From the Laboratory of Molecular Oncology and
¶ Laboratory of Molecular Genetics and Immunology, The Rockefeller
University, New York, New York 10021, the
Department of Pathology, University of
Western Australia, Nedlands, Western Australia 6907, Australia, and
** Osaka Bioscience Institute, 6-2-4 Furuedai, Suita,
Osaka 565-0874, Japan
Received for publication, June 30, 2000, and in revised form, November 3, 2000
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ABSTRACT |
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CMS/CD2AP is a cytoplasmic protein critical for
the integrity of the kidney glomerular filtration and the T cell
function. CMS contains domains and motifs characteristic for
protein-protein interactions, and it is involved in the regulation of
the actin cytoskeleton. We report here that the individual SH3 domains
of CMS bind to phosphotyrosine proteins of ~80, 90, and 180 kDa in cell lysates stimulated with epidermal growth factor. The second SH3
domain of CMS bound specifically to a tyrosine-phosphorylated protein
of 120 kDa, which we identified as the proto-oncoprotein c-Cbl. The
c-Cbl-binding site for CMS mapped to the carboxyl terminus of c-Cbl and
is different from the proline-rich region known to bind SH3-containing
proteins. CMS binding to c-Cbl was markedly attenuated in a tyrosine
phosphorylation-defective c-Cbl mutant indicating that this interaction
is dependent on the tyrosine phosphorylation of CMS. It also implies
that CMS interacts with c-Cbl in an inducible fashion upon stimulation
of a variety of cell-surface receptors. Immunofluorescence analysis
revealed that both proteins colocalize at lamellipodia and leading
edges of cells, and we propose that the interaction of CMS with c-Cbl
offers a mechanism by which c-Cbl associates and regulates the actin cytoskeleton.
Adapter type molecules are composed of noncatalytic
protein-protein interaction domains. They are important components of integrated signal transduction pathways and of the cytoskeleton that
organizes the structure of eukaryotic cells (reviewed in Refs. 1 and
2). These molecules selectively control the spatial and temporal
assembly of multiprotein complexes that transmit intracellular signals
that regulate cell proliferation, differentiation, and survival
(reviewed in Refs. 3-5).
CMS/CD2AP (p130Cas ligand with multiple
SH31 domains/CD2-associated
protein) is an adapter-type molecule composed of three SH3 domains, a
proline-rich region, and a coiled-coil domain. Originally, we
identified CMS as a molecule that binds via its proline-rich region to
the SH3 domain of p130Cas (6). We have also shown that CMS
can associate with and become phosphorylated by nonreceptor tyrosine
kinases. These interactions are mediated also by the SH3 domain of the
tyrosine kinase and the proline-rich region in CMS. The coiled-coil
domain in the carboxyl terminus of CMS mediates its dimerization
(6).
CMS plays a role in the regulation of the actin cytoskeleton.
Overexpression studies in COS-7 cells demonstrated that full-length CMS
colocalizes with F-actin to membrane ruffles and leading edges of cells
(6). Interestingly, the first SH3 domain of the mouse homologue of CMS,
named CD2AP, has been shown to bind to the cell adhesion molecule CD2
(7). This interaction enhances CD2 clustering and anchors CD2 at sites
of cell contacts. Besides CD2, no other molecules have been identified
to date that associate with the SH3 domains of CMS. CMS/CD2AP-deficient
mice exhibit defects in glomeruli of the kidney, develop nephrotic
syndrome, and die of renal failure (8). Glomerular epithelial cells
(podocytes) display effacement of their foot processes accompanied by
mesangial hyperplasia and extracellular matrix deposition.
The proto-oncogene c-cbl is the cellular homologue of
v-cbl, the transforming oncogene of the Cas NS-1 retrovirus
(9). c-Cbl is a component of protein tyrosine kinase signaling pathways where it has been established as a negative regulator (10). It is an
adapter protein that contains many structural domains involved in
protein-protein interactions. The amino terminus is highly conserved
among the Cbl family members that harbor a tyrosine kinase binding
domain. This domain binds to the activated nonreceptor tyrosine kinases
ZAP-70 and Syk (11-13) and to the epidermal growth factor (EGF),
platelet-derived growth factor, and colony-stimulating growth factor-1
receptor tyrosine kinases (14-18). The center region of c-Cbl contains
a conserved RING finger domain, which recruits and activates an E2
ubiquitin-conjugating enzyme (19, 20). c-Cbl contains in its carboxyl
terminus many PXXP motifs that can bind to SH3 domains as
well as tyrosine residues, which when phosphorylated form binding sites
for SH2 domains. These motifs serve as binding sites for Grb2, 14-3-3, phosphatidylinositol 3-kinase, Crk, Nck, Vav, and Src-like tyrosine
kinases (reviewed in Ref. 10). A leucine zipper is located in its
carboxyl terminus, which could mediate oligomerization.
