From the Department of Oncology, Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007
Received for publication, August 3, 2000, and in revised form, September 26, 2000
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ABSTRACT |
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Cdc42, a Rho GTPase, regulates the organization
of the actin cytoskeleton by its interaction with several distinct
families of downstream effector proteins. Here, we report the
identification of four new Cdc42-binding proteins that, along with
MSE55, constitute a new family of effector proteins. These molecules,
designated CEPs, contain three regions of homology, including a Cdc42
binding domain and two unique domains called CI and CII.
Experimentally, we have verified that CEP2 and CEP5 bind Cdc42.
Expression of CEP2, CEP3, CEP4, and CEP5 in NIH-3T3 fibroblasts induced
pseudopodia formation. Fibroblasts coexpressing dominant negative Cdc42
with CEP2 or expressing a Cdc42/Rac interactive binding domain mutant of CEP2 did not induce pseudopodia formation. In primary keratinocytes, CEP2- and CEP5-expressing cells showed reduced F-actin localization at
the adherens junctions with an increase in thin stress fibers that
extended the length of the cell body. Keratinocytes expressing CEPs
also showed an altered vinculin distribution and a loss of E-cadherin
from adherens junctions. Similar effects were observed in keratinocytes
expressing constitutively active Cdc42, but were not seen with a
Cdc42/Rac interactive binding domain mutant of CEP2. These results
suggest that CEPs act downstream of Cdc42 to induce actin filament
assembly leading to cell shape changes.
Cdc42 is a member of the p21 Rho family of small GTPases that act
downstream of cell surface receptors to regulate the formation of
different F-actin-containing structures (1). Cdc42 regulates actin
polymerization to induce the assembly of filopodia, which are thin
actin filaments that project from cells (2, 3). Cdc42 can also regulate
other biological processes such as cytokinesis (4, 5) and cell polarity
(6-13). As to its role in cell polarity, Cdc42 regulates polarization
of Drosophila wing epithelial cells by inducing actin
polymerization at the apical cortex of these cells (6). In mammalian
cells, Cdc42 regulates a number of polarized responses, including actin
localization (7, 8), nuclear displacement (7), protein trafficking (9),
and directed cell movement (10-13). These studies suggest that Cdc42
regulates cell shape and polarity changes by coordinating actin changes with altered cell membrane assembly.
The ability of Cdc42 to regulate the actin cytoskeleton and cell
polarity in mammalian cells stems from its interaction with a large
number of downstream effector proteins (14). One such effector protein,
N-WASP, induces actin polymerization leading to filopodia formation
(15). N-WASP mediates these actin changes by directly binding actin
(15), the Arp2/3 complex (16), and by interacting with the
actin-binding protein profilin (17). The p21-activated protein kinases
(PAK)1 also mediate
actin reorganization required for filopodia formation (18, 19), cell
motility (20, 21), and neurite-outgrowth (22, 23). Unlike N-WASP, PAKs
mediate their biological activities by phosphorylating a number of
cytoskeletal targets (20, 21). Despite what is known about Cdc42 and
effector interactions, the molecular details of how Cdc42 regulates
actin and cell polarity changes in different cell types are only
beginning to emerge.
We recently characterized MSE55 as a nonkinase Cdc42 effector that
mediates actin reorganization leading to the formation of pseudopodia
(24). In the present study, we identify four additional proteins CEP2,
CEP3, CEP4, and CEP5 that share homology with MSE55. These proteins
contain a CRIB domain that is homologous to that of MSE55 (24, 25). Two
other domains, designated CI and CII, are also found in MSE55/CEP1,
CEP2, CEP3, and CEP4, whereas CEP5 lacks a CI domain but instead
contains a proline-rich region. Using in vitro binding
assays, CEP2 and CEP5 interacted with Cdc42, but not Rac. Similar to
our previous results with MSE55 (24), expression of all the CEP members
induced long pseudopodia in NIH-3T3 fibroblasts. In contrast, only
background levels of pseudopodia were observed in fibroblasts
expressing a CRIB mutant of CEP2 or coexpressing dominant negative
Cdc42 with CEP2. In primary keratinocytes, expression of Cdc42-Q61L,
CEP2, and CEP5 all decreased F-actin staining in the adherens junction,
but increased the number of cytoplasmic actin stress fibers. Unlike
Rho-induced stress fibers, the cytoplasmic stress fibers seen in the
Cdc42- and CEP-expressing cells were greater in number, were thinner,
and transversed the entire cell body. In many cases, keratinocytes
expressing CEPs also showed cell elongation in the direction of the
actin filaments. These results suggest that CEPs act downstream of
Cdc42 to induce actin filament assembly leading to cell shape changes.
