Visceral glomerular epithelial cells (GEC) are critical fornormal permselectivity of the kidney. Nephrin is a moleculethat is expressed specifically in GEC in a structure calledthe slit diaphragm and is required for normal morphology andpermselectivity of GEC. However, the mechanisms of action ofnephrin are not understood precisely. The intracellular domainof nephrin has six conserved tyrosine residues. It was hypothesizedthat these tyrosine residues are phosphorylated by Src-familykinases and that this phosphorylation modulates the functionof nephrin. A transient transfection system was used to studythe role of tyrosine phosphorylation of the cytoplasmic domainof nephrin in its function. When nephrin was co-transfectedwith Src-family kinases Fyn or Src in Cos-1 cells, nephrin wasstrongly tyrosine phosphorylated by Fyn and less so by Src.The results with tyrosine-to-phenylalanine mutations suggestedthat multiple tyrosine residues contribute to phosphorylationmediated by Src-family kinases. The intracellular domain ofnephrin is known to interact with another slit diaphragm protein,podocin. When nephrin and podocin were transfected with Fyn,the interaction between nephrin and podocin was augmented significantly.Podocin was not tyrosine phosphorylated by Fyn; thus, the increasedinteraction is likely to be secondary to tyrosine phosphorylationof nephrin. Fyn also significantly augmented the activationof the AP-1 promoter induced by nephrin and podocin. In summary,Fyn phosphorylates the cytoplasmic domain of nephrin on tyrosine,leading to enhanced association with podocin and downstreamsignaling of nephrin.
Visceral glomerular epithelial cells (GEC) or podocytes arecritical for structural integrity as well as permselectivityof the glomerulus. In 1998, Kestila et al. (1) identified anew molecule, nephrin, which localizes in the slit diaphragmof podocytes. Mutations of nephrin cause severe congenital proteinuriaand renal failure (Finnish-type nephrotic syndrome). This breakthroughdiscovery was followed by identification of a number of otherpodocyte-associated molecules essential for glomerular permselectivity,such as CD2AP, podocin, and -actinin-4 (2). These moleculesare believed to maintain the normal morphology and permselectivityof podocyte by interacting with the actin cytoskeleton, buttheir precise mechanisms of action are largely unknown.
After the initial cloning of nephrin in 1998, substantial amountsof information were obtained about the expression level anddistribution pattern of nephrin in various kidney diseases.However, information about the signal transduction of nephrinis only beginning to accumulate. Nephrin belongs to the Ig superfamily.It is a transmembrane protein, and its extracellular domainhas eight Ig-like domains and a fibronectin-like domain. Itis believed that nephrin molecules from adjacent foot processesbind to each other in a homophilic manner, which serves as abackbone for the slit diaphragm (3). When the homophilic bindingof nephrin was disrupted, intracellular actin organization wasdisrupted (4). Thus, in addition to its function as an adhesionmolecule, nephrin is likely to transmit extracellular signalsinto the cells (4). Podocin, which was identified in 2000, isa membrane-associated protein that belongs to the stomatin proteinfamily. Mutations of podocin cause autosomal recessive steroid-resistantnephrotic syndrome in childhood (5). Podocin interacts withnephrin via its C-terminus (amino acids 125 to 385) (6,7). Nephrin-podocininteraction is believed to be important for the recruitmentof nephrin to lipid raft (8). Also, podocin augments the activationof the AP-1 promoter by nephrin (6). Thus, it is likely thatthe nephrin–podocin interaction has an important rolein glomerular permselectivity (2).
Nephrin has a short cytoplasmic domain (150 amino acids), whichcontains six tyrosine residues conserved in human, mouse, andrat sequences (Figure 1A). Recently, several groups reportedthat nephrin is tyrosine phosphorylated in the cytoplasmic domain(9–11). The injection of a monoclonal antibody that causesmorphologic changes of podocyte induced marked tyrosine phosphorylationof nephrin (10). Clustering of nephrin by antibodies causedstrong tyrosine phosphorylation of nephrin in the cytoplasmicdomain (9). Also, it was shown that the Src-family tyrosinekinase Fyn bound and phosphorylated nephrin (11). However, thesignificance of nephrin tyrosine phosphorylation has yet tobe elucidated. It was recently reported that the cytoplasmicdomain of nephrin binds to the regulatory p85 subunit of phosphoinositide3-kinase (PI3K) and together with CD2AP and podocin activatesthe Akt signaling pathway (12). It was suggested that this bindingis dependent on tyrosine phosphorylation of nephrin (12).
