THE JOURNAL OF BIOLOGICAL CHEMISTRY

Accelerated Publication Vol. 276, No. 45, Issue of November 9, pp. 41543–41546, 2001
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.

Interaction with Podocin The steroid-resistant nephrotic syndrome caused by muta-

tions of NPHS2, the gene encoding for podocin, follows a milder
Facilitates Nephrin Signaling* clinical course. Proteinuria often starts several months after
birth but can be delayed for several years (5, 6). NPHS2 en-
Received for publication, August 13, 2001, codes for podocin, a protein exclusively expressed in the podo-
and in revised form, September 18, 2001 cytes of the developing and mature glomeruli. Podocin is a
Published, JBC Papers in Press, September 18, 2001,
member of the stomatin protein family with a short N-terminal
DOI 10.1074/jbc.C100452200
domain, a transmembrane region, and a cytosolic C-terminal
Tobias B. Huber, Michael Köttgen, Birgit domain. Podocin is predicted to form a membrane-associated
Schilling, Gerd Walz‡, and Thomas Benzing hairpin-like structure with a cytosolic N- and C-terminal do-
From the Renal Division, University Hospital Freiburg, main that is typical for stomatin-like proteins and caveolins.
Freiburg 79106, Germany CD2-associated protein (CD2AP) is an adapter molecule that
Mutations of NPHS1 or NPHS2, the genes encoding was first identified as an SH3-containing protein that binds to
for the glomerular podocyte proteins nephrin and podo- the cytoplasmic domain of CD2 (7). Surprisingly, mice lacking
cin, cause steroid-resistant proteinuria. In addition, CD2AP exhibit a nephrotic syndrome that resembles NPHS1
mice lacking CD2-associated protein (CD2AP) develop a mutations (8). Because CD2AP appears to interact with the
nephrotic syndrome that resembles NPHS mutations cytoplasmic tail of nephrin, these findings suggested that
suggesting that all three proteins are essential for the CD2AP and nephrin participate in a common signaling path-
integrity of glomerular podocytes. Although the precise way necessary to maintain crucial podocyte functions.
glomerular function of either protein remains unknown, Using human embryonic kidney (HEK) 293T cells, a cell line
it has been suggested that nephrin forms zipper-like that lacks nephrin, podocin, and CD2AP, we demonstrated that
interactions to maintain the structure of podocyte foot nephrin is a signaling molecule. Expression of nephrin in HEK
processes. We demonstrate now that nephrin is a signal- 293T cells triggers cellular activation that involves stimulation
ing molecule, which stimulates mitogen-activated pro- of the stress-activated protein kinase p38 and the c-Jun N-
tein kinases. Nephrin-induced signaling is greatly en- terminal kinase (JNK). Cellular activation is greatly enhanced
hanced by podocin, which binds to the cytoplasmic tail
by podocin, which binds to the C-terminal cytoplasmic domain
of nephrin. Mutational analysis suggests that abnormal
of nephrin but fails to bind and augment nephrin R1160X, a
or inefficient signaling through the nephrin-podocin
nephrin mutation truncated by a nonsense mutation at Arg1160
complex contributes to the development of podocyte
dysfunction and proteinuria. (9). Our findings suggest that nephrin and podocin form a
signaling complex that supports the functional and structural
integrity of glomerular podocytes.
Hereditary nephrotic syndrome is a heterogeneous disease, MATERIALS AND METHODS
characterized by heavy proteinuria and renal failure. The most Cloning of Mouse Podocin—The human podocin cDNA sequence was
severe disorder is the congenital nephrotic syndrome of the used to search for mouse EST clones containing podocin nucleotide se-
quence (standard nucleotide BLAST, www.ncbi.nlm.nih.gov/BLAST/).
