The intracellular domain of nephrin is connected indirectly to the actin cytoskeleton, is tyrosine phos-phorylated, and mediates signalling from the slit diaphragm into the podo-cytes..
Trang 12 Medical Chemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
3 Ludwig Institute for Cancer Research, Uppsala, Sweden
4 Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki, Finland
5 Department of Pediatrics, Kyorin University School of Medicine, Mitaka, Tokyo, Japan
Keywords
Fyn, IQGAP1, phosphoinositide 3-kinase,
podocyte, slit diaphragm
Correspondence
K Tryggvason, Karolinska Institutet
Department of Medical Biochemistry and
Biophysics, Division of Matrix Biology,
Scheeles Va¨g 2 B1, Plan 4 SE-17177,
Stockholm, Sweden
Fax: +46 8 316165
Tel: +46 8 5248 7720
E-mail: karl.tryggvason@mbb.ki.se
(Received 22 July 2004, revised 21 September
2004, accepted 22 September 2004)
doi:10.1111/j.1432-1033.2004.04408.x
Nephrin is a signalling cell–cell adhesion protein of the Ig superfamily and the first identified component of the slit diaphragm that forms the critical and ultimate part of the glomerular ultrafiltration barrier The extracellular domains of the nephrin molecules form a network of homophilic and heterophilic interactions building the structural scaffold of the slit dia-phragm between the podocyte foot processes The intracellular domain of nephrin is connected indirectly to the actin cytoskeleton, is tyrosine phos-phorylated, and mediates signalling from the slit diaphragm into the podo-cytes CD2AP, podocin, Fyn kinase, and phosphoinositide 3-kinase are reported intracellular interacting partners of nephrin, although the biologi-cal roles of these interactions are unclarified To characterize the structural properties and protein–protein interactions of the nephrin intracellular domain, we produced a series of recombinant nephrin proteins These were able to bind all previously identified ligands, although the interaction with CD2AP appeared to be of extremely low stoichiometry Fyn phosphory-lated nephrin proteins efficiently in vitro This phosphorylation was required for the binding of phosphoinositide 3-kinase, and significantly enhanced binding of Fyn itself A protein of 190 kDa was found to associate with the immobilized glutathione S-transferase–nephrin Peptide mass finger-printing and amino acid sequencing identified this protein as IQGAP1, an effector protein of small GTPases Rac1 and Cdc42 and a putative regula-tor of cell–cell adherens junctions IQGAP1 is expressed in podocytes at significant levels, and could be found at the immediate vicinity of the slit diaphragm However, further studies are needed to confirm the biological significance of this interaction and its occurrence in vivo
Abbreviations
CD2AP, CD2-associated protein; CHAPS, 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate; FITC, fluorescein-isothiocyanate; HEK293, human epidermal kidney cells; HT1080, human fibrosarcoma; HT1080 L, mouse fibroblast; pAb 2, polyclonal antibody 2; PI 3, phosphoinositide 3.
Trang 2We have seen in recent years the rapid unravelling of
the molecular components of the slit diaphragm, a
spe-cialized structure connecting the podocyte foot
proces-ses An important finding in this regard was the
identification of nephrin as a gene mutated in
congen-ital nephrosis NPHS1 [1,2] NPHS1 patients show a
massive proteinuria starting in utero and their
podo-cyte foot processes are effaced Identical symptoms can
be brought about in mice by inactivating the nephrin
gene by homologous recombination [3] These mice die
from renal failure within 24 h after birth, which
under-lines the role of nephrin as a key component of the slit
diaphragm
The nephrin protein has an apparent molecular mass
of 180 kDa [1] The extracellular part of nephrin has
eight Ig-like modules, one fibronectin type III-like
module, and an unknown number of N-linked
carbo-hydrate moieties [4,5] The intracellular domain of 156
amino acids has no homology with other known
pro-teins but contains nine tyrosine residues, some of
which are phosphorylated [6] The domain structure
and biochemical properties of nephrin brought about
the immediate suggestion that nephrin molecules may
form dimers through homophilic interactions spanning
the slit diaphragm [1] Very recently, our electron
tomography studies showed that the slit diaphragm is
a network of intertwined strands containing nephrin
[7] Consistently, biochemical studies have indicated
that nephrin has an ability to form homo- and
hetero-dimers with NEPH1 [8–10], another component of
the slit diaphragm NEPH1 belongs to the Ig
super-family, has five extracellular Ig domains, and its
dele-tion leads to heavy proteinuria and early postnatal
death [11] P-cadherin and FAT, a large cadherin
pro-tein, have been localized at the slit diaphragm area or
close to it [12,13] Their participation and role in the
formation of the slit diaphragm structure is not
known Inactivation of the FAT gene in mice leads to
severe renal phenotype with the fusion of foot
proces-ses and loss of slit junctions [14], but
P-cadherin-knockout mice do not show any abnormalities in the
kidney [15,16]
The best-documented intracellular interaction
part-ner of nephrin is podocin [17,18] It is a member of the
stomatin protein family and is predicted to form a
membrane-associated hairpin-like structure with
cyto-solic N- and C-terminal domains Podocin has been
localized to the slit diaphragm area [19], and it is
mutated in a form of autosomal recessive familial focal
segmental glomerulosclerosis (SRN1) [20] Podocin is
