Department of Pathology, Haartman Institute and Helsinki University Central Hospital, University of Helsinki, 00014 Helsinki, Finland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CD2-associated protein (CD2AP) is an adapter protein associating with several membrane proteins, including nephrin, mutated in congenital nephrotic syndrome of the Finnish type, and polycystin-2, mutated in type 2 autosomal dominant polycystic kidney disease. Both proteins have critical roles in the maintenance of the integrity of the nephrons. Previous studies have suggested a role for CD2AP in the regulation of the organization of the actin cytoskeleton, but it has not been known whether the postulated association between CD2AP and actin is direct or mediated by other proteins. In this study, we address this question by using various cellular and biochemical approaches. We show that CD2AP and F-actin partially colocalize in cultured cells and that disruption of the actin cytoskeleton results in disorganization of endogenous CD2AP. Using cytoskeletal fractionation by differential centrifugation, we demonstrate that a proportion of CD2AP associates with the actin cytoskeleton. Furthermore, using pure actin and purified CD2AP fusion proteins in an F-actin coprecipitation assay, we show that CD2AP directly associates with filamentous actin and that this interaction is mediated by means of the COOH terminus of CD2AP. The present results suggest that CD2AP is involved in the regulation of the actin cytoskeleton and indicate that CD2AP may act as a direct adapter between the actin cytoskeleton and cell membrane proteins, such as nephrin and polycystin-2. Alterations in these interactions could explain some of the pathophysiological changes in congenital nephrotic syndrome and polycystic kidney disease.
METS-1; microfilaments; nephrotic syndrome; polycystic kidney disease; SH3 domain
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN THE SEARCH FOR NOVEL developmentally regulated genes in the developing mouse kidney, we used differential hybridization to identify a gene that is strongly upregulated during mesenchyme-to-epithelium transition (13, 18). The encoded protein, originally named METS-1 (mesenchyme-to-epithelium transition protein with multiple SH3 domains) (18), is identical to CD2-associated protein (CD2AP), which has been identified from mouse T cells as a CD2 cell adhesion protein-binding partner (6). The human homolog of CD2AP, Cas ligand with multiple SH3 domains (CMS), was identified as a focal adhesion protein p130Cas-associated protein (16).
CD2AP is an 80-kDa protein that is widely expressed in tissues (6, 16, 19). The protein includes several putative protein-protein interaction domains, suggesting that CD2AP is an adapter molecule. Thus CD2AP contains three SH3 domains in its NH2-terminal region, a proline-rich region in the midregion, and a coiled-coil domain and actin-binding sites in the COOH terminus (6, 16, 18). SH3 domains are conserved protein modules known to interact with proline-rich sequences (21, 24) that are present in a variety of signaling and cytoskeletal proteins. The proline-rich region of CD2AP provides several putative binding sites for SH3 domains, and coiled-coil domains are known to mediate protein-protein interactions.
In accord with its proposed function as an adapter molecule, CD2AP has been shown to have several interaction partners. In the kidney in vivo, CD2AP associates with polycystin-2 (18) and nephrin (21a, 27, 28), which are essential for maintaining the integrity of the nephrons (15, 20). In T cells, CD2AP binds the adhesion molecule CD2, enhancing its clustering at the T cell-antigen-presenting cell contact area, thereby affecting the cytoskeletal polarity of the cells (6). In addition, CD2AP interacts with the protoncogene product c-Cbl (17), which is involved in tyrosine kinase signaling and regulation of lamellipodia formation and cell morphology (26). Collectively, CD2AP may thus act as a scaffolding protein in various signaling cascades controlling cellular adhesion, motility, and morphology, all processes depending on the actin cytoskeleton.
Interestingly, mice lacking CD2AP develop nephrotic syndrome resembling
the human disease. These mice show effacement of the podocyte foot
processes and accumulation of mesangial deposits (28).
Podocyte foot process effacement and the subsequent development of
proteinuria is characterized by disaggregation and redistribution of
podocyte actin microfilaments. This suggests an important role for
actin and/or the associated proteins in the maintenance of the foot
process organization (29). In line with this, mutations in
several actin-associated proteins have been shown to lead to glomerular
disease. Thus mutations in the ACTN4 gene, encoding -actinin-4,
cause familial focal segmental glomerulosclerosis (14),
and mutations in the podocin gene NPHS2 lead to autosomal recessive
steroid-resistant nephrotic syndrome (3).
Immunofluorescence studies on cultured podocytes showed that CD2AP
colocalizes with F-actin at the leading edge of lamellipodia and in
small spots, which were unevenly distributed in the cytoplasm. The
spot-shaped F-actin structures were also positive for the Arp2/3
protein complex and cortactin (36). The Arp2/3 complex has
been shown to be activated by cortactin and to be involved in the
regulation of actin assembly (32, 35). In addition,
transient transfection of COS-7 cells with CMS, the human homolog of
CD2AP, has been shown to decrease actin fiber formation and lead to
concentration of actin into small dots (16). CD2AP may
thus have a role in the regulation of the actin cytoskeleton, and the
effacement of the podocyte foot processes in CD2AP-deficient mice could
result from dysregulation of the podocyte actin cytoskeleton organization.