Finnish type, caused by mutations in NPHS1,1 the gene encod-
Clone AW106985 was identified to contain the complete coding region of
ing for nephrin. The disease manifests itself with massive mouse podocin (GenBankTM accession number AY050309).
proteinuria in utero and nephrosis at birth (1). The nephrotic Plasmids—To generate C-terminally FLAG-tagged full-length hu-
syndrome usually leads to death during the first 2 years of life man nephrin the 5-prime sequence was isolated by polymerase chain
unless the patient undergoes renal transplantation (2). Neph- reaction from a human kidney library (CLONTECH, Palo Alto, CA) and
rin is an integral membrane protein located at opposing sites of inserted into clone NPHS902 kindly provided by Dr. Karl Tryggvason
(Karolinska Institute, Stockholm, Sweden); the FLAG-tag was inserted
the secondary foot processes formed by podocytes, a specialized
at the 3⬘ end of the coding sequence to create a C-terminally FLAG-
epithelial cell that ensures size- and charge-selective ultrafil- tagged construct (denoted as Nephrin.F). Membrane-bound fusion pro-
tration (reviewed in Ref. 3). The precise function of nephrin is teins of the C-terminal and N-terminal cytoplasmic domains of podocin
unknown; however, it appears to form a zipper-like filter struc- and the C-terminal cytoplasmic domain of nephrin were generated
ture within the ⬃40-nm-wide slit between two foot processes using a pCDM8 cassette that contained the leader sequence of CD5
(4). fused to the CH2 and CH3 domains of human IgG1 followed by the
transmembrane region of CD7 (10).
Co-immunoprecipitation—Co-immunoprecipitations were performed
* This study was supported by Deutsche Forschungsgemeinschaft as described (11). Briefly, HEK 293T cells were transiently transfected
Grants Be2212/3-1 and Wa517/5-1. The costs of publication of this article by the calcium phosphate method. After incubation for 24 h, cells were
were defrayed in part by the payment of page charges. This article must washed twice and lysed in a 1% Triton X-100 lysis buffer. After centrif-
therefore be hereby marked “advertisement” in accordance with 18 U.S.C. ugation (15,000 ⫻ g, 15 min, 4 °C) cell lysates containing equal amounts
Section 1734 solely to indicate this fact. of total protein were incubated for 1 h at 4 °C with the appropriate
The nucleotide sequence(s) reported in this paper has been submitted
antibody, followed by incubation with 40 ␮l of protein G-Sepharose
to the GenBankTM/EBI Data Bank with accession number(s) AY050309.
beads for ⬃3 h. The beads were washed extensively with lysis buffer,
‡ To whom correspondence should be addressed: Renal Division, Uni-
versity Hospital Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Ger- and bound proteins were resolved by 10% SDS-PAGE.
many, Tel.: 49-761-270-3250; Fax: 49-761-270-3245; E-mail: walz@ Luciferase Assay—HEK 293T cells seeded in 12-well plates were
med1.ukl.uni-freiburg.de. transiently transfected with a luciferase reporter construct, a ␤-galac-
1
The abbreviations used are: NPHS1, congenital nephrotic syndrome tosidase expression vector (kindly provided by C. Cepko), and vectors
of the Finnish type; NPHS2, steroid-resistant nephrotic syndrome; directing the expression of the proteins as indicated. Total DNA amount
CD2AP, CD2-associated protein; PAGE, polyacrylamide gel electro- was 1.5–2.0 ␮g/well. Cells were serum-starved for 12 h, harvested in
phoresis; HEK, human embryonic kidney; JNK, c-Jun N-terminal cold phosphate-buffered saline, and lysed in 100 ␮l of reporter lysis
kinase. buffer (Applied Biosystems, Norwalk, CT) for 10 min at 4 °C. Lysates

This paper is available on line at http://www.jbc.org 41543

This is an Open Access article under the CC BY license.