required for recruitment of nephrin into lipid rafts
[21], and podocin-knockout mice die from renal failure
within a few days of birth [22] In addition to nephrin,
it interacts with the NEPH proteins and CD2-associ-ated adaptor protein CD2AP [18,23] Furthermore, nephrin itself has been reported to coimmunoprecipi-tate with CD2AP [24] that connects several membrane proteins to the actin cytoskeleton [25] The importance
of CD2AP for glomerular ultrafiltration is emphasized
by the fact that CD2AP-knockout mice develop neph-rotic syndrome 1–2 weeks after birth that leads to renal failure and death at 6–7 weeks of age [24] More-over, heterozygous CD2AP+⁄ –, mice are haploinsuffi-cient and display glomerular changes at 9 month’s of age with a histological pattern similar to that in human focal segmental glomerulosclerosis [26] CD2AP has been localized close to the attachment site
of the slit diaphragm [27], and its interaction with nephrin has been examined also in vitro, although the mapping of interacting domain for nephrin binding resulted in controversial reports [27,28] Studies employing gradient centrifugation and immunofluores-cence techniques have demonstrated that nephrin is indeed either directly or indirectly associated with the actin cytoskeleton [29–31]
Altered morphological characteristics of podocytes such as foot process effacement are typical to many experimental and human glomerulopathies [32,33] All these changes are reversible and recovery from these anomalies can only be achieved by the reformation of foot processes and slit diaphragms Nephrin expression levels and subcellular localization are affected in sev-eral renal diseases and proteinuric diseases such as dia-betes [34] This implies that there has to be signalling from the slit diaphragm into the podocytes, and vice versa Nephrin is very probaly a major player in this signalling Transfection of nephrin into HEK293 cells activates protein kinase p38 and c-jun aminoterminal kinase (JNK), thereby activating transcription factor AP-1 [17] Nephrin has been found to be dislocated to the apical pole of the narrowed filtration slits and tyro-sine phosphorylated, when the slit diaphragm is dis-rupted by in vivo injection of antibodies recognizing a podocyte-specific 9-O-acetylated GD3 ganglioside [6] Fyn kinase has been shown to bind and phosphorylate nephrin [35], and we have observed that tyrosine phosphorylation of nephrin is induced robustly, when nephrin is clustered on the cell surface, by using anti-nephrin Igs [36] In addition, a central signalling molecule phosphoinositide 3 (PI 3)-kinase has been reported to associate with nephrin [37]
These observations demonstrate how a complicated network of interactions and signalling is needed to organize and maintain the slit diaphragm They also indicate how important the elucidation of the protein– protein interactions formed by nephrin are for the
Trang 3of small GTPases Rac1 and Cdc42, as a potential new
interacting partner of nephrin Nephrin and IQGAP1
were found to colocalize both in cultured cells and in
kidney sections, but the demonstration of the
biologi-cal significance requires further studies
Results
The intracellular domain of mouse nephrin is
com-posed of 156 amino acids, of which 116 are conserved
in human and rat nephrin [38] However, it is not
possible to show any homology to other known
proteins in databases Nevertheless, there are several
putative docking and phosphorylation sites in
neph-rin (Table 1) Prediction of secondary structures
was carried out with several programs that were
able to recognize only two a-helical segments (http://
www.expasy.ch, http://xray.bmc.uu.se/sbnet/prosal.html)
The first one, RRRLRRLAEE(1100–1110), follows
immediately the transmembrane domain, and was
detected by all programs employed, while the second,
EEDRIRNEY(1120–1128), was suggested by most
programs
Using these in silico analyses as a background, we
proceeded to analyse the properties of the recombinant
intracellular domain For these studies, we produced
indicating that the C-terminal half of the intracellular domain is sensitive to bacterial proteases (Fig 1A) Western blot analysis with the antibody pAb 2 gener-ated against the intracellular domain of human neph-rin confirmed that all polypeptides detected in Fig 1A originate from nephrin (results not shown) We first tested whether these fusion proteins were able to pull-down proteins found in previous studies to associate with nephrin As demonstrated in Fig 1B, both podo-cin and Fyn kinase readily associated with GST–C ter-minus and GST–nephrincyt CD2AP did not interact with the fusion proteins in the initial experiments, and
we therefore investigated its association with nephrin
in various conditions We were able to detect a weak, occasional interaction in the lysis buffer containing 1% (v⁄ v) Chaps as a detergent, but not in 1% (v ⁄ v) digito-nin, 1% (v⁄ v) NP-40, or 0.5% (v ⁄ v) Triton X-100 Recently, we and others have reported that nephrin
is tyrosine phosphorylated, probably by Fyn kinase
in vivo [35,36] Here we show direct phosphorylation
by Fyn kinase in vitro Fyn was able to phosphory-late the untagged full-length intracellular domain,
as well as all GST–nephrin fusion proteins indicating that there are phosphorylation sites on both halves
of the intracellular domain (Fig 2A) Furthermore, several degradation fragments of GST–C terminus
Table 1 Amino acid sequence analysis of nephrin Sequences presented are of mouse nephrin Only sites conserved in human, mouse and rat are mentioned.