The above studies on CD2AP and actin cytoskeleton organization rely on immunohistochemistry and do not reveal whether the postulated association between CD2AP and actin is direct or involves mediator proteins. In the present investigation, we have analyzed the association of F-actin and CD2AP at both the cellular and the biochemical levels. Our results indicate that the intracellular localization of CD2AP depends on an intact actin cytoskeleton and that CD2AP associates with the actin cytoskeleton. Most importantly, by direct coprecipitation assays utilizing pure polymerized actin and purified CD2AP fusion proteins, we show, for the first time, that the COOH terminus of CD2AP interacts directly with F-actin. These results suggest that CD2AP functions as a scaffolding protein connecting the actin cytoskeleton to plasma membrane proteins, such as CD2, nephrin, and polycystin-2.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture. Mouse M-1 kidney cortical collecting duct epithelial cells (CRL-2038, American Type Culture Collection) were cultured in a 1:1 mixture of Ham's F-12 medium/Dulbecco's modified Eagle's medium containing 5 µM dexamethasone and supplemented with 5% fetal calf serum.
Production of CD2AP antisera. The rabbit antiserum (R1774) generated against amino acid residues 6-574 of CD2AP has been described (18). An independent rabbit antiserum (R211) was raised against a glutathione S-transferase (GST)-tagged fusion protein carrying amino acid residues 1-330 of CD2AP. The fusion protein was produced in Escherichia coli and purified as recommended by the manufacturer (Amersham Pharmacia Biotech). To show the specificity of the antibody reactivity, the antisera were diluted to working concentration and preincubated with the corresponding purified recombinant protein at 1 or 10 µg/ml at room temperature for 30 min, followed by Western blotting and immunofluorescence microscopy, respectively; this totally competed the signal (Lehtonen S, unpublished observations). Affinity-purified antibodies were obtained by incubating the antisera with the corresponding fusion protein immobilized on filter, followed by thorough washes. The antibodies were eluted using 0.2 M glycine-HCl, pH 2.9, and neutralized with unbuffered 1 M Tris. The affinity-purified antiserum R211 was used for immunoelectron microscopy; all other analyses were performed with antiserum R1774. The results with affinity-purified antibodies and antisera were the same.
Immunocytochemistry.
Cells grown on glass coverslips were fixed in 3.5% paraformaldehyde in
PBS for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 15 min or with 20°C methanol for 10 min. Oregon green 514 phalloidin
and rhodamine phalloidin for detection of F-actin were purchased from
Molecular Probes, and the mouse monoclonal antibodies against actin and
paxillin were from Amersham Pharmacia Biotech and Zymed, respectively.
The mouse monoclonal antibodies against E-cadherin and protein
disulfide isomerase (PDI) were from Transduction Laboratories and
StressGen, respectively. Primary antibodies and the
FITC/TRITC-conjugated secondary antibodies (Jackson ImmunoResearch
Laboratories) were diluted in PBS supplemented with 0.5% saponin and
5% fetal calf serum. Microscopy was performed with a Zeiss Axiophot 2 microscope and a Leica TCSSP1 confocal microscope.
Immunoelectron microscopy. Nickel grids placed on coverslips were covered with Formvar and coated with carbon. The grid assemblies were dipped into 1% poly-L-lysine (molecular weight 114,700; Sigma) and rinsed with PBS, followed by incubation in a moist chamber overnight to make the surface wettable. The assemblies were sterilized with ethanol and rinsed with PBS and culture medium. M-1 cells were plated on the grid assemblies at a density allowing them to reach 70% confluency in 3 days to ensure proper attachment and spreading on the grids. The cells were rinsed with 0.1 mM PIPES, pH 6.9, 1 mM EGTA, 4% polyethylene glycol 8000 [microfilament stabilizing buffer (MSB)], followed by incubation with 1 mM short-arm crosslinker dithiobis (succinimidyl propionate) (Pierce) in MSB. Cells were extracted with 0.2% Triton X-100, 1 mM dithiobis (succinimidyl propionate) in MSB before fixation with 3.5% paraformaldehyde in PBS (2). Filamentous actin was stabilized by 20 µM phalloidin (Molecular Probes) in PBS, and the cells were stained with affinity-purified rabbit polyclonal CD2AP antibodies, a rabbit preimmune serum, and mouse monoclonal anti-actin antibodies (Sigma). Cells were first washed with PBS, followed by a rinse in 20 mM Tris · HCl, pH 7.5, 5 mM NaCl, and 0.01% gelatin [Tris-buffered saline (TBS) buffer]. Colloidal gold-conjugated secondary antibodies (5-nm gold anti-rabbit and 10-nm gold anti-mouse, Sigma) were diluted in TBS. Cells were washed with TBS and postfixed with 2.5% glutaraldehyde, followed by washing with H2O and dehydration in ascending concentrations of acetone. The samples were critical point-dried in a CPD-30 apparatus (Balzers) and observed under a JEOL JEM 1200EX electron microscope.