Identification of CEP Clones--
Clones of human CEP2 were
identified from a TBLASTN search of the expressed sequence tag (EST)
data base at the National Center for Biotechnology Information using
the 16-amino acid sequence of the CRIB domain of MSE55 as a query (24,
25). An EST clone corresponding to human CEP2 was obtained from the
IMAGE Consortium (clone identification number 22978). This clone was
sequenced on an Applied Biosystems 377 DNA sequencer and has the
GenBankTM Accession number AF098290. Clones containing related genes, human CEP3 (hCEP3), human CEP4 (hCEP4), and mouse CEP5 (mCEP5) were
also obtained from the IMAGE Consortium (clones 488206, 360199, and
408069, respectively) and sequenced. The nucleotide and amino acid
sequences of hCEP3, hCEP4, and mCEP5 have GenBankTM accession numbers
AF104857, AF099664, and AF102773, respectively.
Mammalian Expression Vectors--
N-terminal Myc-tagged
expression vectors of pRK5-Cdc42T17N, pcDNA3-Cdc42,
pRK5-Cdc42-Q61L, and EXV-RhoB-G14V were used (24, 26, 27). The coding
sequence of CEP2 was amplified by PCR from the corresponding cDNA
clone using the two primers 5'-GAGGGATCCACCAAGGTGCCCATC-3' and
5'-GAGCTCGAGCTATGTGGGGATCTGCAT-3' and then subcloned in-frame into the
BamHI/XhoI sites downstream of a CMV-driven
N-terminal FLAG epitope-tagged pCAF1 mammalian expression vector and
into the CMV-driven N-terminal Myc epitope-tagged pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA) (24, 26). The CEP3 coding
sequence was amplified by PCR using two
BglII/XhoI linker primer pairs,
5'-GAGAGATCTCCAGCCAAGACCCCAATT-3' and
5'-GAGCTCGAGTTACTTATTTTTATCCATTAC-3', and subcloned in-frame into the
BamHI/XhoI sites of the CMV-driven N-terminal Myc
epitope-tagged pcDNA3 mammalian expression vector. The coding
sequence of CEP4 was amplified using the following linker primers:
5'-GAGGGATCCCCAATCCTCAAGCAACTG-3' and
5'-GAGCTCGAGTCACACGATTTCATCCTC-3', whereas mCEP5 was amplified using
5'-GAGGGATCCCCGGTAATGAAGCAGCTG-3' and 5'-GAGCTCGAGCTACAGACCGATGACGTC-3'
linker primers. hCEP4 and mCEP5 PCR products were subcloned
in-frame into the BamHI/XhoI sites of the pCAF1
mammalian expression vector. A pCAF-CEP2 mutant containing double
alanine substitutions in the CRIB domain (CEP2-H39A, H42A) was
generated using the QuikChange mutagenesis kit (Stratagene, La Jolla,
CA) according to the manufacturer's instructions. CEP2-H39A, H42A was
also subcloned in-frame into the Myc-tagged pcDNA3 vector. The
integrity of all constructs was confirmed by DNA sequencing.
GST Capture Experiments--
Approximately 5 µg of either
recombinant glutathione S-transferase (GST), Rac-Q61L, or
Cdc42-G12V expressed as fusion proteins in Escherichia coli
were purified on glutathione-agarose resin and loaded with GTP NIH-3T3 Fibroblast Transfection and Immunofluorescence--
Cell
culture and transfection of NIH-3T3 fibroblasts were performed
essentially as described (24, 26) except that LipofectAMINE Plus (Life
Technologies, Inc., Rockville, MD) was used as the transfection
reagent. Cells were grown overnight on polylysine-coated coverslips and
then transfected with one of the following epitope-tagged constructs:
pCAF-CEP2, pcDNA3-CEP3, pCAF-CEP4, pCAF-CEP5, pcDNA3-Cdc42, or
pcDNA3-Cdc42-T17N. Twenty-four hours post-transfection, NIH-3T3 fibroblasts were fixed, stained, and analyzed using the following primary antibodies: mouse anti-FLAG M2 at a 1:1000 dilution to detect
CEP2, CEP4, and CEP5 protein expression or mouse anti-c-Myc (9E10)
antibody (Sigma) at a 1:500 dilution to detect CEP3, Cdc42, and
Cdc42-T17N protein expression. Epitope-tagged proteins were then
detected using FITC-conjugated goat-anti-mouse IgG at a 1:500 dilution
(Rockland, Gilbertsville, PA). Texas Red-conjugated Phalloidin (Sigma)
was used at a 1:200 dilution to elucidate F-actin. For Cdc42 and CEP
cotransfection experiments, cells were simultaneously stained with a
rabbit anti-Myc polyclonal antibody (Santa Cruz Biotechnology, Santa
Cruz, CA) at a 1:200 dilution to detect Cdc42 and with the mouse
anti-FLAGTM antibody to detect CEP expression. The cells
were then incubated with FITC-conjugated goat anti-rabbit (Rockland)
and Texas Red-conjugated goat anti-mouse (Jackson ImmunoResearch
Laboratory, Westgrove, PA) antibodies to detect Cdc42 and CEP2, respectively.