Putative site Sequence in nephrin Consensus sequence and ⁄ or reference Docking and phosphorylation site for Src kinase LYDEV(1207–1211) YE ⁄ D ⁄ TE ⁄ N ⁄ DI ⁄ V ⁄ M ⁄ L [42]
Docking site for Nck adaptor proteins LYDEV(1207–1211) YDEP ⁄ D ⁄ V [42]
Proline-rich sequence for SH3 domain binding PQLPP(1160–1164) PXXP [63]
a-Helical region RRRLRRLAEE(1100–1109) http://www.expasy.ch
EEDRIRNEY(1120–1128) http://xray.bmc.uu.se/sbnet/prosal.html Repeated sequences GHLYDEVE(1188–1195) http://www.ebi.ac.uk/Radar/
GPLYDEVQ(1205–1212) Protein kinase C phosphorylation site SEK(1112–1114) S ⁄ TXR ⁄ K
SMR(1155–1157) http://c.expasy.org/tools/scanprosite/ Casein kinase II phosphorylation site STAE(1145–1148) S ⁄ TXD ⁄ E
SMRD(1155–1158) http://c.expasy.org/tools/scanprosite/ TLEE(1166–1169)
Most probable tyrosine phosphorylation sites Y1153, Y1208, Y1225, Y1232 http://www.cbs.dtu.dk/services/NetPhos/ Most probable serine or threonine
phosphorylation sites
S1119, S1142, S1145, S1155, S1160, S1171 http://www.cbs.dtu.dk/services/NetPhos/
Trang 4were phosphorylated, whereas only the full-length
GST-1173 was phosphorylated at a detectable level
Prolonged incubation or increased amount of Fyn
kinase did not significantly increase phosphorylation
suggesting that the in vitro phosphorylation was
efficient and close to quantitative (Fig 2B) Next, we
wanted to investigate whether in vitro phosphorylation
provides new properties for the nephrin intracellular
domain First, we examined whether the
phosphoryl-ated GST–nephrin fusion proteins were able to bind
phosphatidylinositol 3-kinase that has been reported recently to associate with nephrin [37] We found that
PI 3-kinase bound only to the phosphorylated GST– nephrincyt and GST-1173 implying that one of the SH2 domains in the p85 subunits of PI 3-kinase is involved in the interaction (Fig 2C) These findings are also well in line with the prediction that the segment YYSM(1153–1156) constitutes a docking site for the SH2 domains of PI 3-kinase
Having demonstrated the functionality of the phos-phorylated GST–nephrin fusion proteins, we proceeded
to investigate how the phosphorylation effects on the interaction of nephrin with Fyn and podocin Interest-ingly, we observed that the tyrosine phosphorylation enhanced association of Fyn with nephrin, but did not have any significant effect on the interaction with podocin (Fig 3)
Identification of IQGAP1 as a putative interacting partner for nephrin
The above described findings encouraged us to utilize GST–nephrincyt in a search for novel interactors of nephrin Indeed, incubation of metabolically labelled podocyte, HEK293, HT1080, and L-cell lysates with the fusion protein conjugated to the glutathione-Seph-arose beads resulted in the strong and reproducible binding of a 190 kDa protein, as demonstrated by ana-lysing the eluates by SDS⁄ PAGE and autoradiography (Fig 4A)
To identify the 190 kDa protein, it was purified in quantities sufficient for peptide mapping by MALDI-TOF-MS (Fig 4B) Pooled fractions from three separ-ate purifications were concentrsepar-ated, and loaded on a one-dimensional SDS⁄ PAGE gel, which was then stained by silver The bands containing the 190 kDa protein were excised, and the gel pieces treated as described in Experimental procedures After overnight in-gel digestion with trypsin, the resulting peptide mixture was analysed by MALDITOF-MS Fifty-seven peptide masses were determined, and used to search the database Up to 30 masses corresponded with com-puted masses of tryptic peptides of mouse IQGAP1 (Table 2), a 189 kDa effector protein of small GTPases Rac1 and Cdc42 These peptides covered 19% of the mouse IQGAP1 sequence (GenBank accession number AF240630) These results allowed an unequivocal iden-tification of the 190 kDa protein as IQGAP1 In addi-tion, we sequenced three of the mapped peptides and two other peptides, which were not detected in the ori-ginal MALDITOF-MS analysis All obtained peptide sequences could be positioned in the mouse IQGAP1 sequence, confirming the identification
Fig 1 The C-terminal half of the nephrin intracellular domain is
pro-tease-sensitive and interacts with Fyn and podocin (A) GST fusion
proteins containing various parts of the nephrin intracellular domain
were produced and purified by standard methods in the presence
of protease inhibitors The purified proteins were analysed by
SDS ⁄ PAGE and Coomassie Brilliant Blue-staining (B) Cell lysates
prepared from parental HEK293 cells, or from those stably
expres-sing recombinant human podocin, were incubated with immobilized
GST fusion proteins, after which equal amounts of bound proteins
were separated on two SDS ⁄ PAGE gels Gels were subjected
either to Western blot analysis with anti-Fyn or anti-podocin Igs, or
to Coomassie Brilliant Blue-staining to control the loading of the
GST proteins to the beads.