c-Cbl is a widely expressed molecule, and it is localized exclusively
in the cytoplasm. Endogenous c-Cbl is localized in osteoclasts to some
vesicular structures in the perinuclear areas and at the cell periphery
(21), whereas overexpressed c-Cbl in NIH 3T3 fibroblasts is targeted to
membrane ruffle-associated actin lamellae (22). This association
requires specific SH3-binding sequences localized in the
carboxyl-terminal half of c-Cbl.
We report here that tyrosine-phosphorylated c-Cbl associates with CMS
in vitro and in vivo. This interaction was
further characterized, and the binding region for CMS in c-Cbl was
determined. Moreover, we found in our studies that both molecules
colocalize to actin structures in membrane ruffles, suggesting that CMS
may link c-Cbl to the actin cytoskeleton.
Plasmid Construction--
Construction of FLAG-tagged
full-length CMS (639 aa) and the GST fusion protein constructs for the
individual SH3 domains of CMS were described (6). The full-length CMS
construct was subcloned in frame with a Myc tag in the retroviral
vector pCX (a gift from T. Agaki) (23). The CMS Cell Lines, Retroviral Infection, Transient Transfection, EGF
Treatment of 293T Cells, and Cell Lysis--
Human 293T kidney
epithelial (293T) cells and COS-7 cells were grown in Dulbecco's
modified Eagle's medium (DMEM, Cellgrow) containing 10% fetal calf
serum (FCS, HyClone). Immortalized mouse podocytes (a gift from P. Mundel) were cultured in RPMI 1640 supplemented with 10% FCS and 10 ng/ml interferon Expression of GST Fusion Peptides and in Vitro Binding
Assay--
Expression and affinity purification of GST fusion proteins
as well as in vitro binding assay were carried out as
described (6).
Immunoprecipitation and Immunoblotting--
Proteins were
immunoprecipitated by incubating 250 µg of total cell lysate with the
specific antibodies for 2 h and further collected on
GammaBindTM G-SepharoseTM (Amersham
Pharmacia Biotech) for 1 h at 4 °C. The antibodies and antisera
were as follows: anti-FLAG (M2, monoclonal) (Sigma); anti-HA
(polyclonal) and anti-GST (polyclonal), (Santa Cruz Biotechnology); anti-Cbl (17, monoclonal) (Transduction Laboratories); anti-Cbl (C-15,
polyclonal), (Santa Cruz Biotechnology); anti-phosphotyrosine (Tyr(P),
4G10, monoclonal) (Upstate Biotechnology, Inc.). The immunocomplexes
were washed three times in lysis buffer, denatured by boiling for 5 min
in double-strength sample buffer, and resolved by SDS-polyacrylamide
gel electrophoresis (PAGE). Immunoblotting was performed as described
(25).
Immunofluorescence--
COS-7 cells and podocytes grown on
poly-D-lysine-coated coverslips were transiently
transfected with 0.5 µg of the indicated plasmid DNA. For
immunofluorescence analysis 48 h post-transfection, the cells were
prepared as described (6). Cells were inspected by microscopy using a
Nikon Eclipse 800 instrument attached to a digital camera.
In Vitro and in Vivo Association of the SH3 Domains of CMS with
Cbl--
CMS contains multiple protein-protein interaction sites
(shown in Fig. 1A). To
investigate the signaling potential of CMS, we analyzed the ability of
the individual SH3 domains of CMS fused to glutathione
S-transferase (GST) to bind to tyrosine-phosphorylated proteins. We performed an in vitro pull-down assay where the
GST fusion peptides and control GST proteins were incubated with cell lysates from EGF-treated 293T cells. Immunoblotting with an
anti-phosphotyrosine antibody revealed that all three SH3 domains of
CMS bound proteins with the molecular masses of ~180, 90, and
80 kDa. In addition, the second SH3 domain associated with a protein of
~120 kDa (Fig. 1B).