Cytoskeleton disrupting treatments. Mouse M-1 kidney cells growing on glass coverslips were treated with 2 µM cytochalasin D (Sigma) or 3 µM jasplakinolide (Calbiochem) in culture medium at 37°C for 1 h and thereafter processed for immunocytochemistry. In some experiments, the cytochalasin D-treated cells were allowed to recover in the maintenance medium for 1 h before fixation.
Cytoskeleton preparations of cells growing on coverslips. M-1 cells cultured on coverslips were washed twice with PBS and incubated in 4 M glycerol, 25 mM PIPES, 1 mM EGTA, 1 mM MgCl2, and 0.2% Triton X-100, pH 6.9, at room temperature for 5 min (1). Immunocytochemistry was performed as above.
Preparation of cytoskeletal fractions.
Mouse M-1 kidney cells were grown to 90% confluency, washed with cold
PBS, and lysed in 150 µl of precooled (in mM) 100 Tris · HCl,
pH 7.6, 100 NaCl, 10 EGTA, 1 MgCl2, 1 PMSF, 10 NaF, 2 Na3VO4, 10 -glycerophosphate, and 0.2 ATP
(lysis buffer), as well as 2% Triton X-100, 5 µg/ml aprotinin, and 5 µg/ml leupeptin (7) on ice. In some experiments, 2 mg/ml
DNase I (preincubated with 1 mM PMSF on ice for 15 min) were added to
the lysis buffer to depolymerize filamentous actin (7).
After shaking in an ice bath for 15 min, the crude cytoskeleton
fraction was prepared by centrifuging at 10,000 g at 4°C
for 10 min. The supernatant was further centrifuged in a Beckman
Airfuge at 100,000 g at 4°C for 3 h to separate the
membrane skeleton and soluble fractions. The pellets were washed three
times and resuspended in lysis buffer, raising their volumes to
correspond to the volume of the soluble fraction. Equal volumes of the
crude cytoskeleton, membrane skeleton, and soluble fractions were
resolved by SDS-PAGE and Western blotted with antibodies against actin
(Amersham Pharmacia Biotech) or CD2AP.
Actin filament precipitation assays. Truncated forms of CD2AP encompassing the NH2- and COOH-terminal domains of the protein (amino acids 1-330 and 331-637, respectively) were subcloned into pGEX-4T-1 vector (Amersham Pharmacia Biotech) to produce GST-tagged fusion proteins. The fusion proteins were produced in E. coli and purified as recommended by the manufacturer (Amersham Pharmacia Biotech). Rabbit skeletal muscle actin (Cytoskeleton) was diluted in G buffer [(in mM) 5 Tris · HCl, pH 8.0, 0.2 CaCl2, 0.5 dithiothreitol, and 0.2 ATP] to produce a final concentration of 3 µM in the 50 µl polymerization reaction volume. Actin was polymerized by adding 5 µl of 10× actin polymerization initiation buffer [10× buffer (in mM) 500 KCl, 20 MgCl2, and 10 ATP] followed by incubation at room temperature for 30 min. The CD2AP GST fusion proteins, or GST as a control, were diluted in G buffer to produce a final concentration of 1 µM in the 50 µl polymerization volume. The concentration of the intact COOH-terminal fusion protein was estimated from Coomassie blue-stained gels. The fusion proteins were combined with polymerized actin and incubated at room temperature for 30 min. The samples were centrifuged in a Beckman TL100 ultracentrifuge at 100,000 g at 4°C for 1 h. The pellets were resuspended in SDS sample buffer, and equal amounts of the supernatant and pellet fractions were resolved by SDS-PAGE. The Coomassie blue-stained gels were scanned, and the intensities of actin and the fusion protein bands were quantified by using the NIH Image 1.62 program.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Distribution of endogenous CD2AP in cultured cells.
The sequence of CD2AP predicts a cytosolic protein with no apparent
membrane spanning regions (6, 16, 18). Accordingly, the
predominant distribution pattern of CD2AP in various cultured cells is
cytosolic, concentrating around the nucleus in a fine, reticular
pattern (Fig. 1, A-D).