Trang 5Characterization of the IQGAP1–nephrin
interaction
IQGAP1 was purified from L-cells rather than
podo-cytes as L-cells proliferate more rapidly, and are
there-fore more convenient for a large-scale application In
order to demonstrate that the 190 kDa protein bound
to GST–nephrincyt from the podocyte cell lysate was
IQGAP1, we carried out pull-down assays from these
lysates The eluates were analysed by Western blotting
with anti-IQGAP1 Igs, and, as expected, a protein of
190 kDa recognized by the antibodies was found to
bind to GST–nephrincyt (Fig 5B) We also used
var-ious deletion constructs to map the areas of the
neph-rin intracellular domain responsible for IQGAP1
binding All constructs containing the C-terminal half
of the intracellular domain (amino acids 1167–1256)
bound IQGAP1, whereas we could not detect any
interaction between IQGAP1 and N-terminal half on
the nephrin intracellular domain, or between IQGAP1
and GST alone The last 11 residues of the
intracellu-lar domain are fully conserved between human, mouse
and rat nephrin However, a fusion protein lacking this
part did bind IQGAP1, indicating that the region was
not needed for IQGAP1 binding We were not able to
convincingly demonstrate the nephrin – IQGAP1
inter-action in NPH5 cells by the immunoprecipitation
method, as IQGAP1 tended to coimmunoprecipitate
also with the Finn-minor mutant form of nephrin
lack-ing nearly the whole intracellular domain Tyrosine
phosphorylation of nephrin did not appear to effect
significantly on the in vitro interaction of GST–neph-rins and IQGAP1 (Fig 5B)
Nephrin and IQGAP1 colocalize in the HEK293 cell line expressing recombinant nephrin and IQGAP1 can be found at the slit diaphragm
As the binding experiments described above were all
in vitro studies, and the immunoprecipitation experi-ments did not give a definite demonstration of the interaction in intact cells, we performed immunolocali-zation studies to see whether the subcellular distribu-tion of nephrin and IQGAP1 supports the possibility that IQGAP1 is an intracellular binding partner of nephrin First, formaldehyde-fixed NPH5 cells were double-labelled for nephrin and IQGAP1 IQGAP1 is known to localize at the cortical cytoskeleton and cell–cell adhesion sites [39,40] The monoclonal anti-IQGAP1 Ig was found to stain the cortical cytoskele-ton in the NPH5 cells, and also diffusely the plasma membrane (Fig 6B) The strongest nephrin staining was detected at the plasma membrane and the cortical cytoskeleton, and due to overexpression, nephrin could also be found in the cytoplasm (Fig 6A) The expres-sion patterns of IQGAP1 and nephrin were overlap-ping (Fig 6C)
Our pull-down and Western blotting experiments indicated that IQGAP1 is expressed in podocytes at significant levels (Fig 4A; X L Liu & P Kilpela¨inen, unpublished observations) To examine the distribution
of IQGAP1 in podocytes in detail, we carried out
Fig 2 The intracellular domain of nephrin is efficiently phosphorylated by Fyn kinase in vitro Phosphorylation of a tyrosine residue within the N-terminal half of the domain generates a binding site for phosphoinositide 3-kinase (A) After in vitro phosphorylation of the different nephrin proteins with a commercial Fyn kinase preparate, 0.5 lg of each protein was analysed by SDS ⁄ PAGE and Western blotting with anti-phosphotyrosine Igs (B) The untagged full-length intracellular domain of nephrin was in vitro phosphorylated with the Fyn kinase prepa-rate for varying incubation times or by using different amounts of the kinase The results demonstprepa-rate that already incubation with 1· amount of the kinase preparate for 90 min (conditions used in the panel A experiment) results in the efficient phosphorylation of the fusion protein (C) Equal amounts of immobilized, nonphosphorylated and phosphorylated GST fusion proteins were incubated with HEK293 lysates, after which bound proteins were analysed by SDS ⁄ PAGE and Western blotting with a monoclonal anti-PI 3-kinase Ig An identical gel was stained with Coomassie Brilliant Blue A third gel with same samples was analysed by Western blotting with anti-phosphotyrosine Igs.