A candidate protein of 120 kDa was c-Cbl. c-Cbl is a component of
tyrosine kinase signaling pathways, and it becomes highly phosphorylated in response to treatment of cells with EGF. Reprobing of
the same membrane with an anti-c-Cbl antibody did indeed identify the
120-kDa phosphotyrosine protein as c-Cbl (Fig. 1C). In
addition, also the first and the third SH3 domain of CMS had a much
weaker interaction with c-Cbl. In contrast, when we used lysates from cells transiently transfected with c-Src as source for phosphotyrosine proteins in this assay, only the second SH3 domain of CMS associated with c-Cbl.
CMS is highly expressed in podocytes of the kidney suggesting a
specific role in these specialized epithelial cells (8). We generated
stable cell lines of immortalized podocytes expressing Myc-tagged
versions of CMS and CMS CMS and c-Cbl Colocalize to Actin Structures in
Lamellipodia--
The association between CMS and c-Cbl prompted us to
analyze the intracellular localization and distribution of CMS and
c-Cbl. We have previously shown that CMS can be found at the leading edge of cells and that it is colocalized with F-actin in lamellipodia (6). Moreover, immunofluorescence analysis of podocyte cell lines
expressing CMS or CMS The Association of CMS with c-Cbl Is Regulated by Tyrosine
Phosphorylation of c-Cbl--
Since the in vitro
interaction of the individual SH3 domains with c-Cbl varied when we
used different methods to phosphorylate c-Cbl, we were interested to
investigate the importance of c-Cbl phosphorylation in this
interaction. In randomly growing cells, a small fraction of cellular
proteins including c-Cbl is phosphorylated on tyrosine residues.
Moreover, we found that transient expression of c-Cbl in 293T cells
results in its tyrosine phosphorylation and, in these cells, further
phosphorylation by EGF is not required to detect the association
between CMS and c-Cbl (Fig.
4A). We introduced eight Tyr
If only structural changes were responsible for the inducible
interaction of CMS with Cbl, then a smaller peptide such as an
individual SH3 domain might be able to bind to the
phosphorylation-defective c-Cbl. We tested this hypothesis by assessing
the ability of the second SH3 domain of CMS to pull down HA-tagged
wild-type c-Cbl and Cbl8F from cell lysates. In this assay, we found
that the individual SH3 domain of CMS was able to associate with Cbl8F. However, compared with wild-type c-Cbl, this association was much weaker (Fig. 4D). This finding suggests that the inducible
interaction of CMS with c-Cbl is not solely mediated by conformational
changes of c-Cbl.
CMS Associates with the PXXP Motifs in the Carboxyl-terminal Region
in Cbl--
The site of interaction of CMS with c-Cbl was investigated
by assessing the ability of FLAG-tagged CMS to associate with
GST-tagged Cbl constructs coexpressed in 293T cells. Cbl, Cbl(1-436)
(encoding the amino-terminal half of c-Cbl), Cbl(437-647) (encoding
the proline-rich region), Cbl(648-end), and vector control were used for this assay. As expected, full-length Cbl was efficiently associated with CMS (Fig. 5A, upper
panel). We observed similar efficient binding of CMS only to the
Cbl(648-end) construct and not to Cbl(437-647). In contrast, to the
latter we detected only marginal binding of CMS, although this
construct is composed of the entire proline-rich region (aa 480-655)
of c-Cbl defined in the literature for binding SH3 domain-containing
proteins (22). The c-Cbl(468-end) fragment contains six partially
overlapping PXXP motifs that resemble putative SH3 domain
binding sites (Table I). These
PXXP motifs are situated outside the defined c-Cbl
proline-rich domain. Thus our results suggest that the major
interaction site of CMS in c-Cbl lies carboxyl-terminal of the c-Cbl
proline-rich region. No binding of CMS to the amino-terminal fragment
Cbl(1-436) was detectable (Fig. 5C).