Double staining for CD2AP and PDI revealed colocalization of
the two proteins, suggesting that cytosolic CD2AP localizes to
endoplasmic reticulum (data not shown). In addition, accumulation of
CD2AP to certain subcellular locations could be seen. In sparsely
plated, well-spread cells, CD2AP accumulations were seen along the
leading edge formations (Fig. 1A) colocalizing with F-actin
(Fig. 1, D-F). Accumulations of CD2AP were also
detected in intercellular contact regions (Fig. 1B) and in
plaque-like structures often localizing close to cell-cell contacts
(Fig. 1C). These plaque-like accumulations of CD2AP were
found in solitary cells, subconfluent cells, and confluent
cells. The distribution of these structures differed from that
of paxillin-positive focal adhesion plaques, although double staining
for CD2AP and paxillin revealed partial overlap of the specific signals
on rare occasions (Fig. 1, G-I). In subconfluent cells,
paxillin-positive focal adhesions were frequently seen in the leading
edge regions but rarely in cell-cell contact regions (data not shown).
The latter, however, regularly showed concentrations of CD2AP (Fig. 1,
A-C).
|
|
Redistribution of CD2AP after cytochalasin D and jasplakinolide
treatments.
The NH2 terminus of CD2AP contains three SH3 domains
(6, 16, 18). Many SH3 domain proteins are known to
associate with cytoskeletal components and to direct the proteins to
specific subcellular regions (1). The COOH terminus of
CD2AP contains sequences implicated in binding actin (33,
34). To investigate whether CD2AP associates with the actin
cytoskeleton, M-1 cells growing on coverslips were subjected to
cytochalasin D and jasplakinolide treatments (Fig.
3, A-I). Double staining
of nontreated cells with CD2AP antibodies and phalloidin revealed
cytosolic distribution of CD2AP, including perinuclear accumulation,
but also a distinct signal in the cell periphery rich in F-actin (Fig.
3, A-C). Cytochalasin D, which disrupts the actin
cytoskeleton, had a dramatic effect on CD2AP organization. In treated
cells, CD2AP appeared as aggregates largely colocalizing with the
collapsed actin along the cell periphery (Fig. 3, D-F).
On recovery in the maintenance medium, the cytoplasmic and filamentous
staining patterns, respectively, of CD2AP and F-actin were restored
(not shown). Jasplakinolide stabilizes F-actin and induces actin
polymerization (4). Jasplakinolide treatment caused a
redistribution of CD2AP into large aggregates, partially colocalizing
with masses of actin, which was visualized using anti-actin antibodies
(Fig. 3, G-I). The results indicate that the
organization of CD2AP depends on intact actin cytoskeleton.
|
CD2AP is partially retained in detergent-extracted cells. To further investigate the association between CD2AP and the actin cytoskeleton, we used detergent extraction to produce cytoskeleton preparations of adherent M-1 cells for immunofluorescence microscopy. In these preparations, proteins not connected to the cytoskeleton are extracted. In the detergent-extracted M-1 cells, most of the CD2AP-specific fluorescence was lost (Fig. 3J). However, some CD2AP was retained along the cell periphery in the cell contact regions and as diffusely distributed small granular structures (Fig. 3J). The extraction did not alter the organization of the actin cytoskeleton (Fig. 3K). The cytoskeleton preparations exhibited regions positive for both F-actin and CD2AP, but also regions showing either F-actin or CD2AP alone (Fig. 3, J and K). The cells also showed distinct focal adhesion plaques positive for paxillin (not shown). The results indicate that a fraction of CD2AP is resistant to detergent extraction, suggesting that CD2AP may associate with the actin cytoskeleton.
CD2AP and actin colocalize in cortical actin cytoskeleton.
To further confirm the close association of CD2AP and actin,
detergent-extracted whole-mount cells were studied by immunoelectron microscopy. Actin and actin-associated proteins were stabilized by a
cross-linker before detergent extraction, and after the extraction, actin was further stabilized by treatment with phalloidin. As a result,
the actin cytoskeleton was well preserved, and the motile structures of
the cell, such as lamellipodia, leading edges, membrane ruffles, and
microspikes, were clearly discernible (Fig.
4A). In immunoelectron
microscopy, distinct CD2AP-specific staining could be detected in the
cell periphery, especially in leading edges (Fig. 4B). The
CD2AP-specific label often associated with the cortical cytoskeleton
focally as small patches of several gold particles (Fig. 4,
B and D), a pattern consistent with the granular
staining observed by immunofluorescence microscopy (Fig. 3J). Controls using rabbit preimmune serum showed negligible
labeling (Fig. 4C). After double staining for CD2AP and
actin, colabeling of the same structures was seen, suggesting a close
association of the two proteins (Fig. 4D).
|
A fraction of CD2AP pellets with membrane skeleton.
Highly cross-linked actin filaments, representing the majority of
filamentous actin, are pelleted at low-force centrifugation, and free
or loosely cross-linked actin filaments, representing the submembranous
actin filaments, require high-speed centrifugation for sedimentation
(7, 30). To analyze the partition of CD2AP in these two
cytoskeletal structures, we prepared M-1 cell extracts and analyzed
equal volumes of the 10,000 g crude cytoskeleton fraction,
the 100,000-g membrane skeleton fraction, and the remaining Triton X-100-soluble fraction by Western blotting (Fig.