Trang 6immunoelectron microscopic studies of human kidney
sections (Fig 6D–F) In immunogold labelling,
IQ-GAP1 was distributed in podocyte foot-processes
exclusively intracellularly and the cytoplasmic aspect
of the slit diaphragm or its vicinity was often notably
labelled, whereas the label was rarely found near to
other sites of the podocyte surface (Fig 6D,E) In the
cytoplasm the label was quite often found in densities
(Fig 6F) that could correspond to the endoplasmic
reti-culum better resolved in well-fixed nonimmunosamples
Immunogold staining with the anti-nephrin Ig pAb 2
gave fairly abundant labelling that was located like the
label for IQGAP1 but was concentrated to the slit
diaphragm region and not found at other membrane sites (results not shown)
Discussion
The work that resulted in the identification of the neph-rin gene as a gene mutated in congenital nephrotic syn-drome of Finnish type (NPHS1) [2,41], demonstrated also the crucial role of the nephrin intracellular domain for the structure of the slit diaphragm A number of mutations or deletions in the intracellular domain lead
to severe NPHS1 Analysis of the amino acid sequence reveals that this domain is significantly conserved between different species [38], but it does not show any homology to other known proteins It contains a few very typical consensus sequences found in the most proteins, such as two putative phosphorylation sites for protein kinase C and three for casein kinase II However, a much more conspicuous and significant feature are the six conserved tyrosines found in human, mouse and rat [38], some of which (Tyr1191, Tyr1208 and Tyr1232, numbering according to mouse sequence) match with the consensus sequence of Src family kin-ase and Nck adaptor protein SH2 domain docking site [42] These sites correspond also to Src family kinase phosphorylation sites It is worth pointing out that the amino acid sequences around these tyrosines are indeed very similar (LYDEV, LYDEV, IYDQV), and these could be phosphorylated by the same kinase, for exam-ple by Fyn Phosphorylation at multiexam-ple tyrosines pro-vides several docking sites, which could, for example, result in the assembly of large protein complexes involved in the organization and maintenance of the slit diaphragm As the three putative docking sites are similar, one possibility is that such complexes could contain oligomers of same ligand that adjust or lock nephrin at the right location Finally, as a curiosity, Tyr1128 is flanked by the most conserved sequence that can be found around the tyrosines in nephrin However, this site is an unorthodox substrate for non-receptor tyrosine kinases as it has a negatively charged residue at position )1 before Tyr1128 (EYEES) [43]
On the contrary, several receptor tyrosine kinases have this kind of substrate sequences
As the nephrin intracellular domain did not show any homology to other known proteins, molecular modelling was difficult, and we could only predict the conformation for two short sequences that potentially form a-helixes These were both located close to the N-terminus of the intracellular domain When expres-sing and purifying the bacterial recombinant proteins,
we noticed that the intracellular domain appears to consist of two clearly separate parts, the N-terminal
Fig 3 Tyrosine phosphorylation of nephrin enhances association
with Fyn, but does not have an effect on the interaction with
podo-cin Cell lysates prepared from parental HEK293 cells, or from
those stably expressing recombinant human podocin, were
incuba-ted with equal amounts of nonphosphorylaincuba-ted and phosphorylaincuba-ted
GST proteins immobilized to the glutathione-Sepharose beads.
After extensive washings, bound proteins were analysed by
SDS ⁄ PAGE and Western blotting with anti-Fyn or anti-podocin Igs.
The anti-Fyn blot was reprobed with anti-nephrin Igs, and the
podocin pull-down samples were subjected to SDS ⁄ PAGE and
Coomassie Brilliant Blue-staining to show equal loading of the
phosphorylated and nonphosphorylated fusion proteins to the
beads The eluates were also analysed by SDS ⁄ PAGE and Western
blotting with the anti-phosphotyrosine Igs.