Keratinocyte Transfection and
Immunofluorescence--
Human primary keratinocytes were isolated from
human foreskin as described (28) and maintained in keratinocyte serum
free media containing epidermal growth factor (5 ng/ml), bovine
pituitary extract (50 µg/ml), and gentamicin (10 µg/ml) (Life
Technologies, Inc.). Cells were grown on glass coverslips for 2 days
and then transfected with one of the following constructs:
pcDNA3-Myc-Cdc42-Q61L, pEXV-Myc-RhoB-G14V, pcDNA3-Myc-CEP2,
pcDNA3-Myc-CEP5, or pcDNA3-Myc-CEP2-H39A, H42A using the
Fugene6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) per the manufacturer's instructions. Twenty-four hours after transfection, cells were switched to Dulbecco's modified Eagle's medium high calcium media containing 10% heat-inactivated fetal calf serum to induce adherens junction formation (28). One hour
later, the cells were fixed and stained. Transfected cells were
detected using a rabbit polyclonal anti-Myc antibody at a 1:200
dilution (Santa Cruz Biotechnology). For analysis of F-actin and
vinculin localization, cells were stained simultaneously with rabbit
polyclonal anti-Myc antibody and mouse monoclonal anti-vinculin
antibody (Sigma) at a 1:200 dilution. Cells were then stained with a
Texas Red-conjugated donkey anti-rabbit polyclonal antibody at a 1:300
dilution to detect transfected cells, a Cy5-conjugated donkey
anti-mouse antibody at a 1:300 dilution to detect vinculin, and
FITC-conjugated phalloidin at a 1:300 dilution to detect
F-actin. For E-cadherin staining, cells were simultaneously stained
with rabbit polyclonal anti-Myc antibody and with mouse monoclonal anti-E-cadherin antibody (Transduction Laboratories, Lexington, KY) at
a 1:200 dilution. Cells were then incubated with FITC-conjugated goat
anti-Rabbit antibody at a 1:200 dilution to detect transfected cells
and a Texas Red-conjugated goat anti-mouse antibody at a 1:400 dilution
to detect E-cadherin. Confocal microscopy was performed with an Olympus
Fluoview confocal microscope (Olympus America, Inc., Melville, NY)
attached to an Olympus 1 × 70 inverted fluorescent scope equipped
with a 60× oil immersion lens. Digitized images were captured using
the Fluoview software (Olympus America, Inc.).
Identification of Genes for CEPs--
Previously, we identified
MSE55 as a CRIB domain-containing Cdc42 nonkinase effector protein that
induces long F-actin-containing extensions resembling pseudopodia (24).
Using the amino acid sequence of MSE55 to query the EST data base, we
identified and sequenced three different human EST clones that contain
CRIB domains and two additional regions of amino acid homology to
MSE55. These cDNA clones, designated CEP2, CEP3, and CEP4, encode
proteins of 210, 254, and 356 amino acids, respectively (Fig.
1A). We also identified and
sequenced a mouse cDNA clone designated mCEP5, encoding a protein
of 150 amino acids that contains features in common with MSE55 (Fig.
1A). Analysis of the nucleotide sequences of the different
CEP clones confirmed that they contain a Kozak's consensus translation
initiation sequence and an in-frame stop codon 5' to the methionine
start codon (data not shown). Based on the amino acid sequence and
binding data (see below), we conclude that CEPs define a new family of
Cdc42-binding proteins. Because MSE55 is a Cdc42 effector involved in
actin reorganization at the cell membrane (24) and not a major serum
protein (29), we propose that MSE55 be renamed CEP1 to better describe
its close structural and functional relationship to other members of
the CEP family. At the time of submission to GenBankTM, no additional MSE55-like proteins were known, although a recent contemporaneous study
by Joberty et al. (30) identified the same proteins, denoted as the Borgs, as Cdc42-binding proteins. The corresponding nomenclature is as follows: CEP1/MSE55/Borg5, CEP2/Borg1, CEP3/Borg2, CEP4/Borg4, and CEP5/Borg3.
Potential Signaling Domains in CEPs--
Although all CEP members
contain the 16 amino acid CRIB consensus sequence (25), they also show
further homology extending C-terminal to the CRIB domain (Fig.
1B). This expanded CRIB region has the consensus sequence
Ile-Ser-Xaa-Pro-Leu-Gly-Xaa-Phe-Arg-His-Thr-AA-His-AA-Gly-Xaa-Xaa-Gly-(Xaa)0-2-Asp-AA-Phe-Gly-Asp-Xaa-Ser-Phe-Leu, where AA represents an aliphatic amino acid (Fig. 1B).
The expanded CRIB region of the CEP family is different from that found
in other Cdc42 effector proteins except for the Drosophila
DPR2/DACK tyrosine kinase (25, 31). These comparisons suggest that the C-terminal ends of the extended CRIB domain in CEPs and DPR2/DACK could
be involved in regulating the biological effects of these proteins
and/or are involved in Cdc42/Rac binding. Many families of Cdc42
effector proteins contain unique extended regions of homology
C-terminal to the core CRIB domain that are functionally important. In
PAK kinases, this region negatively regulates its kinase activity (32).
In ACK (33) and WASP (34), these regions form an alpha helix that is
involved in Cdc42 binding.