Figure 1. Alignment of the cytoplasmic domains of human, rat, and mouse nephrin (A) and Tac/nephrin construct (B). (A) Six conserved tyrosine residues are indicated by arrows. Numbers are according to the rat sequence (accession no. NM022628). (B) The extracellular domain of the human IL-2 receptor (Tac) was connected to the transmembrane/cytoplasmic domain of rat nephrin to form a chimera Tac/nephrin. Full-length nephrin is shown for comparison.
Src-family kinases are a large family of nonreceptor tyrosinekinases, which are known to localize in the lipid rafts andare involved in a variety of signal transduction pathways (13,14).For example, c-Src phosphorylates intracellular molecules thatinteract with and transmit signals from adhesion molecules,such as integrins and cadherins, leading to rearrangement inthe cytoskeleton (15). Yu et al. (16) reported that in Fyn-deficientmice (fyn–/–), effacement of podocyte foot processeswas observed, accompanied by significant proteinuria. Similarfindings were reported in fyn–/–yes–/–double knockout mice (11). Thus, it is tempting to speculatethat tyrosine phosphorylation by Src-family kinases may regulateprotein–protein interactions and signal transduction ofnephrin. The current study demonstrates that the cytoplasmicdomain of nephrin is a substrate for the Src-family kinase Fynand that tyrosine phosphorylation of nephrin modulates its interactionwith its molecular partner, podocin.
Tissue culture media, isopropylthio--galactoside, and lipofectamine2000 reagent were purchased from Invitrogen-Life Technologies(Burlington, ON, Canada). Sodium orthovanadate and other standardchemicals were from Sigma Chemical Co. (St. Louis, MO). Proteaseinhibitor cocktail, reagents for PCR, and calf intestinal alkalinephosphatase were from Roche Diagnostics (Laval, QC, Canada).Antiphosphotyrosine antibody Py20 was from BD Biosciences (SanJose, CA). Rabbit anti-CD2AP antibody, rabbit anti-Fyn antibody,mouse anti-Myc antibody, and protein A-Sepharose were from SantaCruz Biotechnology (Santa Cruz, CA). Anti-Src [pY418] phospho-specificantibody was from Biosource International (Camarillo, CA). Srcphosphorylated at Y418 is known to be active. Dual-LuciferaseReporter Assay System was from Promega (Madison, WI). cDNA encodingSrc, T lymphocyte Fyn (FynT), kinase-negative FynT, and C-terminalSrc kinase (Csk; all in the mammalian expression vector pME18S)were gifts from Dr. Junichi Abe (University of Rochester, Rochester,NY) (17–19). cDNA Tac/zeta/zeta (20) was a gift from Dr.Sylvain Latour (Hôpital Necker Enfants-Malades, Paris,France). Isolation of full-length rat nephrin was reported earlier(21), and the coding region was subcloned into the EcoRV/XbaIsites of pcDNA3.1/HisA (Invitrogen-Life Technologies). Plasmidsfor AP-1 luciferase, nephrin-F, and F-podocin were gifts fromDr. Thomas Benzing (Freiburg, Germany) (6).
Cells and Transfection
Cos-1 cells and HEK293T cells were grown in DMEM supplementedwith 10% FBS. Cells were transfected with the indicated amountsof plasmid using lipofectamine 2000 reagent (Invitrogen-LifeTechnologies) following the manufacturer’s instructions.
Induction of Passive Heymann Nephritis
Passive Heymann nephritis (PHN) was induced in male Sprague-Dawleyrats (150 to 175 g body wt; Charles River, St. Constant, QC,Canada) by intravenous injection (400 µl/rat) of sheepanti-Fx1A antiserum as described previously (22). Significantproteinuria was observed 14 d after injection (160 mg/d; normalrats excrete <10 mg protein/d).