Trang 7half (amino acids 1102–1173 in mouse nephrin) and
the C-terminal half (1167–1256) The N-terminal part
was resistant to proteolysis during production,
purifi-cation and storage implying that it may form a tightly
packed domain On the contrary, the C-terminal half
was very sensitive to proteolysis occurring already
dur-ing production in Escherichia coli BL21 It contains
most of the putative consensus sites for tyrosine
phos-phorylation and protein–protein interactions, and as
we show in this study, all known interacting partners
of the intracellular domain except PI 3-kinase, bind to
this region The C-terminal half may form a less
tightly folded domain that has a flexible structure This
might be needed to create several interaction sites, or
to render the area more accessible to the interacting
proteins
Nephrin has been found to be associated and likely
phosphorylated by Src family kinase Fyn in vivo
[35,36] In this study, we showed that Fyn kinase
directly phosphorylates nephrin in vitro (Fig 2A,B)
Phosphorylation was efficient and apparently close to
quantitative Both N- and C-terminal halves were
phosphorylated Phosphorylation at the N-terminal
part never reached the same intensity as that at the
C-terminal half implying that the anti-phosphotyrosine
Ig may detect phosphotyrosines in the C-terminus
more efficiently, or that the C-terminal half may be
more accessible to kinases, or simply that the
C-ter-minal half contains more phosphorylation sites than the N-terminal half The last possibility is the most likely one, as the three highly potent tyrosine phos-phorylation sites for Src family kinases, Tyr1191 Tyr1208 and Tyr1232, are all within this region Fur-thermore, judging from the phosphorylation of degra-dation fragments, the C-terminal half contains at least two phosphorylation sites
Progress with the investigations of the nephrin intra-cellular domain have been relatively rapid, and have to date resulted in the identification of several putative interaction partners for nephrin [17,18,24,35–37] The biological significance and function of some interactions
is obvious, whereas those of others are more difficult to predict Podocin is able to interact with nephrin, NEPH1, and CD2AP, all components of the slit dia-phragm [17,18,23] It may function as a scaffolding pro-tein, and it augments nephrin signalling by facilitating recruitment of nephrin to the slit diaphragm area [17,21] An adapter protein CD2AP has been reported
to interact both with podocin and nephrin, and puta-tively connects the slit diaphragm to the actin cytoskele-ton [18,24,25] Src family kinase Fyn phosphorylates nephrin, and associates with it [35,36] This phosphory-lation may be critical for the integrity of the glomerular filtration barrier, as the Fyn kinase-knockout mice display a renal phenotype having structurally distorted
or coarsened podocyte foot processes, and in some,
Fig 4 The intracellular domain of nephrin interacts with a 190-kDa protein (A)35 S-labeled extracts from podocytes, HT1080 cells, L -cells, and HEK293 cells were incu-bated with immobilized GST or the GST fusion protein containing the full-length nephrin intracellular domain Bound proteins were resolved by SDS ⁄ PAGE, and analysed by autoradiography (B) The 190-kDa protein was purified from the L-cell extract using GST–nephrin affinity chromatography An aliquot corresponding
to a 1 ⁄ 50 portion of the purified protein is analysed here by SDS ⁄ PAGE and silver staining Three similar large-scale preparations were performed to obtain enough protein for MALDITOF-MS.
Trang 8podocytes foot processes are completely effaced [35,44].
PI 3-kinase has been found to be able to bind to
nephrin via the p85 subunit, and in a cell culture model
this binding is related to activation of antiapoptotic
PI 3-kinase⁄ AKT pathway [37] However, further
char-acterization of nephrin–ligand interactions is required,
and especially their regulation is nearly completely unknown In this study, we could detect binding of podocin, Fyn, and PI 3-kinase to GST–nephrin We tested binding of CD2AP to GST–nephrin using four different detergent conditions and two cell lines in pull-down assays Two different antibodies were employed
to visualize CD2AP in Western blot analysis Nonethe-less, we were able to detect CD2AP-binding only occa-sionally, and only in a lysis buffer containing 1% Chaps Neither did in vitro phosphorylation of GST– nephrin induce CD2AP-binding It is likely that neph-rin–CD2AP interaction is of very low stoichiometry also
in vivo It has not been detected in all previous studies [17], and there are two contradictory reports on which domains of CD2AP are involved in the nephrin-binding [27,28] Consequently, it is possible that the biologically more critical interaction occurs between podocin and CD2AP, and it may be mainly the lack of this inter-action that contributes to the phenotype found in the CD2AP-knockout mice [24]
Surprisingly, more Fyn kinase bound to the phos-phorylated GST–nephrin than to the unphosphory-lated fusion protein This may imply that nephrin is phosphorylated processively by Fyn In this process, Fyn at first would phosphorylate a site in nephrin that becomes a high affinity binding site for the SH2 domain Interaction between this site and the SH2 domain of Fyn facilitates phosphorylation of subse-quent tyrosines in nephrin – alternatively Fyn could phosphorylate other substrates that are forming a pro-tein complex with the intracellular domain of nephrin The processive phosphorylation by Src family kinases has been demonstrated with several multiphosphory-lated substrates [45,46]
Interestingly, another important signalling molecule,
PI 3-kinase bound only to the N-terminal half of the intracellular domain The classical motif for the binding
of PI 3-kinase SH2 domains is YXXM [42] The match-ing sequence YYSM is conserved in mouse and rat (Tyr1153 in mouse), but is replaced in human nephrin
by the sequence YYRSL This segment is the most probable candidate for the PI 3-kinase binding site also as the phosphorylated tyrosine in GST-1173 is obviously located very close to the C-terminal end of the fusion protein The PI 3-kinase binding site is apparently needed mainly for signalling purposes, and this same tyrosine may not be involved in the forma-tion of an intracellular protein complex around nephrin
or in the regulation of contacts with the cytoskeleton Tyrosine phosphorylation in the C-terminal half of nephrin has more likely this kind of functions
Having confirmed the functionality of GST–nephri-ncyt, we employed GST–nephrincyt affinity
chromato-Table 2 Peptide masses obtained by MALDITOF-MS analysis after
in-gel tryptic digestion of the 190-kDa protein identify IQGAP1 as
an interacting partner for nephrin Measured masses are obtained
by MALDITOF-MS analysis after in-gel tryptic digestion of the
190-kDa protein Data base searches with this set of peptide masses
assigned a total of 30 peptides to mouse IQGAP1 corresponding to
a sequence coverage of 19% The peptide mass error was less
than 100 p.p.m with all except one peptide The identification was
confirmed by sequencing three peptides shown in bold and two
other peptides not detected in the original MALDITOF-MS analysis.
