visualize F-actin.
In serum-deprived N1E-115 cells, endogenous p116Rip
colocalizes with F-actin structures, especially the actin-rich microspikes
(Fig. 1, top). After
stimulation with LPA, a potent activator of RhoA, N1E-115 cells rapidly round
up and neurites retract (17,
18). In those contracted
cells, p116Rip is found relocalized to the contractile
actomyosin ring at the cell cortex (Fig.
1). In NIH3T3 cells, maintained either in serum-free medium or
stimulated with LPA, p116Rip colocalizes with F-actin-rich
structures, particularly along stress fibers, at cortical microfilaments, and
at the leading edge of lamellipodia (Fig.
1, bottom). Of note, p116Rip staining
is also detected in the cytoplasm and the nucleus
(Fig. 1, bottom).
Specificity of the observed immunostaining was confirmed by using the GST-RBD
polypeptide antigen (previously termed 2; ref.
13), which blocked the
p116Rip fluorescence signal. Furthermore,
p116Rip transfected into COS-7 or N1E-115 cells showed the
same subcellular distribution pattern as endogenous
p116Rip: colocalization with F-actin-rich structures as
well as nuclear and cytoplasmic staining
(Fig. 2B and results
not shown). No colocalization with endogenous RhoA was detected in either
N1E-115 or NIH3T3 cells (results not shown).
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The NT Region of p116Rip Dictates Subcellular LocalizationThe subcellular localization of p116Rip raises the possibility that p116Rip is an F-actin-binding protein. To test this notion, we examined the intracellular localization of distinct domains of p116Rip and determined their detergent solubility. p116Rip contains several putative protein and phospholipid interaction motifs, including a central PH domain, an N-terminal PH domain (aa 43152; not noted previously (13)); two proline-rich regions, and a C-terminal coiled-coil region (Fig. 2A). The putative "RhoA-binding domain" (RBD) that was isolated in yeast two-hybrid screens (13) overlaps with the coiled-coil region, as indicated in Fig. 2A.
We generated HA-tagged p116Rip and three truncated versions (HA-tagged) encoding the CT coiled-coil region, the RBD and the NT half (NT-p116Rip; Fig. 2A). The various cDNA constructs were transiently transfected into N1E-115 cells and the subcellular localization and detergent solubility of the resulting proteins were analyzed. Transfected HA-p116Rip, like endogenous p116Rip, localizes to cortical F-actin (and the nucleus; data not shown). In contrast, the p116Rip-CT and RBD polypeptides display nuclear and cytoplasmic localization (Fig. 2B). In keeping with these results, the CT and RBD truncation mutants are largely Triton-soluble, whereas full-length p116Rip (transfected and endogenous) is about 50% insoluble (Fig. 2C and results not shown), consistent with association with the cytoskeleton.
Similar to full-length p116Rip, the N terminus of p116Rip (NT-p116Rip) colocalizes with F-actin and is also detectable in the nucleus (Fig. 2D and results not shown). When the NT-p116Rip-expressing cells were analyzed at >48 h after transfection, however, the F-actin cytoskeleton was largely disrupted (see below). Collectively, these results indicate that the N-terminal part of p116Rip (aa 1382) determines its subcellular localization.
Binding of p116Rip to F-actinF-actin associates with the motor protein myosin-II to generate contractile forces in non-muscle cells. In metabolically labeled N1E-115 cells, we found that endogenous p116Rip as well as the purified polypeptide NT-p116Rip (fused to GST) coprecipitated with proteins of 43 and 200 kDa (Fig. 3A, lanes 2 and 4, respectively). Immunoblot analysis confirmed that the 43-kDa protein is actin (not shown), and revealed that the 200 kDa protein represents the heavy-chain of non-muscle myosin-II (Fig. 3B). Although the reciprocal precipitations yielded variable results, our data support the notion that p116Rip associates with actomyosin in vivo.
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We next investigated the actin-binding properties of
NT-p116Rip. As shown in
Fig. 4A,
NT-p116Rip (fused to GST) cosediments with purified
F-actin, as did -actinin, whereas GST alone and BSA did not. Fusion
proteins containing the C-terminal regions of p116Rip (CT
and RBD) failed to cosediment with F-actin (results not shown). It thus seems
that the N-terminal region of p116Rip contains an
F-actin-binding domain. We next examined the binding affinity of full-length
p116Rip for F-actin. Increasing concentrations of purified
F-actin (03.5 µM) were mixed with a fixed amount of
p116Rip (1 µM). After high-speed
centrifugation, the amount of p116Rip cosedimenting with
F-actin was determined. From the resulting binding curve we estimate that
p116Rip binds to F-actin with an apparent dissociation
constant (Kd) of about 0.5 µM
(Fig. 4B).