5, lanes marked DNase I). Most of the
CD2AP protein was found in the Triton X-100-soluble fraction [100,000
soluble (S), DNase I
]. Of the cytoskeleton-associated CD2AP, most
was found in the high-speed membrane skeleton fraction [100,000 pellet
(P), DNase I
] and a negligible part in the crude cytoskeleton
fraction (10,000 P, DNase I
). As expected, most of the actin pelleted
in the crude cytoskeleton fraction (10,000 P, DNase I
).
|
The COOH terminus of CD2AP directly associates with F-actin.
To investigate whether CD2AP and F-actin interact directly, we
performed an F-actin precipitation assay using COOH- and
NH2-terminal GST-fusion proteins of CD2AP. For this, pure,
polymerized actin was mixed with the purified CD2AP fusion proteins.
The samples were then centrifuged to pellet filamentous actin and the
possible bound fusion proteins. Analysis of equal amounts of the
soluble and pelleted fractions revealed that under the conditions used, about one-half of the COOH-terminal fusion protein pelleted in the
presence of F-actin (Fig. 6A,
lanes marked actin+). In the absence of actin, the COOH-terminal fusion
protein pelleted at a negligible level, suggesting that it does not
precipitate by itself (Fig. 6A, lanes marked actin).
Densitometric analysis showed that ~60% of the COOH-terminal CD2AP
used in the assay cosedimented with F-actin (Fig. 6B,
C+actin). In contrast, the NH2-terminal fusion protein
pelleted at a similar level in the absence (Fig. 6C, lanes
marked actin
) and presence (Fig. 6C, lanes marked actin+)
of F-actin. This suggests that the NH2 terminus of CD2AP
does not bind actin but that a part of the protein precipitates by
itself. GST alone did not pellet in the absence (Fig. 6D,
lanes marked actin
) or presence (Fig. 6D, lanes marked
actin+) of F-actin.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we show that endogenous CD2AP and F-actin partially colocalize in cultured epithelial M-1 cells and that disruption of the actin cytoskeleton results in disorganization of CD2AP. Using cytoskeletal fractionation combined with actin depolymerization, we show that a fraction of CD2AP associates with the membrane skeleton. Finally, using an F-actin coprecipitation assay, we show that the COOH terminus of CD2AP directly binds F-actin.
Our immunofluorescence and immunoelectron microscopy analysis showed that the distribution of CD2AP is related to the locomotory status of the cells. The prevailing localization of CD2AP was cytosolic, colocalizing with a marker (PDI) for endoplasmic reticulum. However, in motile cells, CD2AP accumulations were also found along the leading edge, colocalizing with F-actin, and in plaque-like structures, which were distinct from paxillin-positive focal adhesion plaques. CD2AP has previously been shown to associate with the protoncogene product c-Cbl (17), which is involved in tyrosine kinase signaling and regulation of lamellipodia formation and cell morphology (26). Localization of CD2AP in the leading edge of lamellipodia (this study and Refs. 16 and 36) suggests that CD2AP might also have a role in these processes. Furthermore, targeting of c-Cbl to the actin cytoskeleton of NIH3T3 fibroblasts has been shown to require interaction with an actin-associated SH3 domain-containing protein (26), such as CD2AP. CD2AP could thus act as a scaffolding protein in specific signaling cascades controlling cellular motility and morphology.
The above immunofluorescence colocalization studies suggest a close association between CD2AP and a fraction of cellular F-actin. In the present study, we also obtained findings suggesting that CD2AP is associated with a distinct cytoskeletal fraction, the membrane skeleton. First, double staining of detergent-extracted cells revealed that although the majority of CD2AP was extracted from these cytoskeleton preparations, a distinct fraction of the protein was retained along the cell periphery, partially overlapping with F-actin in the vicinity of the intercellular contacts. Second, cytoskeletal fractionation confirmed that the majority of CD2AP is soluble but that a fraction of the protein pellets with the loosely cross-linked actin filaments or the membrane skeleton. The crude cytoskeleton fraction, which includes highly cross-linked actin filaments, representing the majority of filamentous actin, contained scarcely any CD2AP.
Our results from actin filament coprecipitation assay, using pure actin and purified CD2AP fusion proteins, demonstrate that the association between CD2AP and F-actin is direct and does not depend on mediator proteins. Furthermore, the results indicate that the interaction between CD2AP and F-actin is mediated by the COOH terminus of CD2AP. These results agree with the observation that the formation of the specialized cell contact between the T cell and the antigen presenting cell, a process involving adhesion molecules and the actin cytoskeleton (9), is disturbed by overexpression of a CD2AP construct lacking the COOH terminus (6).