Their sequences, TLQALQIPAAK and LFQTALQEEIK, correspond,
respectively, to residues 557–567 and 1025–1035 of the mouse
IQ-GAP1 sequence.
Measured
mass
Computed
mass Sequence
Residues in IQGAP1 Start To 725.453 725.386 LQYFR 829 833
735.453 735.373 IPYGMR 1156 1161
746.487 746.444 IQAFIR 842 847
769.434 769.401 MHQARK 818 823
800.439 800.429 ATGLHFR 105 111
800.439 800.425 DIRNQR 1488 1493
826.521 826.527 LIVDVIR 1391 1397
851.431 851.432 MVVSFNR 1054 1060
908.486 908.475 LGNFFSPK 81 88
934.464 934.512 EEYLLLR 1018 1024
934.464 934.454 QDKMTNAK 267 274
1040.576 1040.634 ALQSLALGLR 327 336
1117.543 1117.595 LIFQMPQNK 989 997
1117.543 1117.566 LDNSIRNMR 1131 1139
1123.602 1123.638 IIGNLLYYR 1186 1194
1140.508 1140.563 YGIQMPAFSK 192 201
1148.563 1148.585 TCLDNLASKGK 1533 1543
1258.676 1258.706 MREEVITLIR 892 901
1274.681 1274.701 MREEVITLIR 892 901
1318.665 1318.713 LFQTALQEEIK 1025 1035
1408.769 1408.851 RLAAVAAINAAIQK 388 401
1414.729 1414.781 LGLAPQIQDLYGK 162 174
1482.670 1482.666 ATFYGEQVDYYK 1517 1528
1531.698 1531.719 IFYPETTDIYDR 131 142
1564.613 1564.646 FDVPGDENAEMDAR 1369 1382
1566.757 1566.843 TLINAEDPPMIVVR 857 870
1715.841 1715.908 GVLLEIEDLQANQFK 1572 1586
1723.791 1723.837 EEIQSSISGVTAAYNR 723 738
1752.870 1752.911 IELEKYGIQMPAFSK 187 201
1752.870 1752.961 VDQIQEIVTGNPTVIK 1038 1053
1889.909 1889.948 FALGISAINEAVDSGDVGR 623 641
1953.882 1953.967 LPYDVTPEQALSHEEVK 1112 1128
2020.976 2020.998 NVIFEIGPTEEVGDFEVK 1587 1604
2054.108 2054.079 LEGVLAEVAQHYQDTLIR 568 585
Trang 9graphy and peptide mass fingerprinting to search for
new intracellular interaction partners for nephrin This
work resulted in the identification of IQGAP1, an
effector protein of small GTPases Rac1 and Cdc42
and a putative regulator of cell–cell adheren junctions
[39,40] According to the GST pull-down assays, the
interaction took place strictly and specifically between
the C-terminal half of the nephrin intracellular domain
and IQGAP1 The binding was not lost even after
rel-atively harsh washing (four times in the lysis buffer
containing 0.5 m NaCl), and IQGAP1 could be
detec-ted in the pull-down samples also by silver staining of
SDS⁄ PAGE gels (Fig 5C) Compared to the
inter-actions with the previously identified ligands, that
between nephrin and IQGAP1 appeared to be clearly
the strongest and most specific one However, despite
the fact that these two proteins are according to our
immunoelectron microscopic and immunocytochemical
findings physically located very close to each other,
immunoprecipitation could not demonstrate direct
association between nephrin and IQGAP1 in HEK293
cells This could imply that the nephrin–IQGAP1
interaction is transient, taking place only during certain situations, such as during the formation of the slit structure, and possibly even before nephrin is associated with other intracellular interactors
Although it is not clear whether the nephrin– IQGAP1 interaction is a biologically relevant one, study of the IQGAP1 literature strongly supports the notion that this could be the case Rac1 and Cdc42 belong to the Rho subfamily of small GTPases that has been implicated in the regulation of a wide range
of biological processes, including cell motility and adhesion, cytokinesis, cell morphology and polariza-tion, and cell growth [47] They affect these processes mostly by controlling the reorganization of the actin cytoskeleton, but they also regulate signal transduction pathways affecting gene transcription in the nucleus Rac proteins stimulate the formation of lamellipodia and membrane ruffles and are involved also in actin polymerization, cell–cell adhesion and cell motility, whereas Cdc42-activation triggers the formation of filopodia and affects cell–cell adhesion IQGAP1 accu-mulates at the polarized leading edge and areas of