41544 Nephrin Signaling

FIG. 1. Podocin augments the nephrin-mediated AP-1 activa-

tion. A, HEK 293T cells were transfected with an AP-1-dependent
luciferase construct and expression plasmids as indicated. Podocin trig-
gered a modest (less than 5-fold) increase in luciferase activity, whereas
nephrin stimulated an ⬃20-fold increase. The combination of nephrin
and podocin was synergistic, reaching a nearly 40-fold increase in
luciferase activity (***, p ⬍ 0.001). B, CD2AP had no significant effect
on nephrin-mediated AP-1 activation. HEK293T cells were transfected
with an AP-1-dependent luciferase construct and expression plasmids
as indicated. CD2AP triggered only a marginal increase in luciferase
activity and did not increase the nephrin-mediated AP-1 activation. C,
augmentation of nephrin signaling by podocin was not the result of
altered protein expression. HEK 293T cells were transfected with
FLAG-tagged proteins as indicated. Cellular lysates were separated on
SDS-PAGE; Western blot analysis was performed, using the FLAG-
specific M2 monoclonal antibody. FIG. 2. The nephrin-mediated AP-1 activation involves activa-
tion of the stress-activated protein kinases p38 and JNK. A, the
were centrifuged at 14,000 rpm for 5 min to remove insoluble material. nephrin-mediated AP-1 activation was significantly inhibited by domi-
Luciferase activity was determined using a commercial assay system nant-negative (DN) mutants of Cdc42, Rac1, and RhoA (SGP DN), as
(Applied Biosystems, Norwalk, CT) and normalized for ␤-galactosidase well as MKK4 (MKK4 DN) and a combination of MKK3 and MKK6
activity to correct for transfection efficiency. Equal expression of pro- (MKK3/6 DN). The dominant-negative mutant of MEK1 (MEK1 DN)
teins was ensured by Western blot analysis. did not affect nephrin-mediated AP-1 signaling. These results suggest
that the nephrin-mediated AP-1 activation involves the activation of
Mitogen-activated Protein Kinase Phosphorylation—To determine
p38 and JNK but not ERK1/ERK2. B, nephrin/podocin-mediated AP-1
the phosphorylation status of p38 and c-Jun, dually phosphorylated p38
activation was inhibited by the same set of dominant-negative protein
and phosphorylated c-Jun were visualized by Western blot analysis, kinases, indicating that the signaling pathways activated by nephrin
using phosphospecific antisera (New England Biolabs). Equal loading and the nephrin/podocin combination are identical. C, monitoring of
was confirmed by reprobing the membrane with the non-phospho-anti- phosphorylation of p38 and c-Jun confirms that nephrin activates p38
bodies and by Amido Black staining. The degree of phosphorylation of and JNK. HEK 293T cells were transfected with expression plasmids as
p38 and c-Jun was quantified by densitometry of non-saturated radio- indicated. Cellular lysates were separated on SDS-PAGE, and phospho-
graphs with the NIH Image software. p38 and phospho-c-Jun as well as total amounts of p38 and c-Jun were
determined by Western blot analysis using phospho-specific and phos-
RESULTS AND DISCUSSION pho-independent antisera. The depicted bars (mean ⫾ S.E.) represent
The domain architecture of the extracellular domain of neph- the relative changes of activation status obtained after densitometry of
at least three independent experiments.
rin, containing eight immunoglobulins, a fibronectin type III-
like domain repeat, and a single transmembrane domain, sug-
gests that nephrin is as an adhesion molecule involved in struct. Western blot analysis revealed that this augmentation
cell-cell or cell-matrix interactions. Based on the structure of was not the result of altered protein levels (Fig. 1C). In con-
the glomerular podocyte slit diaphragm (12) and the electron trast, CD2AP had only a marginal effect on nephrin-mediated
microscopic localization of nephrin (4), it was suggested that AP-1 activation (Fig. 1B) and did not further augment the
the N-terminal six immunoglobulin repeats of nephrin form synergism between nephrin and podocin (data not shown).