In addition to the CRIB domain, CEP family members share two other
regions of homology designated the CI and CII domains that span 11 and
14 amino acids, respectively (Fig. 1B). The CI domain is
located C-terminal to the CRIB domain and has the consensus sequence
AA-AA-Lys-Asn-Ala-AA-Ser-Leu-Pro-Xaa-AA (Fig. 1B). The CII
domain is located C-terminal to CI domain and has the consensus sequence of Asp-Leu-Gly-Pro-Ser-AA-Leu-Xaa-Xaa-AA-Leu-Xaa-AA-Met (Fig.
1B). Although CEP1, CEP2, CEP3, and CEP4 have CRIB, CI, and
CII domains, CEP5 is unique in that it lacks a CI domain and instead
contains a polyproline region in its place (amino acids 56-89) (Fig.
1A). Although the functions of the CI and CII domains are
unknown, several other signaling molecules contain similar domains. Two
Caenorhabditis elegans cDNA clones (GenBankTM accession numbers S72575 and F44D12.4) encode proteins that contain
potential CI domains (Fig. 1B). The Drosophila
Cdc42 tyrosine kinase effector DPR2/DACK (25, 31) has a potential CII
domain ~100 amino acids downstream of the CEP-like CRIB region (Fig.
1B). The mouse PSM protein, an SH2-SH3 adapter molecule
(35), also contains a CII-like domain (Fig. 1B). The fact
that the CII domain occurs separately in CRIB-containing DPR2/DACK and
CEP5 proteins suggests that the CI and CII domains function
independently of each other.
CEPs Bind to Cdc42--
Previously, we showed that CEP1/MSE55 (24)
and the Drosophila DPR2/DACK CRIB-containing kinase (25)
bind strongly to Cdc42 but weakly to Rac. In light of these findings
and the fact that the CRIB domain of all the CEP members and DPR2/DACK
are 75% identical, we were interested in determining if other CEP
family members could also interact with Cdc42. We used GST-affinity
chromatography to examine binding of CEP2 and CEP5 to Rac and Cdc42. In
these experiments, both CEP2 and CEP5 bound to Cdc42, but not
detectably to Rac (Fig. 2). Similar
experiments with CEP4 demonstrated a specific interaction with Cdc42,
but not with Rac (data not shown). Yeast two-hybrid analysis also
validated CEP2 and CEP5 interaction with Cdc42 (data not shown).
Furthermore, a CRIB mutant of CEP2 containing alanine substitutions
within two conserved histidine residues (CEP2-H39A, H42A) showed no
detectable binding to Cdc42 (Fig. 2). Taken together these data suggest
that CEPs interact with Cdc42 via their CRIB domain and function as
downstream effector proteins.
CEPs Induce F-actin-containing Pseudopodia in NIH-3T3
Fibroblasts--
FLAG- or Myc-tagged mammalian expression vectors for
CEP2, CEP3, CEP4, and CEP5 were transfected into NIH-3T3 fibroblasts. After transfection, CEP protein expression was observed in both the
cytoplasm and at the plasma membrane (Fig.
3, a, c,
e, and g). Approximately 40% of CEP-expressing
fibroblasts showed one to two pseudopodia that were longer than the
cell body length (Fig. 3, b, d, f, and
h). The number of CEP-expressing cells showing pseudopodia
was two times greater than that of untransfected control cells. Some
cells with pseudopodia also exhibited laterally placed ruffles or
ruffles localized to the distal ends of the pseudopodia (Fig. 3,
f and h). The CEP-expressing cells extending
pseudopodia did not show cell body rounding or collapse of the
cytoplasm, suggesting these structures were distinct from retraction
fibers. Furthermore, at higher magnifications, the CEP-induced
pseudopodia in some of the cells contained long F-actin cables that
extended into the cell body (data not shown). No obvious differences in pseudopodia morphology were seen between any of the CEPs, including CEP1 (24). These data demonstrate that the five CEP family members induce similar pseudopodia formation in NIH-3T3 fibroblasts.
Cdc42 Regulates CEP-induced Pseudopodia Formation--
To more
clearly define the relationship between Cdc42 and CEPs, we tested the
effect of CEP2/Cdc42 coexpression in NIH-3T3 fibroblasts. As previously
reported (2, 3), fibroblasts expressing Cdc42 showed many uniformly
placed filopodia around the cell periphery (Fig.
4, a and b). In
contrast, cells coexpressing Cdc42 and CEP2 induced one or two
pseudopodia with multiple filopodia and/or membrane ruffles emanating
from it (Fig. 4, c and d). Additionally, the
majority of coexpressing cells showed peripherally placed filopodia
similar to those seen in cells only expressing Cdc42 yet showed a more
elongated cell shape (compare Fig. 4, c and d,
with Fig. 4, a and b). Fibroblasts coexpressing
dominant negative Cdc42-T17N and CEP2, failed to produce pseudopodia
(Fig. 4, e and f). However, in many of these
fibroblasts, expression of the dominant negative Cdc42 mutant alone or
in combination with CEP2 appeared to be toxic, inducing cell rounding
in ~50% of the cells.