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To define the N-terminal regions mediating F-actin binding in further
detail, we made various deletion mutants and determined their F-actin
cosedimentation properties, as illustrated in
Fig. 4C. Whereas
p116Rip has no obvious sequence homology to known
actin-binding proteins, potential actin-binding motifs include the N-terminal
PH domain (19) as well as a
stretch of positively charged residues (KKKRK, aa 157161) that could
interact with the highly anionic actin filament
(20). We found that one
deletion mutant (6; aa 1212), comprising both the PH domain and
the positive stretch, can bind F-actin, whereas the other mutants cannot
(Fig. 4C). Thus, the
extreme N terminus (aa 143), the PH domain, and the adjacent cationic
residues are all necessary to mediate F-actin binding. Further definition of
the critical actin-binding motif(s) within the N-terminal region must await
future studies.
p116Rip Induces Bundling of F-actin in Vitro via its
N-terminal RegionWe next examined the ability of
p116Rip and NT-116Rip to induce actin
cross-linking in vitro, using -actinin as a positive control.
Myc-p116Rip and GST-p116Rip were
isolated from transfected COS-7 cells using affinity chromatography, and
protein purity was determined by Coomassie Blue staining.
Myc-p116Rip, like GST-p116Rip, binds
F-actin, as shown by cosedimentation assays using lysates from transfected
COS-7 cells (see Fig.
6A, left). Because the dimeric nature of GST
could mediate artifactual actin cross-linking by
GST-p116Rip, we also used Myc-p116Rip.
Purified GST-p116Rip, Myc-p116Rip,
-actinin, or GST alone were incubated with F-actin, and the samples
were subsequently analyzed by electron microscopy. In the absence of
p116Rip or in the presence of GST alone, long actin
filaments were randomly distributed all over the grid and no organized actin
bundles were observed (Fig.
5A). In the presence of either
GST-p116Rip or Myc-p116Rip, however,
F-actin became organized into thick bundles similar to those formed by the
actin-bundling protein
-actinin
(Fig. 5, B, C, and
D). The bundles consisted of many actin filaments closely
aligned in juxtaposition, with no branching of filaments observed.
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We also tested the isolated N-terminal actin-binding domain (NT-p116Rip; aa 1382) and the C-terminal coiled-coil region (CT-p116Rip; aa 5451024) for bundling activity. In these experiments, GST-fusion proteins were produced in bacteria followed by GST cleavage. As expected, the NT-p116Rip protein induced actin bundling similar to full-length p116Rip, whereas no actin bundles were observed after incubation of F-actin with the CT polypeptide (Fig. 5, E and F). Thus, p116Rip induces bundling of F-actin in vitro through its N-terminal actin-binding domain.
Expression of p116Rip or the N-Terminal Actin-binding Domain Promotes Stress Fiber Disassembly and Process OutgrowthWe investigated the effects of overexpression of p116Rip and its N-terminal region on cell morphology and cytoskeletal organization in NIH3T3 cells. To this end, we used HA-tagged p116Rip and a p116Rip-GFP fusion protein (its direct binding to F-actin was confirmed; Fig. 6A). Contrary to expectations raised by the actin-bundling studies, overexpression of p116Rip in NIH3T3 cells resulted in loss of stress fibers and outgrowth of long dendrite-like processes (Fig. 6B). This phenotype was observed with wild-type p116Rip, Myc-, HA- and p116Rip-GFP (Fig. 6, B and C, and results not shown). Less than 10% of the p116Rip-transfected NIH3T3 cells contained stress fibers, compared with >60% of the GFP-expressing control cells (Fig. 6D). LPA stimulation of NIH3T3 cells leads to rapid RhoA-mediated cell contraction (albeit less dramatic than in N1E-115 cells). However, no contractile response to LPA was seen in the p116Rip-overexpressing NIH3T3 cells, similar to what we previously observed in p116Rip-overexpressing N1E-115 cells (13). Loss of stress fibers was already detectable at 6 to 8 h after transfection, whereas process extension appeared at later time points (>1216 h, when p116Rip levels were more elevated).
Expression of the actin-binding region (HA-NT-p116Rip) in NIH3T3 cells led to the same dramatic loss of stress fibers and induction of dendrite-like extensions. In contrast, cells expressing the C-terminal domain only (HA-CT-p116Rip) displayed a normal stress fiber pattern (Fig. 7, A and B). Thus, the NT region of p116Rip is necessary and sufficient for stress-fiber disruption and consequent loss of contractility in p116Rip-overexpressing cells.
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Finally, we examined the cytoskeletal response of p116Rip-overexpressing NIH3T3 cells to platelet-derived growth factor (PDGF), which is a potent inducer of Rac-mediated lamellipodia formation and membrane ruffling. PDGF induced prominent lamellipodia formation in the control cells but not in the p116Rip-GFP-expressing cells (Fig. 8, A and B). We conclude that although p116Rip has actin-bundling activity in vitro, overexpression of p116Rip in fibroblasts and neuronal cells disrupts F-actin assembly and thereby interferes with Rho/Rac-controlled cytoskeletal remodeling.
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DISCUSSION |
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The N-terminal region p116Rip shows no obvious sequence
similarity to known F-actin-binding proteins. Therefore,
p116Rip does not classify as a member of the superfamily
of actin-binding proteins that includes -actinin/spectrin members,
plectin, filamin, and dystrophin
(2224).