The studies reported here provide strong evidence that CD2AP may link
the actin cytoskeleton to nephrin, earlier shown to interact with CD2AP
(21a, 27, 28). Nephrin has been suggested to form the
framework of the slit diaphragm, the ultrafiltration unit of the
glomerulus (11, 12, 25). In congenital nephrotic syndrome
of the Finnish type (NPHS1 or CNF) (10), nephrin is mutated (15), leading to loss of slit diaphragms and
effacement of the podocyte foot processes (15, 23). As
CD2AP /
mice develop nephrotic syndrome resembling the human
disease, CD2AP apparently has a role in maintaining the integrity of
the slit diaphragm, supposedly as a protein anchoring nephrin to the
cytoskeleton (28). Our present results support this
proposal and show that CD2AP may act as a direct adapter between
nephrin and the actin cytoskeleton. In line with this, while the
present manuscript was under review, it was demonstrated that nephrin
anchors the slit diaphragm to the actin cytoskeleton, possibly by
linkage to CD2AP (37). Collectively, the data suggest that
disturbances in the protein complex, including nephrin, CD2AP, and
actin, could account for the alterations observed on foot process
effacement in CNF and in CD2AP
/
mice.
Our present and earlier (18) findings suggest that CD2AP may have an important role in the signaling cascade involved in the pathogenesis of the type 2 autosomal dominant polycystic kidney disease (ADPKD2) phenotype. In polycystic kidney disease, the kidney tubular cells lose their differentiated function and morphology, and the kidney develops cystic structures lined by flattened epithelial cells, a process apparently involving changes in cytoskeletal organization (5). We have shown earlier that CD2AP interacts in vivo with polycystin-2 (18), mutated in ADPKD2 (20). Mutations in ADPKD2 typically result in the production of truncated forms of polycystin-2 (see Ref. 31), which might be unable to associate with CD2AP, because the interaction involves the COOH-terminal domain of polycystin-2 (18). The data would thus be compatible with a molecular mechanism, in which a mutation in ADPKD2 results in loss of connection, mediated by CD2AP, between polycystin-2 and the actin cytoskeleton. This would then lead to the development of the morphological and pathophysiological changes observed in ADPKD2. The significance of intact cell membrane-actin cytoskeleton interactions for protein complexes containing polycystin-2 is further emphasized by the observation that cytochalasin D disrupts polycystin-1/polycystin-2-containing macromolecular complexes (8). In line with the proposed role of CD2AP in the polycystin-2 protein complex, we also show here that CD2AP partially colocalizes with E-cadherin, a component of this complex (8).
In conclusion, the present findings demonstrate a direct association between CD2AP and the actin cytoskeleton and indicate that CD2AP may act as a direct adapter between the actin cytoskeleton and cell membane proteins, such as nephrin and polycystin-2. Both nephrin and polycystin-2 have been shown to interact with CD2AP in vivo, thus associating CD2AP with CNF and ADPKD2, respectively. Collectively, the data suggest that disturbances in CD2AP-mediated interactions between membrane protein complexes and the actin cytoskeleton may have an important role in the pathogenesis of the changes characteristic to congenital nephrotic syndrome and polycystic kidney disease.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank U. Kiiski for technical assistance and M. G. Farquhar, P. Lappalainen, V.-P. Lehto, V. M. Olkkonen, and I. Virtanen for a critical reading of the manuscript. P. Salmikangas is acknowledged for her advice concerning the F-actin cosedimentation assay.
![]() |
FOOTNOTES |
---|
The work was supported by Academy of Finland Grants 68290 and 71234 (E. Lehtonen), the Emil Aaltonen Foundation (S. Lehtonen), the Wihuri Foundation (E. Lehtonen), the Paulo Foundation (S. Lehtonen), the Finnish Cultural Foundation (S. Lehtonen), the Sigrid Jusélius Foundation (F. Zhao), and the Clinical Research Fund of Helsinki University Central Hospital, Finland (E. Lehtonen).
Address for reprint requests and other correspondence: E. Lehtonen, Dept. of Cellular and Molecular Medicine, Univ. of California, San Diego, Dept. 0651, 9500 Gilman Dr., La Jolla, CA 92093-0651 (E-mail: elehtonen{at}ucsd.edu or eero.lehtonen{at}helsinki.fi).
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.
May 7, 2002;10.1152/ajprenal.00312.2001
Received 10 October 2001; accepted in final form 14 April 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bar-Sagi, D,
Rotin D,
Batzer A,
Mandiyan V,
and
Schlessinger J.
SH3 domains direct cellular localization of signaling molecules.
Cell
74:
83-91,
1993[ISI][Medline].
2.
Bell, PB, Jr,
and
Safiejko-Mroczka B.
Improved methods for preserving macromolecular structures and visualizing them by fluorescence and scanning electron microscopy.