Plasmid Preparations
To construct Tac/nephrin (Figure 1B), we amplified cDNA correspondingto the transmembrane and cytoplasmic domain of rat nephrin (21)by reverse transcription–PCR (RT-PCR) from RNA preparedfrom rat glomeruli. After the sequence was confirmed, this fragmentwas subcloned into Tac/zeta/zeta to replace the zeta/zeta portion,to form a chimera of the extracellular domain of the human IL-2receptor (Tac) and the transmembrane/cytoplasmic domains ofrat nephrin. Finally, Tac/nephrin was subcloned into the mammalianexpression vector pcDNA3.1(+)/hygro (Invitrogen-Life Technologies).cDNA corresponding to the coding region of mouse podocin (5)was amplified by RT-PCR from mouse kidney RNA. After the sequencewas confirmed, this fragment was cloned into pcDNA3.1(–)/Myc-HisB(Invitrogen-Life Technologies) to construct Myc (HIS)-taggedpodocin (Myc/podocin). cDNA corresponding to the coding regionof mouse CD2AP (23) was amplified by RT-PCR from mouse liverRNA. After the sequence was confirmed, the fragment was subclonedinto pcDNA3.1(+)/hygro (Invitrogen-Life Technologies). Tyrosineto phenylalanine mutations of nephrin were carried out by PCR-basedmutagenesis. To construct mutants in full-length rat nephrin,we transferred the fragments that contained mutations from Tac/nephrinto full-length rat nephrin using BglII and XbaI sites. As partof the subcloning process, His-tag was removed from the originalfull-length rat nephrin and the expression vector was changedto pcDNA3.1 (Invitrogen-Life Technologies). All sequences wereconfirmed by automated sequencing at Sheldon Biotechnology Centre(McGill University, Montreal, QC, Canada).
Preparation of Antinephrin Antiserum
cDNA corresponding to the cytoplasmic domain of rat nephrinwas amplified by RT-PCR from rat glomerular RNA and was subclonedinto pGEX-5X-2 (Pharmacia) to construct a glutathione S transferase(GST)-fusion protein. The construct was expressed in bacteriaXL-10-Gold (Stratagene), and GST-fusion protein was inducedby isopropylthio--galactoside. The GST-fusion protein was separatedby SDS-PAGE, purified, and used to immunize rabbits. Specificityof the antiserum was verified using cell lysates from Tac/nephrin-transfectedCos-1 cells and rat glomerular lysates.
Immunoprecipitation and Immunoblotting
Cells or glomeruli were lysed in ice-cold IP buffer (1% TritonX-100, 125 mM NaCl, 10 mM Tris [pH 7.4], 1 mM EDTA, 1 mM EGTA,2 mM Na3VO4, 10 mM sodium pyrophosphate, and 25 mM NaF) thatcontained a protease inhibitor cocktail (Roche Diagnostics).After insoluble components were removed by centrifugation (14,000rpm, 5 min, 4°C), protein concentrations of supernatantswere quantified using a commercial reagent (Bio-Rad). Immunoprecipitationwas performed with 5 µl of antiserum and 20 µl ofprotein A-Sepharose per sample. Equal amounts of protein (25to 50 µg of total lysates) or immunoprecipitates wereseparated by 7.5% SDS-PAGE under reducing conditions. Proteinswere electrophoretically transferred to nitrocellulose membrane,blocked with 5% dry milk (or 5% BSA for Py20), and incubatedwith first antibodies for 16 h at 4°C. After three washes,membranes were incubated with secondary antibodies conjugatedwith horseradish peroxidase, and horseradish peroxidase activitywas detected by enhanced chemiluminescence (Amersham PharmaciaBiotech, Baie d’Urfé, QC, Canada). Protein contentwas quantified using scanning densitometry (NIH Image software).
Luciferase Assay
AP-1 luciferase assay was performed as reported by Huber etal. (6) with minor modification. HEK293T cells were plated in24-well plates and transfected with plasmids using lipofectamine2000 reagent in Opti-MEM. Dual-Luciferase Reporter System wasused to normalize transfection efficiencies. One day later,cells were harvested and cell lysates were subjected to luciferaseassay following the manufacturer’s instructions.