We also examined whether cells transfected with the CRIB mutant of CEP2
could induce pseudopodia. By cell counting, CEP2 was found to induce
pseudopodia formation 2-fold over untransfected cells (Fig.
5). In contrast, cells expressing the
CRIB mutant of CEP2 (CEP2-H39A, H42A) showed levels of pseudopodia
similar to untransfected cells (Fig. 5), although the expression level and localization of the mutant was similar to that of wild type CEP2
protein. Fibroblasts coexpressing Cdc42 with CEP2 also showed a slight
increase in pseudopodia formation over CEP2 expression alone (Fig. 5).
Together these data are consistent with a model whereby Cdc42 is
required for CEP-mediated pseudopodia formation in fibroblasts.
Constitutively Active Cdc42 Induces Thin F-actin Stress Fiber
Formation in Keratinocytes--
Based on the CEP-induced phenotype
observed in fibroblasts, we were interested in determining whether CEPs
might function in Cdc42-mediated actin reorganization in other cell
types. Although Madin-Darby canine kidney cells have been used to study
Cdc42-induced actin changes (36, 37), we were unable to detect
CEP-induced changes in actin polymerization or cell shape in these
cells (data not shown). As an alternative epithelial cell type, we
chose to study the function of Cdc42 and CEPs in primary keratinocytes, which form well-defined adherens junctions driven by actin filament polymerization following a switch to calcium-containing media (38).
Unlike wild type Cdc42 that showed no effect on F-actin (data not
shown), keratinocytes expressing Cdc42-Q61L (Fig.
6a) showed low levels of
F-actin staining at the adherens belt and contained thin F-actin
filaments that extended the length of the cell body (Fig.
6b). By confocal analysis, we found that these actin
filaments localized at the ventral surface and within the cytoplasm of
the cell (Fig. 6b). This Cdc42-induced phenotype differed
from untransfected neighboring cells that showed high levels of F-actin
staining at the adherens junction (Fig. 6b). Cdc42-Q61L-expressing cells showed vinculin staining at the tips of the
actin filaments, indicating that these actin filaments are stress
fibers (Fig. 6c). This pattern of vinculin staining was
different from that of untransfected cells in that it was less
organized around the cell periphery.
We also examined the effect of expressing a constitutively active Rho
mutant in keratinocytes. Although a RhoA expression vector expressed
poorly in these cells, a constitutively active RhoB construct (Fig.
6d) expressed at levels similar to Cdc42. These
Rho-expressing cells showed fewer stress fibers that were thicker in
width and shorter in length (Fig. 6e) than those observed in
cells expressing Cdc42-Q61L (Fig. 6b). RhoB-expressing cells showed fewer vinculin-staining complexes that were more pronounced than
the fine punctate vinculin staining observed in Cdc42-Q61L-expressing cells (compare Fig. 6f with Fig. 6c). These
results suggest that expression of constitutively active Cdc42 in
primary keratinocytes induced the assembly of fine actin stress fibers
that are distinct from stress fibers induced by constitutively active Rho.
CEP2 and CEP5 Alter F-actin and Vinculin Organization in
Keratinocytes--
We next expressed CEP2 and CEP5 in primary
keratinocytes. CEP5 (Fig. 7a)
and CEP2 (Fig. 7d) showed membrane and cytoplasmic localization with high levels of staining in a distinct cytoplasmic compartment surrounding the nucleus. This localization differed from
the cytoplasmic and membrane localization of CEPs found in fibroblasts
(see Fig. 3). CEP5- and CEP2-expressing keratinocytes showed less
F-actin staining at the adherens belt and had an increase in fine
F-actin filaments that ran along the length of the cell (Fig. 7,
b and e). These effects resembled the F-actin
changes observed in Cdc42-Q61L- expressing cells except that
CEP-expressing cells tended to be more elongated than cells expressing
Cdc42-Q61L. Expression of CEP2 and CEP5 prior to calcium switch also
showed these F-actin changes, although they were less pronounced (data not shown). The tips of CEP-induced actin filaments stained positive for vinculin indicating that these filaments were stress fibers (Fig.
7, c and f). CEP-expressing cells often showed
the highest vinculin levels in regions of the cell that lacked contact
with neighboring cells. This differed from untransfected cells that contained uniform vinculin staining around the cell periphery even when
not neighbored by other cells. The CEP2 CRIB mutant showed a similar
expression pattern (Fig. 7g) as wild type CEP2, but did not
show an increase in actin filaments (Fig. 7h) or altered vinculin distribution (Fig. 7i). Instead, cells expressing
the CEP2-CRIB mutant showed F-actin staining (Fig. 7h) and
vinculin staining (Fig. 7i) at the cell periphery that
resembled that of untransfected cells. Taken together these results
suggest that CEP5 and CEP2 mediate Cdc42-like actin changes by inducing
the actin and vinculin reorganization that may contribute to epithelial cell shape changes.