interdigitating zipper-like homophilic interactions (4, 13). The AP-1 is composed of homodimers and heterodimers of Jun,
C-terminal domain of nephrin is relatively short but contains Fos, or activating transcription factor family members and
several potential tyrosine phosphorylation sites. In particular, modulates a variety of cellular programs, including prolifera-
Tyr1176, Tyr1193, and Tyr1210 are predicted to serve as potential tion, differentiation, and apoptosis (reviewed in Ref. 16). Using
docking sites for SH2 domain-containing adapter and signaling AP-1 as a downstream target of nephrin signaling in combina-
molecules (cansite.bidmc.harvard.edu/, medium stringency). tion with dominant-negative mutations of protein kinases and
Because overexpression of surface receptors can initiate signal- small G-proteins, we determined that nephrin activates stress-
ing in a ligand-independent fashion (14, 15), we assayed the activated p38 protein kinase as well as c-Jun N-terminal pro-
activity of AP-1 transcription factor in HEK 293T cells express- tein kinase JNK (17, 18). As demonstrated in Fig. 2A, domi-
ing nephrin. Fig. 1 demonstrates that nephrin caused a sub- nant-negative mutants of small G-proteins (Cdc42, Rac1,
stantial increase (⬃20-fold) in AP-1-dependent luciferase activ- RhoA) and protein kinases that activate JNK (MKK4) and p38
ity. In contrast, serum triggered a 3-fold increase (data not (MKK3, MKK6) inhibited the nephrin-mediated AP-1 activa-
shown), whereas podocin and CD2AP alone had only marginal tion. In contrast, a dominant-negative MEK1 had no effect on
effects on AP-1 activation (Fig. 1). Interestingly, podocin syn- the nephrin-mediated AP-1 activation, indicating that nephrin
ergistically augmented nephrin-mediated AP-1 activation, stimulates p38 and JNK but not ERK1/ERK2. Similar results
yielding a nearly 40-fold transactivation of the luciferase con- were obtained for the AP-1 activation triggered by nephrin in
 Nephrin Signaling 41545
combination with podocin (Fig. 2B), suggesting that podocin
increases the efficiency of nephrin signaling without recruiting
additional signaling molecules. To confirm these results, we
monitored the phosphorylation status of p38 and c-Jun, using
phosphospecific antisera. As demonstrated in Fig. 2C, nephrin
triggers phosphorylation of p38 and c-Jun that is significantly
augmented by podocin. Taken together, these results strongly
suggest that nephrin is a signaling molecule that can activate
canonical mitogen-activated protein kinase cascades.
The structural similarities of podocin to other stomatin-like
proteins suggest that podocin forms homo-oligomers to facili-
tate recruitment of nephrin to specialized membrane domains.
We speculated therefore that the synergistic effect of podocin
on nephrin signaling results from a direct interaction between
podocin and nephrin in vivo. Fig. 3A demonstrates that the
cytoplasmic tail of nephrin binds podocin. This interaction is
mediated by the C-terminal domain of podocin (Fig. 3B). The
Fin-major (2-base pair deletion in exon 2 that results in a
frameshift and introduces a stop codon within the same exon)
and the Fin-minor (nonsense mutation in exon 26 resulting in
a stop at Arg1109) mutations are the two most common muta-
tions, found in more than 90% of all Finnish patients with
congenital nephrotic syndrome (NPHS1). However, neither the
Fin-major nor the Fin-minor mutant is expressed, and nephrin
is completely absent in patients with either mutation. To de-
termine the significance of AP-1 signaling in congenital ne-
phrotic syndrome, caused by NPHS1 mutations, we truncated
nephrin at amino acid 1160, corresponding to the most C-
terminal nephrin truncation reported in patients to date (9,
19). This truncation deletes the three potential SH2-binding
sites but retains several other potential tyrosine and threonine
phosphorylation sites. As demonstrated in Fig. 3C, the trun-
cated nephrin bound only marginal amounts of podocin, indi-
cating that a sufficient interaction between podocin and neph-
rin requires the C-terminal 81 amino acids of nephrin. In
contrast, neither nephrin nor podocin interacted with CD2AP