At least three distinct mechanisms could account for the actin-bundling
activity of NT-p116Rip. One possibility would be that
NT-p116Rip is able to dimerize and thereby induces actin
bundling. However, using transfected COS cells, we did not detect an
interaction between NT-p116Rip and full-length
p116Rip (results not shown), which argues against the
possibility that the NT domain can form dimers. The second possibility is that
NT-p116Rip might bundle F-actin through the polycationic
KKKRK motif (residues 157161), just after the first PH domain
(20). However, mutational
analysis reveals that neither the first PH domain nor the cationic motif is
sufficient for F-actin binding (Fig.
4C). A third possibility is that
NT-p116Rip may harbor two actin-binding domains, each of
which binds a separate actin filament; as yet, we have no evidence for or
against this notion. Further studies are required to identify the N-terminal
sequence motifs in p116Rip that determine F-actin binding
and bundling.
Contrary to what one would expect for a protein with actin-bundling activity, overexpression of p116Rip in NIH3T3 cells causes loss of stress fibers and produces a dendrite-like morphology. This phenotype, which is reminiscent of cells expressing dominant-negative RhoA (18, 25) requires the N-terminal actin-binding domain of p116Rip but not the C-terminal coiled-coil region. The importance of the N terminus in determining cytoskeletal architecture can also be inferred from the observation that overexpressed p116Rip causes cell flattening in N1E-115 cells, whereas an N-terminally truncated version does not (13). Loss of stress fibers and other actin-rich structures is a common feature of overexpressed actin-monomer (G-actin) sequestering proteins (2628), but our efforts to test whether NT-p116Rip can bind G-actin yielded negative results (not shown). However, there is precedent for actin cross-linking proteins to cause F-actin disassembly in vivo. In particular, overexpression of the actin-binding region of neurabin, an F-actin cross-linking protein, causes collapse of stress fibers and promotes filopodial outgrowth, apparently by recruiting protein phosphatase I to F-actin-rich structures (29). Furthermore, overexpression of villin, a protein that can bundle, cap, nucleate, or sever actin in vitro, results in the disappearance of stress fibers and enhanced microvilli elongation, a phenotype that strictly correlates with the actin-bundling activity of villin (30).
Overexpressed p116Rip not only induces an inactive RhoA
phenotype but also interferes with PDGF-induced lamellipodia formation, which
is a typical Rac-mediated response. The present findings lead us to suggest
that, rather than being a negative regulator of Rho/Rac,
p116Rip can destabilize F-actin-rich structures by
competing with and displacing other actin-cross-linking proteins. An
alternative or additional possibility is that p116Rip may
recruit regulatory proteins that disassemble the F-actin network (such as
actin-severing proteins or protein phosphatases; see Ref.
29). As for the displacement
model, the neuronal F-actin-binding protein drebrin induces the formation of
highly branched processes, similar to that observed with
p116Rip. It does so by interfering with the actin binding
and bundling activities of fascin, -actinin, and tropomyosin
(31,
32). A similar mechanism might
underlie the p116Rip overexpression phenotype.
Finally, we note that a recently identified actin-binding protein named Tara (593 residues) shows a high degree of similarity to p116Rip (46% overall amino acid identity (33)). In common with p116Rip, Tara contains an N-terminal PH domain and a C-terminal coiled-coil region, but it lacks the N-terminal actin-binding region of p116Rip. No actin cross-linking activity has been reported for Tara until now; nevertheless, overexpression of Tara in HeLa cells leads to enhanced formation of stress fibers and cortical F-actin (33). Thus, despite their structural similarities, p116Rip and Tara have opposing actions on F-actin organization.
In conclusion, our studies specify p116Rip as a novel F-actin-binding protein with bundling activity in vitro and demonstrate that p116Rip can affect, directly or indirectly, the integrity and contractility of the actomyosin-based cytoskeleton. Further insight into the physiological role of p116Rip in cytoskeletal regulation will rely on the identification of additional binding partners of p116Rip as well as on interference approaches by using RNA interference-expressing vectors. These studies are currently in progress.
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FOOTNOTES |
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Present address: University Medical Center Utrecht, 3584 CX Utrecht, The
Netherlands.
|| To whom correspondence should be addressed. Tel.: 31-20-512-1971; Fax: 31-20-512-1989; E-mail: w.moolenaar{at}nki.nl.
1 The abbreviations used are: F-actin, filamentous actin; LPA,
lysophosphatidic acid; PH, pleckstrin homology; aa, amino acids; HA,
hemagglutinin; FL, full-length; GST, glutathione S-transferase; GFP,
green fluorescent protein; NT, N terminus; CT, C terminus; BSA, bovine serum
albumin; RBD, RhoA-binding domain; PBS, phosphate-buffered saline; PDGF,
platelet-derived growth factor.
2 J. Mulder, O. Kranenburg, and M. Poland, unpublished results.
3 F. van Horck and J. Mulder, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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