Cdc42-Q61L- and CEP-expressing Keratinocytes Show Reduced
E-cadherin Staining at Adherens Junctions--
Because both Cdc42 and
CEP expression resulted in the loss of F-actin at adherens junctions,
we next examined the effect of Cdc42 and CEPs on E-cadherin
distribution at adherens junctions. One hour following calcium switch,
cells expressing Cdc42-Q61L (Fig. 8,
a and b), CEP2 (Fig. 8, c and
d), or CEP5 (Fig. 8, e and f) showed
reduced levels of E-cadherin at adherens junctions (Fig. 8,
b, d, and f). Cdc42-expressing cells
also showed an increase in E-cadherin localized within vesicles. In
contrast, untransfected cells showed strong E-cadherin staining at
cell-cell junctions (Fig. 8, b, d, and
f). These data suggest that overexpression of Cdc42 or CEPs
interfere with the assembly of normal adherens junctions.
Here we report the structure and function of a new family of Cdc42
effector proteins that we have called CEPs. This family contains five
members: CEP1/MSE55, CEP2, CEP3, CEP4, and CEP5. CEP1 through CEP4 all
contain a previously identified CRIB domain (25) and two new domains,
designated CI and CII. CEP5 is structurally different because it lacks
a CI domain. Transfection experiments indicate that CEPs induce actin
reorganization and cell shape changes that lead to pseudopodia
formation in fibroblasts. Interestingly, pseudopodia normally occur in
NIH-3T3 fibroblasts and are increased in cells overexpressing v-Fos
(39), CD-44 (40), or an F-actin-bundling protein (41). Although the
biological relevance of the CEP-induced pseudopodia with relation to
Cdc42 signaling is still unclear, there are several possible
explanations. The CEP-induced pseudopodia in fibroblasts may represent
an exaggerated activity of these molecules, producing a phenotype that
is not generally observed with endogenous levels of CEPs. Previous
studies have shown that inducible recruitment of Cdc42 to the membrane
induces a single filopodium (42) rather than the array of filopodia
that is conventionally seen with expression of wild type Cdc42. Based
on these findings, CEPs might function in fibroblasts at specific
membrane sites resulting in a net effect of directed membrane
protrusion. CEPs may also represent targets of other Cdc42-related
GTPases that normally function in fibroblasts to generate pseudopodia
formation rather than filopodia formation. Consistent with this model,
Cdc42 homologues such as TC10 (43) and TCL (44) interact with
CRIB-containing proteins in vitro and induce actin
structures that are distinct from Cdc42.
Recently Joberty et al. (30) identified and characterized a
set of Cdc42-binding proteins, termed Borgs, which are identical to the
CEPs. Although they observed similar morphological changes following
overexpression in fibroblasts, they suggested that Borgs/CEPs function
to inhibit Rho activity, leading to the production of retraction
fibers. Our results are difficult to reconcile with this retraction
fiber model. First, CEP-induced pseudopodia and retraction fibers are
morphologically distinct and are formed in different ways. Retraction
fibers appear as dendritic-like processes (45) and are formed following
collapse of the cytoskeletal architecture. The pseudopodia formed in
our studies were produced by an active process leading to the formation
of single or bipolar membrane protrusions, as visualized by time-lapse
videography (24). The CEP-induced pseudopodia are also not
dendritic-like, but are limited to one or two extensions per cell
without much branching. CEP-expressing cells did not show cell body
rounding or collapse, which is normally seen in cells exhibiting
retraction fibers. Second, in Madin-Darby canine kidney epithelial
cells, CEPs had no noticeable effect on stress fiber formation. Third, we were unable to reproduce the finding (30) that coexpression of
constitutively active Rho reverses CEP-induced morphological changes in
NIH-3T3 fibroblasts.2 The
reason for this is unknown, but could be due to differences in cell
culture conditions. Fourth, in keratinocytes, CEPs induced thin actin
stress fibers that were similar to what was observed following
transfection with an activated mutant of Cdc42. Thus, we propose an
alternative model in which CEPs, acting as classical Cdc42 effector
molecules, increase actin filament assembly to cause cell shape changes.