(Fig. 3D), and CD2AP did not affect the binding between neph-
rin and podocin (data not shown). Surprisingly, the nephrin
R1160X mutation promotes a high level of AP-1 activation that FIG. 3. The cytoplasmic tail of nephrin interacts with the C-
was not significantly different from wild-type nephrin (Fig. terminal domain of podocin. A, the cytoplasmic, C-terminal domain
of nephrin was fused to human immunoglobulin and the transmem-
3E). Because this nephrin mutation failed to bind podocin, we brane domain of CD7 (sIg.7.Nephrin) and co-transfected into HEK 293T
speculated that one important function of podocin is the aug- cells with FLAG-tagged podocin (F.Podocin). After immunoprecipita-
mentation of nephrin signaling. To test this hypothesis, the tion with protein G, podocin is detectable in the precipitate containing
nephrin R1160X truncation was co-expressed with podocin. As nephrin but not the control protein (sIg.7) lacking the cytoplasmic
domain of nephrin. Western blot analysis was performed using the
demonstrated in Fig. 3E, podocin failed to significantly aug- FLAG-specific M2 monoclonal antibody. B, the C-terminal domain of
ment the AP-1 activation mediated by the nephrin R1160X podocin binds nephrin. The sIg.7 fusion proteins containing either the
mutant, whereas both podocin and the nephrin mutation were N-terminal 104 amino acids or the C-terminal 258 amino acids of
well expressed (Fig. 3F). podocin were co-transfected into HEK 293T cells with nephrin, contain-
ing a C-terminal FLAG-tag. The C-terminal, but not the N-terminal,
Our results suggest that the function of nephrin is contin- domain of podocin precipitated nephrin. Protein G was used to precip-
gent on three requirements: expression of functionally intact itate podocin, whereas nephrin was detected using the FLAG-specific
protein, targeting of nephrin to the secondary foot processes, M2 monoclonal antibody. C, the nephrin R1160X truncation fails to
and efficient signaling of nephrin originating at the slit dia- efficiently bind podocin. Co-immunoprecipitation of FLAG-tagged podo-
cin with either a control (sIg.7) or Ig-fusions with wild-type C-terminal
phragms. Most nephrin mutations, including the Fin-major nephrin (sIg.7.Nephrin) or C-terminal nephrin truncated at amino acid
and Fin-minor mutations, result in the absence of protein (20), 1160 revealed that podocin binds to the C-terminal 81 amino acids of
thereby completely abrogating nephrin function. In contrast, nephrin. Only a weak interaction was detectable between podocin and
the nephrin R1160X mutation appears to be well expressed, at the nephrin R1160X mutant. D, CD2AP fails to interact with nephrin or
the nephrin-podocin complex. HEK 293T cells were transfected with ex-
least in a heterologous expression system, and retains the pression plasmids as indicated. Immunoprecipitation of sIg.7.Nephrin
ability to activate AP-1. However, it fails to interact with podo- was performed using protein G. FLAG-tagged proteins were detected by
cin. Based on the predicted structure of podocin and its poten- Western blot analysis, using the FLAG-specific M2 monoclonal antibody.
E, podocin does not augment the AP-1 activation mediated by the nephrin
tial to form membrane-associated homo-oligomers, podocin
R1160X truncation. HEK 293T cells were transfected with the AP-1
may serve two closely related functions: the recruitment and/or luciferase construct and expression plasmids as indicated. Although the
stabilization of nephrin at the podocyte foot process, and the AP-1 activation mediated by the nephrin R1160X truncation is not af-
augmentation of nephrin signaling, perhaps by organizing spe- fected in comparison to wild-type nephrin, podocin fails to augment the
AP-1 activation, triggered by mutant nephrin. F, Western blot analysis of
cialized nephrin-containing microdomains. It is therefore con-
cellular lysates revealed that both proteins, the nephrin R1160X trunca-
ceivable that nephrin signaling is preserved in patients with tion and podocin, were well expressed.
NPHS2 mutations but greatly reduced. We assayed the forma-
41546 Nephrin Signaling
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