Statistical Analyses
Data are presented as mean ± SEM. The t statistic wasused to determine significant differences between two groups.One-way ANOVA was used to determine significant differencesamong groups. When significant differences were found, individualcomparisons were made between groups using the t statistic.
Fyn Tyrosine Phosphorylates the Cytoplasmic Domain of Nephrin
To test the hypothesis that the cytoplasmic domain of nephrinis a substrate for Src-family kinases, we first used a chimericconstruct in which the transmembrane and cytoplasmic domainof rat nephrin is connected to the extracellular domain of thehuman IL-2 receptor Tac (Tac/nephrin; Figure 1B). Tac has beenused in the past to study the function of the cytoplasmic domainof transmembrane molecules and the effect of cross-linking ofthe extracellular domain by the monoclonal anti-Tac antibody7G7 (20,24). When Tac/nephrin was transiently transfected inCos-1 cells and immunoprecipitated by antinephrin antiserum,no tyrosine phosphorylation was observed (Figure 2A). However,when Tac/nephrin was co-transfected with the Src-family kinaseFyn, Tac/nephrin was strongly tyrosine phosphorylated (Figure 2A).This tyrosine-phosphorylated band at 90 kD was not observedin the absence of nephrin (Figure 2A). Tac/nephrin was expressedas multiple bands ranging from 70 to 90 kD presumably becauseof differential glycosylation in the extracellular Tac domainand/or by ubiquitination/degradation (20); however, tyrosine-phosphorylatednephrin appeared as a single band of 90 kD (Figure 2A). Similartyrosine phosphorylation was observed when the cytoplasmic domainof nephrin (not shown) or the full-length rat nephrin (Figure 2E)was used instead of Tac/nephrin. Another Src-family kinaseSrc also tyrosine phosphorylated Tac/nephrin; however, phosphorylationwas significantly weaker as compared with Fyn (Figure 2B). Moreover,there was a second tyrosine-phosphorylated band (80 kD) observedonly with Src (Figure 2B). A similar difference in electrophoreticmobility between Fyn and Src was observed with full-length nephrin(Figure 2F). Although the precise nature of the lower band isnot clear, it seems that the lower band represents hypophosphorylatednephrin. These results suggest that the cytoplasmic domain ofnephrin is a substrate of Fyn and Src. Fyn phosphorylates nephrinmore strongly, as compared with Src, and there may be a qualitativeand/or quantitative difference between tyrosine phosphorylationby Fyn and Src.
Figure 2. Nephrin is tyrosine phosphorylated in Cos-1 cells. Cos-1 cells were transfected in 10-cm plates (A, B, C, E, and F) or in 35-mm plates (D) with the indicated amounts of plasmids using lipofectamine 2000 reagent. One to 2 d later, cell lysates were immunoprecipitated with rabbit antinephrin antiserum (A, B, C, E, and F) and immunoprecipitates (A, B, C, E, and F) or total cell lysates (25 µg; D) were blotted for phosphotyrosine (Py20) and nephrin. Note that Tac/nephrin is detected as multiple bands ranging from 70 to 90 kD. Tyrosine-phosphorylated Tac/nephrin migrated at 90 kD as a single band with Fyn (A through D) and at 90 and 80 kD as double bands with Src (B and D). Full-length nephrin (FL-nephrin) migrated at 180 kD as a doublet (E and F). When FL-nephrin was phosphorylated by Fyn, phosphorylated FL-nephrin co-migrated predominantly with the top band of the doublet, whereas when it was phosphorylated by Src, it co-migrated equally with the two bands of the doublet (F).