We identified the carboxyl-terminal region of c-Cbl as a major binding
site for CMS. This prompted us to investigate the intracellular localization of coexpressed CMS and Cbl(648-end). Immunofluorescence analysis of mouse podocytes revealed that both molecules did
colocalize. The expression pattern observed for Cbl(648-end) was
similar to that observed for full-length c-Cbl (Fig.
6). Cbl(648-end) was expressed throughout
the cytoplasm and at the cell periphery. Moreover, we also noticed
prominent colocalization of CMS and Cbl peptides to vesicular
structures formed in cells overexpressing CMS and different versions of
Cbl. In our previous studies (6), we found that ectopic expression of
CMS in COS-7 cells induces vesicle formation. We noticed increased
vesicle formation also in podocytes coexpressing Cbl(648-end) with CMS.
These structures were spread throughout the entire cell (not
shown).
Initially we observed differences in the binding potential of the
individual SH3 domains toward c-Cbl. Moreover, we found that the major
recognition site for CMS in c-Cbl lies outside of the ascribed c-Cbl
proline-rich region. In an in vitro pull-down assay, we
analyzed the binding of the individual SH3 domains to FLAG-tagged
Cbl(437-647) and Cbl(648-end) (Fig. 5B). This assay also
revealed the region of aa 648-906 as a major interaction site. The
second SH3 domain bound predominantly, and by this assay we established
the second SH3 domain of CMS as a major structural element for the
CMS/c-Cbl association. We obtained similar results when we coexpressed
in 293T cells c-Cbl with mutant CMS constructs containing point
mutations in the individual SH3 domains that abolish their binding
capacity (not shown).
To characterize further the CMS-binding motif in c-Cbl, we mutated the
core PXXP sequences by introducing Pro The modular structure of CMS/CD2AP indicates a function in
assembling intracellular molecules into selective complexes. Previously we have shown that CMS interacts with signaling molecules such as
p130Cas, Src-like kinases, and the p85 subunit of the
phosphatidylinositol 3-kinase via PXXP-SH3 interactions (6).
Furthermore, CD2AP, which is the mouse homologue of CMS, was first
identified as CD2-associated protein (7) where the binding to CD2 was
mediated by the first SH3 domain of CMS.
Treatment of cell lines with growth factors or phorbol ester is known
to activate tyrosine kinases that result in an overall increase in
tyrosine-phosphorylated cellular proteins and to induce the
reorganization of the actin cytoskeleton. A prominent feature of
cytoskeletal rearrangements is the induction of membrane ruffles and
lamellipodia (sheet-like extensions of the plasma membrane that contain
a meshwork of F-actin). We have shown that CMS colocalizes with F-actin
in such phorbol ester-induced membrane ruffles (6). To identify further
molecules of the signal transduction pathways involving CMS, we
searched for EGF-induced tyrosine-phosphorylated proteins that interact
with the three individual SH3 domains of CMS. One of the molecules
identified was the proto-oncogene c-cbl, which is a
substrate of protein tyrosine kinases that is rapidly phosphorylated
following stimulation by growth factors, antigen, and integrins
(28-31). c-Cbl is a widely expressed protein with higher expression in
the thymus and hematopoietic cell lines (9). Rather like c-Cbl, the
mRNA expression level of CMS is elevated in the thymus (6).
Interestingly, in T cells, Dustin et al. (7) suggested a
function for CMS/CD2AP in the process antigen recognition in
particular in the formation of the "immunological synapse," which is a specialized junction between a T
lymphocyte and an antigen-presenting cell. The immunological synapse
consists of a central cluster of T cell receptors and costimulatory
molecules surrounded by a ring of adhesion molecules. The first SH3
domain of CMS/CD2AP has been shown to bind to CD2. We found here that the second SH3 domain of CMS binds to c-Cbl. c-Cbl has been shown to
associate with the T cell receptor complex (reviewed in Refs. 10 and
32) and to be localized to the immunological synapse (33). It is likely
that CMS could act as a bridging molecule between the cell adhesion
molecule CD2 and the ternary complex formed by c-Cbl, the T cell
receptor, and ZAP-70. Overexpression of a dominant-negative form of CMS
formed by the first two SH3 domains blocked CD2-triggered cytoskeletal
polarization and the formation of the immunological synapse. CMS has
the potential to homodimerize via its carboxyl-terminally located
leucine zipper (6). We suggest that dimerization of CMS mediates
the clustering of CD2 molecules and that the truncated CMS acts as a
dominant-negative molecule by titrating out CD2 and c-Cbl from the
central region of the immunological synapse. However, further work is
needed to study this hypothesis.