Scanning Microsc
9:
843-860,
1995[ISI][Medline].
3.
Boute, N,
Gribouval O,
Roselli S,
Benessy F,
Lee H,
Fuchshuber A,
Dahan K,
Gubler MC,
Niaudet P,
and
Antignac C.
NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome.
Nat Genet
24:
349-354,
2000[ISI][Medline].
4.
Bubb, MR,
Senderowicz AM,
Sausville EA,
Duncan KL,
and
Korn ED.
Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin.
J Biol Chem
269:
14869-14871,
1994
5.
Calvet, JP.
Polycystic kidney disease: primary extracellular matrix abnormality or defective cellular differentiation?
Kidney Int
43:
101-108,
1993[ISI][Medline].
6.
Dustin, ML,
Olszowy MW,
Holdorf AD,
Li J,
Bromley S,
Desai N,
Widder P,
Rosenberger F,
van der Merwe PA,
Allen PM,
and
Shaw AS.
A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts.
Cell
94:
667-677,
1998[ISI][Medline].
7.
Fox, JE.
Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib.
J Clin Invest
76:
1673-1683,
1985[ISI][Medline].
8.
Geng, L,
Burrow CR,
Li HP,
and
Wilson PD.
Modification of the composition of polycystin-1 multiprotein complexes by calcium and tyrosine phosphorylation.
Biochim Biophys Acta
1535:
21-35,
2000[ISI][Medline].
9.
Grakoui, A,
Bromley SK,
Sumen C,
Davis MM,
Shaw AS,
Allen PM,
and
Dustin ML.
The immunological synapse: a molecular machine controlling T cell activation.
Science
285:
221-227,
1999
10.
Hallman, N,
Hjelt L,
and
Ahvenainen EK.
Nephrotic syndrome in newborn and young infants.
Ann Pediatr Fenn
2:
227-241,
1956.
11.
Holthöfer, H,
Ahola H,
Solin ML,
Wang S,
Palmen T,
Luimula P,
Miettinen A,
and
Kerjaschki D.
Nephrin localizes at the podocyte filtration slit area and is characteristically spliced in the human kidney.
Am J Pathol
155:
1681-1687,
1999
12.
Holzman, LB,
John PL,
Kovari IA,
Verma R,
Holthofer H,
and
Abrahamson DR.
Nephrin localizes to the slit pore of the glomerular epithelial cells.
Kidney Int
56:
1481-1491,
1999[ISI][Medline].
13.
Jansson, S,
Olkkonen V,
Martin-Parras L,
Chavrier P,
Stapleton M,
Zerial M,
and
Lehtonen E.
Mouse metanephric kidney as a model system for identifying developmentally regulated genes.
J Cell Physiol
173:
147-151,
1997[ISI][Medline].
14.
Kaplan, JM,
Kim SH,
North KN,
Rennke H,
Correia LA,
Tong HQ,
Mathis BJ,
Rodríguez-Pérez JC,
Allen PG,
Beggs AH,
and
Pollak MR.
Mutations in ACTN4, encoding -actinin-4, cause familial focal segmental glomerulosclerosis.
Nat Genet
24:
251-256,
2000[ISI][Medline].
15.
Kestilä, M,
Lenkkeri U,
Männikkö M,
Lamerdin J,
McCready P,
Putaala H,
Ruotsalainen V,
Morita T,
Nissinen M,
Herva R,
Kashtan CE,
Peltonen L,
Holmberg C,
Olsen A,
and
Tryggvason K.
Positionally cloned gene for a novel glomerular proteinnephrin
is mutated in congenital nephrotic syndrome.
Mol Cell
1:
575-582,
1998[ISI][Medline].
16.
Kirsch, KH,
Georgescu MM,
Ishimaru S,
and
Hanafusa H.
CMS: an adapter molecule involved in cytoskeletal rearrangements.
Proc Natl Acad Sci USA
96:
6211-6216,
1999
17.
Kirsch, KH,
Georgescu MM,
Shishido T,
Langdon WY,
Birge RB,
and
Hanafusa H.
The adapter type protein CMS/CD2AP binds to the proto-oncogenic protein c-Cbl through a tyrosine phosphorylation-regulated src homology 3 domain interaction.
J Biol Chem
276:
4957-4963,
2001
18.
Lehtonen, S,
Ora A,
Olkkonen VM,
Geng L,
Zerial M,
Somlo S,
and
Lehtonen E.
In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2.
J Biol Chem
275:
32888-32893,
2000
19.
Li, G,
Ruotsalainen V,
Tryggvason K,
Shaw AS,
and
Miner JH.
CD2AP is expressed with nephrin in developing podocytes and is found widely in mature kidney and elsewhere.
Am J Physiol Renal Physiol
279:
F785-F792,
2000
20.