We next studied whether tyrosine phosphorylation of nephrinis dependent on the kinase activity of Src-family kinases. First,a kinase-negative mutant of Fyn (K299M) (19) failed to phosphorylateTac/nephrin, indicating that tyrosine phosphorylation of nephrinis dependent on the kinase activity of Fyn (Figure 2C). It isknown that phosphorylation of carboxyl terminal tyrosine (Tyr527in Src) by C-terminal Src kinase (Csk) inhibits activity ofSrc-family kinases. Thus, we also tested the impact of Csk onFyn/Src-medicated nephrin phosphorylation. When the same amountsof Fyn or Src (0.2 µg) were transfected into Cos-1 cellswith Tac/nephrin (0.2 µg), Tac/nephrin was tyrosine phosphorylatedmore strongly by Fyn, as compared with Src (Figure 2D, lanes3 and 6), consistent with the results in Figure 2B. When Cskwas co-transfected, tyrosine phosphorylation of Tac/nephrinwas markedly attenuated (Figure 2D, lanes 8 and 9). In fact,tyrosine phosphorylation by Src was not apparent in the presenceof Csk (Figure 2D, lane 9). These results support that tyrosinephosphorylation of nephrin is dependent on the kinase activityof the Src-family kinases. Although these results do not provethat nephrin is a substrate of Src-family kinases in vivo, Vermaet al. (11) reported that nephrin phosphorylation is markedlyreduced in fyn–/– mice. Together, it is reasonableto conclude that nephrin is tyrosine phosphorylated, at leastin part, by Src-family kinases.
Identification of Tyrosine Residues Phosphorylated by Fyn
We next studied which among the six conserved tyrosine residueswithin the nephrin cytoplasmic domain are phosphorylated bySrc-family kinases. Because Fyn phosphorylated nephrin morestrongly than Src, subsequent studies focused on Fyn. We firstevaluated the possibility of tyrosine phosphorylation of thesix conserved residues using NetPhos 2.0 prediction programprovided by the Center for Biologic Sequence Analysis. (www.cbs.dtu.dk/services/NetPhos)(25). This program predicts tyrosine residues that are likelyto be phosphorylated in eukaryotic cells. On the basis of theresults shown in Figure 3A, we chose to focus on Y1152, Y1171,Y1204, and Y1238, which were predicted to be the most likelysites of phosphorylation. When these tyrosine residues weremutated in Tac/nephrin individually to phenylalanine (Figure 3B),tyrosine phosphorylation decreased as follows: wild type,100%; Y1152F, 72 ± 10%; Y1171F, 68 ± 1%; Y1204F,53 ± 8%; Y1228F, 89 ± 8%; wild type without Fyn,0 ± 0% (Figure 3C). When these tyrosine residues weremutated in combinations, tyrosine phosphorylation decreasedmore markedly (compared with wild type [100%]; double [Y1204/1228F],43 ± 11%; triple [Y1152/1204/1228F], 27 ± 13%;and quadruple [Y1152/1171/1204/ 1228F], 8 ± 5%). Analogousexperiments were performed using full-length rat nephrin withsimilar results (Figure 3D). Two additional tyrosine residues,which had low probability of phosphorylation (Y1127 and Y1194),were also tested. As compared with wild type (100%), Fyn-mediatedtyrosine phosphorylation of Y1127F and Y1194F were 100 ±6% (n = 6; NS) and 79 ± 6% (n = 5; P < 0.05), respectively.Phosphorylation of Y1127F and Y1228F were not significantlydifferent from wild type, suggesting that these two residuesdo not contribute to phosphorylation by Fyn. All other mutantswere phosphorylated significantly less than wild type, indicatingthat multiple tyrosine residues of nephrin contribute to phosphorylationby Fyn. These residues may be phosphorylated directly by Fynor may facilitate the phosphorylation of the other residues.