Although higher expression levels in the thymus have been reported for
CMS and c-Cbl, both molecules are ubiquitously expressed. A specialized
role for CMS in the dynamic regulation of the actin cytoskeleton has
been underscored by the phenotype of CMS/CD2AP-deficient mice (8) and
overexpression studies in COS-7 cells (6). Interestingly, CMS/CD2AP
Most SH3 domains bind proline-rich motifs with the core consensus
sequence PXXP (34). It is widely believed that this
interaction is rather constitutive, and preformed complexes exist
within the cell. In contrast, the binding of CMS to c-Cbl seems to be
regulated by the phosphorylation of c-Cbl. Phosphorylation of c-Cbl
generates binding sites for SH2 domain-containing proteins such as Vav, p85 subunit of phosphatidylinositol 3-kinase (p85), and Crk (35-37). One possibility could be that the interaction of CMS with c-Cbl is
indirect by binding to one of the above-mentioned proteins. However,
this is very unlikely since we have also shown that isolated SH3
domains of CMS are capable of binding c-Cbl. The possibility that we
favor is an induced conformational change of c-Cbl from a closed to an
open conformation and thereby unmasking putative SH3-domain binding
motifs in the carboxyl terminus of c-Cbl. Support for this idea comes
from the recently identified molecule CIN85, which is structurally
related to CMS (38) and as CMS interacts with c-Cbl in a
phosphorylation-dependent manner. Furthermore, phosphorylation-induced changes of the c-Cbl conformation have been
suggested (38). Recently, it has been demonstrated that c-Cbl can form
a closed structure that prevents Src for binding to it (24). These
structural restrictions can be overcome by binding of Abl to c-Cbl
which then allows Src binding. Conformational changes of c-Cbl could
also lead to the generation of a nonlinear ligand-binding site for CMS
as described for the p53-p53BP2 interaction (39). The interaction of
the SH3 domain of p53BP2 with p53 is determined by the global structure
of the p53 core domain.
Although we cannot rule out the contribution of another protein, we
clearly demonstrated that the tyrosine phosphorylation of c-Cbl
positively regulates its association with CMS. In addition, it could
also be conceivable that the SH3 domains of CMS recognize a novel
consensus sequence that contains a tyrosine phosphorylation site in a
linear sequence motif as identified for the SH3 domain of Eps8
(40).
Cbl contains in its center a proline-rich region with at least nine
overlapping SH3 domain recognition motifs for SH3 domains to which the
binding of Grb2, Nck, CAP, and Src family kinases has been demonstrated
(37, 41-45). Here we provide evidence that CMS binds preferentially to
consensus sequences outside the defined proline-rich region in c-Cbl.
Experiments using deletion mutants of c-Cbl clearly identified the
region of aa 648-906 as a major interaction site. CMS is the first
protein identified to associate via SH3-PXXP interaction
with the carboxyl terminus of c-Cbl.
In summary, we have identified c-Cbl as CMS-associated protein. Our
results suggests that CMS can bind to multiple sites in c-Cbl and thus
be able to contribute to the assembly of larger protein networks upon
stimulation by growth factors and survival signals. It will be of great
interest to examine the role of the CMS·c-Cbl complex formation
during lymphocyte activation and for the integrity of the podocytes in
the kidney glomerulus. Findings derived from such studies will have
substantial implications for the understanding of important
physiological processes such as cell differentiation and apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PP construct lacking
the proline-rich region (aa 332-426) was constructed by standard
polymerase chain reaction (PCR) of the 5' and 3' cDNA fragments
flanking the sequences encoding the proline-rich region. A
PstI restriction site was introduced resulting in a single
amino acid change at position 427 from asparagine to glutamine to
ligate both amplified fragments and cloned in frame with the Myc tag
into the pCX vector. The generations of HA-tagged Cbl and GST-tagged
Cbl, Cbl(437-647), and Cbl(648-end) were described (24). GST-tagged
Cbl(1-436) was created by introducing a stop codon at aa position 436. HA-tagged Cbl8F was generated by introducing Tyr to Phe changes at
positions Tyr-552, Tyr-674, Tyr-700, Tyr-731, Tyr-735, Tyr-774,
Tyr-869, and Tyr-871. The FLAG-tagged constructs F-Cbl(437-647) and
F-Cbl(648-end) were generated by standard PCR and cloned into the
pFLAG-CMV2 vector (Eastman Kodak Co.). Proline to alanine mutations
were introduced into F-Cbl(684-end) as described above. All PCR
products were verified by DNA sequencing.