Fig 5 Characterization of the nephrin–IQGAP1 interaction (A) Mouse podocyte lysates were incubated for 4 h with various immobilized GST proteins, after which bound proteins were analysed by SDS ⁄ PAGE and Western blotting with monoclonal anti-IQGAP1 Ig The results show that the binding site for IQGAP1 resides within the C-terminal half of the nephrin intracellular domain (B) L-cell lysates were incubated with equal amounts of nonphosphorylated and phosphorylated GST proteins immobilized to glutathione-Sepharose beads The bound proteins were analysed, as in panel A, with monoclonal anti-IQGAP1 Ig An identical SDS ⁄ PAGE gel was run to show equal loading of the phosphorylated and nonphosphorylated fusion proteins to the beads The eluates were also analysed by SDS ⁄ PAGE and Western blotting with the anti-phosphotyrosine Igs The results demonstrate that IQGAP1-binding to nephrin is not affected by the nephrin tyrosine phos-phorylation (C) GST–nephrin immobilized to glutathione-Sepharose was incubated with the L -cell lysate, after which the beads were washed four times with the lysis buffer containing indicated concentrations of NaCl Bound proteins were fractionated by SDS ⁄ PAGE and visualized
by silver staining A significant fraction of bound IQGAP1 remains associated even after washing with 0.5 M NaCl.
Trang 10membrane ruffling, as well as at the cell–cell adheren
junctions [39,40,48] It can bind to the activated
GTP-bound Rac1 and Cdc42 maintaining them in an
activated state, but not to the inactive GDP-bound
forms [39,48] Calmodulin is another signalling
mole-cule IQGAP1 binds to [48], and it has also a
well-documented actin-binding activity [49,50] Thus
IQGAP1 is a scaffolding protein connecting Ca2+⁄
calmodulin and Rac1⁄ Cdc42-mediated signalling and
cytoskeleton [51]
Very interesting is the recent observation that
IQGAP1 interacts with CLIP-170, a member of a
protein family that specifically accumulates at the
plus ends of growing microtubules [52] The
CLIP-170–IQGAP1 complex appears to function as a linker
between the plus ends of microtubules and cortical
actin meshwork It may recruit the microtubules at
special cortical sites leading to cell polarization
Acti-vated Rac1 or Cdc42, both known as key regulators
of cell polarization [53], is present as a third member
in the CLIP-170–IQGAP1 complex Furthermore,
IQGAP1 was reported recently to interact with S100B [54] that colocalizes with IQGAP1 at the polarized leading edge and areas of membrane ruf-fling in glioma cells The strongest S100B immuno-reactivity was found in cells that are characterized
by long processes In these cells, a colocalization of S100B and IQGAP1 was evident at plasma mem-brane and in the growing processes However, maybe the most notable issue is that IQGAP1 is involved
in the formation and maintenance of cell–cell adheren junctions It has been suggested to associate both with E-cadherin–catenin and nectin–afadin systems, and to regulate E-cadherin-mediated cell–cell sion [40,55] IQGAP1 may, together with cell adhe-sion proteins, organize F-actin to form specific structures and morphology at the cell–cell adhesion sites in epithelial cells It is possible that the neph-rin–IQGAP1 interaction plays a similar role in the slit diaphragm, which, in fact, has been suggested to
be a modified adherens junctions, as a-, b- and c-catenins as well as P-cadherin have been found in
Fig 6 Nephrin and IQGAP1 colocalize in cultured cells IQGAP1 is found at the slit diaphragm in human kidneys HEK293 cells stably expressing human nephrin were double-stained for nephrin (A) and IQGAP1 (B) Panel C shows the merged picture, indicating that nephrin and IQGAP1 colocalize at the sites of cell–cell contacts Some of the cells had already lost nephrin expression (D–F) IQGAP1-immunogold label in podocyte foot-processes FP, foot processes; SD, slit diaphragm; GBM, glomerular basement membrane; arrowheads, gold label In
D, shorter and longer stretches of slit diaphragm are seen in oblique tangential view in filtration slit between foot processes of human kidney podocytes Note intracellular gold-label for IQGAP1 (arrowheads) mostly along SD In higher magnification (E) gold label for IQGAP1 is seen close to cell surface both at the SD area and elsewhere In F, IQGAP1-label deeper in the podocyte cytoplasm is associated with cytoplas-mic density at the level of cross-cut SD.