Figure 3. Identification of the tyrosine residues that are phosphorylated by Fyn. (A) Probability of tyrosine phosphorylation of the six conserved tyrosine residues were studied using the NetPhos 2.0 prediction program provided by the Center for Biologic Sequence Analysis (www.cbs.dtu.dk/services/ NetPhos). This program predicts tyrosine residues that are likely to be phosphorylated in eukaryotic cells with sensitivity in the range of 69 to 96% (25). Numbers are in the range of 0 to 1, 1 being the highest possibility for phosphorylation. (B) Four conserved tyrosine residues in the cytoplasmic domain of nephrin are indicated (Y1152, Y1171, Y1204, and Y1228). In Tac/nephrin, each residue was mutated to phenylalanine individually (Y1152F, Y1171F, Y1204F, and Y1228F) or in combinations (double [Y1204/1228F], triple [Y1152/1204/1228F], and quadruple [Y1152/1171/1204/1228F]). (C) One microgram each of wild-type (WT) or mutant Tac/nephrin was transfected into Cos-1 cells with Fyn (1 µg). One to 2 d later, cell lysates were immunoprecipitated with rabbit antinephrin antiserum and immunoprecipitates were blotted for phosphotyrosine (Py20). In control, wild-type Tac/nephrin was transfected without Fyn. (Top) Immunolot. (Bottom) densitometry. Values are normalized for expression of Tac/nephrin; n = 4 to 7. For expression of Tac/nephrin, all bands between 70 and 90 kD were quantified. All mutants except Y1228F are significantly different from wild type with Fyn (*P < 0.05 versus wild type with Fyn). (D) The same experiments as in C were repeated with FL-nephrin and its mutants with similar results. *P < 0.05 versus wild type with Fyn.
Tyrosine Phosphorylation of the Cytoplasmic Domain of Nephrin Facilitates Its Interaction with Podocin
The intracellular domain of nephrin is known to interact withanother slit diaphragm protein, podocin. We next studied whetherthis interaction would be affected by tyrosine phosphorylationof nephrin. When Tac/nephrin and Myc/podocin were co-transfectedin Cos-1 cells and nephrin was immunoprecipitated, podocin wasco-immunoprecipitated with nephrin weakly/inconsistently (Figure 4A).When Fyn was transfected with Tac/nephrin and Myc/podocin,co-immunoprecipitation of nephrin and podocin increased significantly(Figure 4A). Similar association of nephrin and podocin wasalso observed when the full-length rat nephrin was used insteadof Tac/nephrin (Figure 4D). Podocin was not tyrosine phosphorylatedby Fyn (Figure 4B); thus, the increased co-immunoprecipitationis likely to be secondary to tyrosine phosphorylation of nephrin.Nephrin is also known to interact with the adapter moleculeCD2AP (15,16); however, we could not demonstrate co-immunoprecipitationof nephrin and CD2AP in this experimental system (data not shown).Moreover, the addition of CD2AP had no consistent impact onnephrin–podocin interaction (Figure 4A, left). We nextstudied the dose-response effect of Fyn on nephrin–podocinassociation (Figure 4C). When 0 to 1 µg of Fyn was transfectedwith Tac/nephrin and Myc/podocin, tyrosine phosphorylation ofnephrin increased in parallel with the amount of Fyn. Nephrin–podocininteraction was most evident with 1 µg of Fyn at both24 and 48 h after transfection. Smaller amounts of Fyn alsomildly increased the interaction when tested at 48 h after transfection(Figure 4C). Thus, within the range tested, Fyn facilitatesnephrin–podocin interaction in a dose-dependent manner.Kinase negative mutant of Fyn did not augment nephrin–podocininteraction, suggesting that the effect of Fyn is dependenton its kinase activity (Figure 4, E and F)
Figure 4. Fyn augments nephrin–podocin interaction. (A) Cos-1 cells were transfected with the indicated amounts of plasmids in 10-cm plates. One to 2 d later, cell lysates were immunoprecipitated with rabbit antinephrin antiserum. Immunoprecipitates were blotted for myc to study co-immunoprecipitation of nephrin with podocin. Total cell lysates (25 µg) were analyzed for expression of Tac/nephrin and Myc/podocin. Note that different amounts of Myc/podocin were used in the left and right panels. In general, when 2 µg of Myc/podocin was used, there was more baseline (without Fyn) co-immunoprecipitation of nephrin and podocin and co-immunoprecipitation was observed in a more consistent manner, as compared with when 1 µg of Myc/podocin was used. (B) Cos-1 cells were transfected with Myc/podocin with or without Fyn. One day later, cell lysates were immunoprecipitated with mouse anti-Myc antibody and immunoprecipitates were blotted for phosphotyrosine (Py20). Expression of Myc/podocin was confirmed in total cell lysates. Myc/podocin was not tyrosine phosphorylated by Fyn. (C) Cos-1 cells were transfected with different amounts of Fyn, and nephrin–podocin interaction was studied 24 or 48 h later. (D) Full-length rat nephrin was transfected with Myc/podocin and Fyn and analyzed as in A. Effect of kinase negative Fyn (kn) was tested with Tac/nephrin (E) and FL-nephrin (F).