(Life Technologies, Inc.) at 33 °C.
Immortalized mouse podocytes were used for generating cell lines stably
expressing CMS and CMS
PP. Bosc23 retrovirus-packaging cells were
maintained in DMEM supplemented with 10% FCS and were cotransfected in
6-cm dishes by using FugeneTM 6 transfection reagent (Roche
Molecular Biochemicals) with 1 µg of the various CMS-containing
plasmids and 1 µg of the pCL-Eco plasmid (Imgenex, San Diego). After
48 h, the virus-loaded supernatants were transferred to
exponentially growing podocytes and supplemented with 10 ng/ml
interferon
and Polybrene (4 µg/ml; Sigma). Infected podocytes
were transferred to new culture dishes and grown in selection medium
containing 10 µg/ml blasticidin (Invitrogen). Stably transfected
podocytes were obtained after ~8 days and further cultured in medium
containing 5 µg/ml blasticidin. 293T cells seeded in 6-cm plates were
transiently transfected as described above with 1 µg each of the
plasmids indicated. After 36 h the cell were lysed as described
(25). Subconfluent 293T cells were washed twice with serum-free medium
and cultured in DMEM containing 0.5% FCS for 16 h. Subsequently
the cells were treated for 5 min with 50 ng/ml EGF (Life Technologies,
Inc.), washed, and lysed as described.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of c-Cbl as binding
partner for the CMS SH3 domains. A, schematic
representation of human CMS. PR, proline-rich region;
CC, coiled-coil domain. Amino acid positions are indicated.
B, in vitro interaction of the CMS SH3 domains
with phosphotyrosine-containing proteins. 293T cells were stimulated
with 50 ng/ml EGF or transfected with c-Src. Clarified cell lysates
were used in an in vitro pull-down assay. Precipitated
proteins were subjected to SDS-PAGE and probed with an
anti-phosphotyrosine antibody (shown only for EGF-treated lysates).
C, interaction of the CMS SH3 domains with c-Cbl. The blots
were stripped and reprobed with an anti-c-Cbl antibody. IB,
immunoblot.
PP (lacking the proline-rich region), and we
were also able to detect in vivo interaction of endogenous
c-Cbl with CMS and CMS
PP (Fig. 2). We
estimate that 5% of the endogenous c-Cbl was associated with CMS in
this assay.
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Fig. 2.
In vivo interaction of CMS with
c-Cbl. Lysates of podocytes stably expressing Myc-tagged wild-type
CMS and CMS PP (lacking the proline-rich region) were incubated with
anti-Myc antibodies. Immunoprecipitates (IP) were collected
on Sepharose beads, subjected to SDS-PAGE, and analyzed by Immunoblot
(IB) with anti-c-Cbl antibodies (upper panel).
Precipitated CMS proteins were monitored with anti-Myc antibodies
(middle panel). c-Cbl expression was assayed in total cell
lysates with anti-Cbl antibodies (lower panel).
PP revealed that both peptides are localized to
membrane ruffles and leading edges of cells (not shown). The expression
pattern of HA-tagged c-Cbl was investigated by immunofluorescence
analysis. As expected, c-Cbl is expressed in the cytoplasm, and it
could be found rather like CMS at the leading edge of migrating cells.
There it colocalized with F-actin similarly to CMS (Fig.
3). In addition, we investigated whether CMS and c-Cbl colocalize to these structures by coexpressing
FLAG-tagged CMS with HA-tagged c-Cbl. Indeed both molecules could be
found simultaneously in membrane ruffles and leading edges of motile cells (Fig. 3).