Mochizuki, T,
Wu G,
Hayashi T,
Xenophontos SL,
Veldhuisen B,
Saris JJ,
Reynolds DM,
Cai Y,
Gabow PA,
Pierides A,
Kimberling WJ,
Breuning MH,
Deltas CC,
Peters DJM,
and
Somlo S.
PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.
Science
272:
1339-1342,
1996[Abstract].
21.
Musacchio, A,
Gibson T,
Lehto VP,
and
Saraste M.
SH3an abundant protein domain in search of a function.
FEBS Lett
307:
55-61,
1992[ISI][Medline].
21a.
Palmén, T,
Lehtonen S,
Ora A,
Kerjaschki D,
Antignac C,
Lehtonen E,
and
Holthofer H.
Interaction of endogenous nephrin and CD2-associated protein in mouse epithelial M-1 cell line.
J Am Soc Nephrol
13:
1766-1772,
2002
23.
Patrakka, J,
Kestilä M,
Wartiovaara J,
Ruotsalainen V,
Tissari P,
Lenkkeri U,
Mannikko M,
Visapaa I,
Holmberg C,
Rapola J,
Tryggvason K,
and
Jalanko H.
Congenital nephrotic syndrome (NPHS1): features resulting from different mutations in Finnish patients.
Kidney Int
58:
972-980,
2000[ISI][Medline].
24.
Ren, R,
Mayer B,
Cicchetti P,
and
Baltimore D.
Identification of a ten-amino acid proline-rich SH3 binding site.
Science
259:
1157-1161,
1993[ISI][Medline].
25.
Ruotsalainen, V,
Ljungberg P,
Wartiovaara J,
Lenkkeri U,
Kestilä M,
Jalanko H,
Holmberg C,
and
Tryggvason K.
Nephrin is specifically located at the slit diaphragm of glomerular podocytes.
Proc Natl Acad Sci USA
96:
7962-7967,
1999
26.
Scaife, RM,
and
Langdon WY.
c-Cbl localizes to actin lamellae and regulates lamellibodia formation and cell morphology.
J Cell Sci
113:
215-226,
2000
27.
Shih, NY,
Li J,
Cotran R,
Mundel P,
Miner JH,
and
Shaw AS.
CD2AP localizes to the slit diaphragm and binds to nephrin via a novel C-terminal domain.
Am J Pathol
159:
2303-2308,
2002
28.
Shih, NY,
Li J,
Karpitskii V,
Nguyen A,
Dustin ML,
Kanagawa O,
Miner JH,
and
Shaw AS.
Congenital nephrotic syndrome in mice lacking CD2-associated protein.
Science
286:
312-315,
1999
29.
Smoyer, WE,
and
Mundel P.
Regulation of podocyte structure during the development of nephrotic syndrome.
J Mol Med
76:
172-183,
1998[ISI][Medline].
30.
Tohyama, Y,
Yanagi S,
Sada K,
and
Yamamura H.
Translocation of p72syk to the cytoskeleton in thrombin-stimulated platelets.
J Biol Chem
269:
32796-32799,
1994
31.
Torra, R,
Badenas C,
San Millán JL,
Pérez-Oller L,
Estivill X,
and
Darnell A.
A loss-of-function model for cystogenesis in human autosomal dominant polycystic kidney disease type 2.
Am J Hum Genet
65:
345-352,
1999[ISI][Medline].
32.
Uruno, T,
Liu J,
Zhang P,
Fan Y,
Egile C,
Li R,
Mueller SC,
and
Zhan X.
Activation of Arp2/3 complex-mediated actin polymerization by cortactin.
Nature Cell Biol
3:
259-266,
2001[ISI][Medline].
33.
Vandekerckhove, J,
and
Vancompernolle K.
Structural relationships of actin-binding proteins.
Curr Opin Cell Biol
4:
36-42,
1992[Medline].
34.
Van Troys, M,
Dewitte D,
Goethals M,
Carlier MF,
Vandekerckhove J,
and
Ampe C.
The actin binding site of thymosin beta 4 mapped by mutational analysis.
EMBO J
15:
201-210,
1996[Abstract].
35.
Weed, SA,
Karginov AV,
Shafer DA,
Weaver AM,
Kinley AW,
Cooper JA,
and
Parsons JT.
Cortactin localization to sites of actin assembly in lamellibodia requires interactions with F-actin and the Arp2/3 complex.
J Cell Biol
151:
29-40,
2000
36.
Welsch, T,
Endlich N,
Kriz W,
and
Endlich K.
CD2AP and p130Cas localize to different F-actin structures in podocytes.
Am J Physiol Renal Physiol
281:
F769-F777,
2001
37.
Yuan, H,
Takeuchi E,
and
Salant D.
Podocyte slit-diaphragm protein nephrin is linked to the actin cytoskeleton.
Am J Physiol Renal Physiol
282:
F585-F591,
2002