Although less is known about the function of Rho GTPases in epithelial
cells as compared with fibroblasts, recent studies indicate that they
function in the assembly of adherens junctions (36, 37, 46, 47). For
example, overexpression of constitutively active Rho or Rac in
Madin-Darby canine kidney cells increases F-actin at the adherens
junctions while dominant negative constructs block the formation of
adherens junctions (46, 47). Likewise, Madin-Darby canine kidney cells
overexpressing constitutively active Cdc42 show increased F-actin at
adherens junctions along with other associated molecules such as
In fibroblasts and keratinocytes, CEP-induced actin changes were
dependent on Cdc42. In NIH-3T3 fibroblasts, coexpression of dominant
negative Cdc42 with CEP2 or expression of a CEP2 CRIB mutant blocked
pseudopodia formation. Similarly, CEP2-induced cytoskeletal and
morphological changes were also abrogated in keratinocytes expressing
the CEP2-CRIB mutant. Based on these observations, we propose a model
in which Cdc42 binding to the CRIB domain of CEPs induces
conformational changes leading to exposure of the previously
inaccessible CI, CII, and/or other domains. Further clues to the normal
function of these molecules may come from the observation that the
Drosophila CRIB-containing ACK-like tyrosine
kinase, DPR2/DACK, also contains a CII-like domain downstream of a similar CRIB domain and functions to
phosphorylate a SH3-containing adapter molecule (31). These results
suggest the possibility that the CII domain recruits additional
actin-binding proteins or other signaling molecules and/or acts as an
autoinhibitory domain as seen in the WASP proteins (48). In the case of
CEP5, we propose that the unique proline-like adapter sequences replace the function of the CI domain to induce similar biological activities. Like the proline-rich sequences found in N-WASP (17), these sequences
may bind profilin and/or SH3-containing molecules.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S as
described previously (24). N-terminal FLAG-tagged mammalian expression
vectors of CEP2, CEP2-H39A, H42A, and CEP5 were transfected by
electroporation (250 microfarads/400 V) into Cos1 cells, and similar
levels of expression were observed for the different constructs. After
48 h, cell lysates were prepared and used in GST capture
experiments as described (24). Briefly, precleared lysates were
incubated for 10 min with each of the immobilized recombinant proteins
and washed, and bound proteins were separated on an 8%
SDS-polyacrylamide gel electrophoresis gel. Proteins were then
electrophoretically transferred to nitrocellulose. The blots were
blocked and then probed with anti-FLAG M2 monoclonal antibody (Sigma
Chemical Co., St. Louis, MO). FLAG-tagged CEPs were then detected by
incubating with rabbit anti-mouse-horseradish peroxidase followed by
incubation with enhanced chemiluminescence reagents (Pierce, Rockford,
IL) and exposure to x-ray film.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Fig. 1.
The CEP family of proteins.
A, protein sequence alignments of the CEP proteins. The
numbers at the beginning and end of each line indicate the total number
of amino acid residues to that point in the sequence. Optimal alignment
of the different CEP protein sequences was performed manually. Regions
sharing significant homology are highlighted in gray.
Completely conserved amino acids in the CI and CII domains are
indicated by an asterisk. The polyproline region of CEP5 is
underlined. B, sequence alignment of CRIB, CI,
and CII regions with other signaling molecules. A dashed
line or a plus sign identifies identical or
semiconserved amino acids to CEP1, respectively. The consensus
sequences are based on completely conserved amino acids between CEP
family members and are shown in boldface at the top, with
conserved aliphatic residues denoted by A. The CRIB domain,
potential CII domains of DPR2/DACK (25, 31), and a potential CI domain
of two distinct C. elegans clones (F44D12.4 and S72575) are
also shown.
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Fig. 2.
CEP2 and CEP5 bind to Cdc42. Immobilized
GST, GST-Cdc42-G12V, or GST-Rac-Q61L were loaded with GTP S and
incubated with whole cell extracts of Cos1 cells transfected with
either FLAG epitope-tagged CEP5, FLAG epitope-tagged CEP2, or FLAG
epitope-tagged CEP2-H39A, H42A. Following washing, bound proteins were
analyzed by Western blot using the M2 mouse anti-FLAG antibody with
anti-mouse-horseradish peroxidase. The blots were developed using
enhanced chemiluminescence (ECL).
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Fig. 3.
CEPs induce pseudopodia formation in NIH-3T3
fibroblasts. FLAG-tagged CEP constructs were transfected into
NIH-3T3 fibroblasts and fixed 24 h after transfection.
Fluorescence micrographs show CEP2 (a and b),
CEP3 (c and d), CEP4 (e and
f), and CEP5 (g and h) expressing
cells inducing pseudopodia. CEP expression was detected using mouse
monoclonal antibodies against the epitope tag followed by staining with
FITC-conjugated goat anti-mouse IgG (a, c,
e, and g). F-actin was visualized with Texas
Red-conjugated Phalloidin (b, d, f,
and h). Bar, 10 µm.
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Fig. 4.
Cdc42 is required for CEP2-mediated
pseudopodia formation. NIH-3T3 fibroblasts were transfected with
Myc-tagged Cdc42 (a and b) or cotransfected with
FLAG-tagged CEP2 plus either Myc-tagged Cdc42 (c and
d) or Myc-tagged Cdc42-T17N (e and f).
Twenty-four hours after transfection, cells were stained with a rabbit
polyclonal anti-Myc antibody followed by staining with FITC-conjugated
goat anti-rabbit to detect Cdc42 (a and c) and
Cdc42-T17N (e). Texas Red-conjugated Phalloidin was used to
detect F-actin (b). FLAG-tagged CEP2 (d and
f) expressing cells were detected with a mouse monoclonal
anti-FLAG antibody followed by staining with Texas Red-conjugated goat
anti-mouse antibody.
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Fig. 5.
A CEP2-CRIB mutant is inactive for
pseudopodia formation. CEP2, CEP2-CRIB mutant (CEP2-H39A, H42A),
or CEP2 plus wild type Cdc42 were transfected into NIH-3T3 fibroblasts,
fixed, and stained 24 h after transfection. Cells with pseudopodia
greater than the cell body length are expressed as a percentage of the
total number of transfected, or untransfected cells counted. Data are
mean ± S.D. of three independent experiments.