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Fig. 3.
c-Cbl colocalizes with CMS and F-actin
to membrane ruffles. A, randomly growing COS-7 cells,
plated on poly-D-lysine-coated coverslips, were transfected
with HA-tagged c-Cbl. Cells were fixed and stained with
rhodamine-labeled phalloidin for visualizing F-actin and anti-HA
antibodies for monitoring c-Cbl. Membrane ruffles are indicated by
arrows. B, COS-7 cells coexpressing HA-tagged
c-Cbl and CMS were analyzed with anti-HA antibodies and anti-CMS rabbit
serum. The cells were inspected with a Nikon Eclipse 800 instrument (× 60). Colocalizations of CMS and c-Cbl to membrane structure are
indicated by arrows.
Phe point mutations in the carboxyl terminus of c-Cbl (Cbl8F), and
we compared the interaction of this HA-tagged mutant with FLAG-tagged
CMS in 293T cells. In this assay, CMS binding to c-Cbl was abolished in
the phosphorylation-defective mutant protein (Fig. 4B).
Increased tyrosine phosphorylation of c-Cbl by coexpressing the protein
tyrosine kinase Fyn did not alter significantly the binding capacity
(Fig. 4B). SH3 domains constitute a family of
protein-protein interaction domains that bind peptides displaying a
PXXP consensus motif. Since SH3 domain PXXP-mediated interactions are independent of tyrosine
phosphorylation of the polyproline ligand, our result suggests that the
interaction of CMS with c-Cbl is rather regulated than constitutive.
Moreover, the tyrosine phosphorylation of c-Cbl proposes the
involvement of structural changes for the association of CMS with
c-Cbl.
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Fig. 4.
CMS associates with c-Cbl in a
tyrosine-dependent manner. 293T cells were
cotransfected with FLAG-tagged CMS and vector control, HA-tagged Cbl,
or with HA-tagged Cbl8F. Tyrosine phosphorylation was induced by
cotransfecting Fyn. A, tyrosine phosphorylation was analyzed
by immunoprecipitation with anti-phosphotyrosine (Tyr(P)) antibodies
and immunoblotting with anti-Tyr(P), anti-HA, anti-FLAG and anti-Fyn
antibodies. B, cell lysates were incubated with anti-FLAG
antibodies, subsequently the precipitates were collected on Sepharose
beads and subjected to SDS-PAGE. Coprecipitated (IP)
wild-type and mutant c-Cbl were identified with anti-HA antibodies
(upper panel). We controlled for immunoprecipitation of CMS
with anti-FLAG antibodies (lower panel). C, the
expression levels were analyzed in total cell lysates by immunoblotting
(IB) with anti-HA, anti-FLAG, and anti-Fyn antibodies.
D, GST-SH3/2 fusion peptide coupled to glutathione beads was
incubated with increasing amounts (20-320 µg) of clarified lysates
of 293T cells expressing HA-tagged c-Cbl or Cbl8F. Precipitates were
analyzed by SDS-PAGE and probed with an anti-HA antibody (upper
right panel). The blot was reprobed with an anti-GST antibody
(lower right panel). The expression levels were analyzed in
total cell lysates by immunoblotting with anti-HA antibody (left
panel). wt, wild type.
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Fig. 5.
CMS binds to proline-rich sequences in the
carboxyl terminus of c-Cbl. A, 293T were transfected
with the vectors alone or vectors containing cDNAs of FLAG-tagged
CMS and GST-tagged Cbl, Cbl(1-436), Cbl(437-647), or Cbl(648-end).
Precipitation was carried out by capturing the GST fusion peptides on
glutathione beads. Precipitates were subjected to SDS-PAGE, and blots
were probed with anti-FLAG antibodies to detect bound CMS (upper
panel) and with anti-GST antibodies to monitor the amounts of
captured GST-Cbl peptides (middle panel). Expression levels
of transiently expressed CMS were analyzed in 25 µg of total cell
lysate (lower panel). B, GST fusion peptides
coupled to glutathione-Sepharose beads were incubated with lysates of
293T cell-expressing FLAG-tagged Cbl(437-647), Cbl(648-end), or vector
control. Precipitates were analyzed for c-Cbl interaction with
anti-FLAG antibodies (left panel). Immunoblot analysis of 25 µg of total lysate was carried out for monitoring the expression
levels of the transiently expressed c-Cbl peptides with anti-FLAG
antibodies. C, schematic representation of the GST-tagged
expression constructs used in the binding assay. Amino acid residues of
the boundaries are indicated. TKB, tyrosine kinase binding
domain; RF, RING finger domain; PR, proline-rich region;
LZ, leucine zipper; arrows indicate Tyr Phe
point mutations introduced in the Cbl8F construct. Multiple + signs indicate strong binding to CMS; +/
, weak binding;
, no
binding of full-length CMS to the Cbl peptides indicated in
A.
Alignment of SH3 domain binding motifs located in the carboxy-terminal
half of c-Cbl
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Fig. 6.
CMS colocalizes with the carboxyl-terminal
region of c-Cbl. Randomly growing podocytes, plated on
poly-D-lysine-coated coverslips, were cotransfected with
FLAG-tagged CMS and GST-tagged Cbl or GST-tagged Cbl(648-end). Cells
were fixed and stained with anti-FLAG antibodies for visualizing CMS or
anti-Cbl for monitoring the expression of the Cbl peptides. Membrane
ruffles and vesicles are indicated by arrows. The cells were
inspected with a Nikon Eclipse 800 instrument (× 60).
Ala point mutants
in FLAG-tagged Cbl(648-end). The mutants were introduced at positions
P684A, P706A, P778A/P779A, and P822A, which are underlined in Table I.
We disrupted the PXXP motifs 10-14 individually. These
constructs and the wild-type counterpart were transiently expressed in
293T cells, and total cell lysates were used in a GST pull-down assay
by using the second SH3 domain of CMS, since this domain initially gave
the strongest signal in the GST pull-down assay. However, we did not
see any change in the binding capacity of CMS to the mutated constructs
compared with the wild-type positive control (not shown). Alignment of
SH3-binding motifs comprising the proline-rich region in c-Cbl (aa
347-647) and the carboxyl-terminal peptide sequence of c-Cbl (aa
648-906) are displayed in Table I. This alignment revealed great
similarity in the PXXP-binding motifs in the
carboxyl-terminal fragment. Five out of six partially overlapping
binding motifs contain a positively charged amino acid. It has been
shown that amino acids adjacent to the core consensus binding motif
contribute to the specificity of SH3-PXXP interactions (26,
27). However, this high similarity may explain why the disruption of
the individual SH3-binding motifs in c-Cbl did not abolish the binding
of the second SH3 domain of CMS to c-Cbl. This suggested that the
second SH3 domain of CMS can bind to more than one PXXP
motif in the carboxyl-terminal region of c-Cbl.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice develop congenital nephrotic syndrome due to the impaired
function of specialized epithelial cells, podocytes, in the glomerulus
of the kidney. The dynamic regulation of the actin cytoskeleton seems
to be affected, because of the observed effacement of the actin-rich
foot processes formed by the podocytes. Interestingly, recently c-Cbl
has also been identified as an important regulator of the actin
cytoskeleton (22). Scaife and Langdon (22) have shown that c-Cbl
localizes to lamellipodia and leading edges of cells. Furthermore, they suggested that c-Cbl localization to actin-rich structures requires the
interaction with a SH3 domain-containing molecule. Here we found that
CMS interacts with c-Cbl via SH3 domain-PXXP motif binding,
and CMS might be the link between c-Cbl and F-actin since at least four
putative actin-binding sites are localized in the carboxyl terminus of
CMS (6).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. P. Mundel for providing us with the immortalized podocytes, to Dr. T. Agaki for the pCX vector, and to Dr. J.E. Fajardo for assistance with the manuscript. We also thank P. Kaloudis for assistance with microscopy. We are grateful to Dr. M. Nussenzweig for accommodating KHK in his group.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA44356 and GM55760.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: The Rockefeller University, Box 220, Tel.: 212-327-8068; Fax: 212-327-8370; E-mail: kirschk@mail.rockefeller.edu.
Supported by NCI Fellowship CA09673 from the National
Institutes of Health.
Published, JBC Papers in Press, November 6, 2000, DOI 10.1074/jbc.M005784200
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ABBREVIATIONS |
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The abbreviations used are: SH, Src homology; CD, cluster of differentiation; aa, amino acid residue; EGF, epithelial growth factor; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; HA, hemagglutinin; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.
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