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Fig. 6.
Constitutively active Cdc42 induces thin
actin stress fibers in keratinocytes. Keratinocytes were
transfected with Myc-tagged Cdc42-Q61L (a-c) or RhoB-G14V
(d-f). Twenty-four hours after transfection, the cells were
changed to high calcium complete media for 1 h. Myc-tagged
Cdc42-Q61L (a) and RhoB-G14V (d) were detected
with rabbit anti-Myc antibody followed by staining with Texas
Red-conjugated donkey anti-rabbit antibody. FITC-conjugated Phalloidin
was used to detect F-actin (b and e). Vinculin
was detected by staining with mouse anti-vinculin followed by
Cy5-conjugated donkey anti-mouse antibody (c and
f). Bar, 10 µm.
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Fig. 7.
Keratinocytes expressing CEP2 and CEP5 form
thin actin stress fibers. Keratinocytes were transfected with
Myc-tagged CEP5 (a-c), Myc-tagged CEP2 (d-f),
or Myc-tagged CEP2-H39A, H42A (g-i). Twenty-four hours
after transfection, the cells were changed to high calcium complete
media for 1 h. Myc-tagged CEP constructs were detected with rabbit
anti-Myc antibody followed by staining with Texas Red-conjugated donkey
anti-rabbit antibody (a, d, and g).
FITC-conjugated Phalloidin was used to detect F-actin (b,
e, and h). Vinculin was detected using a mouse
anti-vinculin antibody followed by staining with Cy5-conjugated donkey
anti-mouse antibody (c, f, and i).
Bar, 10 µm.
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Fig. 8.
Cdc42 or CEP expression reduces the level of
E-cadherin at adherens junctions. Keratinocytes were transfected
with Myc-tagged Cdc42-Q61L (a and b), Myc-tagged
CEP2 (c and d), or Myc-tagged CEP5 (e
and f). Twenty-four hours after transfection, the cells were
changed to high calcium media for one hour. Myc-tagged Cdc42-Q61L
(a), CEP2 (c), and CEP5 (e) were
detected with rabbit anti-Myc antibody followed by staining with
FITC-conjugated goat anti-rabbit antibody. E-cadherin was detected with
mouse anti-E-cadherin antibody followed by staining with Texas
Red-conjugated goat anti-mouse antibody (b, d,
and f). Arrows point to examples of diminished
E-cadherin staining at the borders of transfected cells.
Bar, 10 µ m.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin and E-cadherin (36, 37). Our studies using primary
keratinocytes implicate CEPs in Cdc42-mediated assembly of thin actin
stress fibers that extend across the cell body length. The phenotype
observed here with both Cdc42 and CEPs resembles that of early stage
adherens junction formation, where actin filaments radiate across the
cell body to drive adherens junction formation and would have been missed using immortalized epithelial cells (38). Although Cdc42-Q61L and CEPs induced the formation of fine actin stress fibers, Rho induced
short stress fibers with thicker actin bundles. Vinculin staining in
the Cdc42- and CEP-expressing cells was also less prominent than in
Rho-expressing cells. These results are in agreement with studies in
fibroblasts, demonstrating that Cdc42 and Rho induce distinct actin and
focal adhesion reorganization (3). CEPs and Cdc42 also interfered with
E-cadherin localization to adherens junctions thus providing further
support that they may function in actin reorganization during the early
stages of adherens junction formation.
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ACKNOWLEDGEMENTS |
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Primary human keratinocytes were a kind gift of the laboratory of Dr. Richard Schlegel. We are grateful to the DNA sequencing and Microscopy core facilities of the Lombardi Cancer Center.
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FOOTNOTES |
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* This work was supported by Grant R29-CA77459-01 (to P. D. B.) from the National Cancer Institute, by a predoctoral scholarship from the ARCs Foundation (to D. S. H.), and by a Department of Defense breast cancer predoctoral fellowship (to D. M. P.). The DNA sequencing and Microscopy core facilities of the Lombardi Cancer Center were funded by Grant P30-CA51008 from the National Institutes of Health.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF098290, AF104857, AF099664, and AF102773.
To whom correspondence should be addressed: Georgetown University
Medical Center, Rm. EG16, New Research Bldg., Lombardi Cancer Center,
3970 Reservoir Rd., NW, Washington, DC 20007. Tel.: 202-687-1444; Fax:
202-687-7505; E-mail: burbelpd@gunet.georgetown.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007039200
2 D. S. Hirsch and P. D. Burbelo, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
PAK, p21-activated
protein kinase;
CEP, Cdc42 effector protein;
CRIB, Cdc42/Rac
interactive binding domain;
EST, expressed sequence tag;
PCR, polymerase chain reaction;
CMV, cytomegalovirus;
GST, glutathione
S-transferase;
GTPS, guanosine
5'-3-O-(thio)triphosphate;
FITC, fluorescein isothiocyanate;
AA, aliphatic amino acid.
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