Tyrosine Residues Are Important for Nephrin–Podocin Interaction
We next tested nephrin–podocin co-immunoprecipitationusing various Tac/nephrin mutants in the presence of Fyn. Whennormalized to the wild-type Tac/nephrin (100%), each mutantco-immunoprecipitated podocin as follows: Y1152F, 84 ±14%; Y1171F, 88 ± 15%; Y1204F, 69 ± 10%; Y1228F,97 ± 20%; double (Y1204/1228F), 67 ± 11%; triple(Y1152/1204/1228F), 69 ± 2%; quadruple (Y1152/1171/1204/1238F),54 ± 6%; wild-type without Fyn, 42 ± 3% (Figure 5A).Y1204F and all of the multiple mutants showed significantlyless podocin co-immunoprecipitation, as compared with wild type.Similar results were obtained for Y1204F and quadruple mutantin full-length nephrin (compared with wild type with Fyn [100%];Y1204F, 75 ± 10%; quadruple, 41 ± 4%; wild typewithout Fyn, 34 ± 7%; Figure 5B). Of note, the wild-typenephrin in the absence of Fyn or the quadruple mutant in thepresence of Fyn still demonstrated significant interaction withpodocin under these experimental conditions (Figure 5), whereastyrosine phosphorylation of these two nephrins was minimal (Figure 3).These results suggest that tyrosine phosphorylation of nephrinfacilitates nephrin–podocin interaction but is not a prerequisitefor this interaction.
Figure 5. Tyrosine mutations of the cytoplasmic domain of nephrin decrease nephrin–podocin interaction. (A) Wild-type and mutant Tac/nephrin as described in Figure 3 were transfected with Myc/podocin and Fyn in Cos-1 cells. One day later, cell lysates were immunoprecipitated with rabbit antinephrin antiserum and immunoprecipitates were blotted for Myc to study co-immunoprecipitation of nephrin with podocin. Total cell lysates (25 µg) were analyzed for expression of Tac/nephrin and Myc/podocin. (Top) Immunoblots. (Bottom) Densitometry. Results were normalized for expression of Tac/nephrin as in Figure 3C. *P < 0.05 versus wild type with Fyn; n = 5. (B) The same experiments as in A were repeated for Y1204F and quadruple mutant prepared in full-length nephrin with similar results. *P < 0.05 versus wild type with Fyn; n = 5 to 8.
Fyn Augments AP-1 Promoter Activation by Nephrin and Podocin
It was reported previously that nephrin activates the AP-1 promoter,which is significantly augmented by podocin (6). Although thephysiologic significance of this pathway is yet to be established,we used this system as a "readout" to address the functionalimpact of Fyn on nephrin/podocin-mediated signaling (Figure 6).The AP-1 promoter was activated by 2.9-fold by nephrin andpodocin. Addition of Fyn significantly augmented this activationto 4.4-fold. Fyn alone mildly activated the AP-1 promoter; however,when Fyn was added to nephrin alone or podocin alone, the increaseof the AP-1 promoter activity was small. These results are consistentwith the hypothesis that Fyn augments nephrin–podocininteraction, thereby augmenting nephrin-mediated signaling.
Figure 6. Fyn augments nephrin-podocin–mediated AP-1 activation. HEK293T cells were plated in 24-well plates and transfected with AP-1 luciferase (50 ng for all wells), nephrin-F (200 ng), F-podocin (200 ng), and Fyn (75 ng) as indicated, and luciferase activity was quantified as in the Materials and Methods section. *P < 0.05 versus nephrin+podocin; n = 3.
Nephrin Is Tyrosine Phosphorylated In Vivo
To begin to address the role of tyrosine phosphorylation ofnephrin by Src-family kinases in vivo, we first studied whethernephrin is tyrosine phosphorylated in normal glomeruli. We isolatedglomeruli from normal rats and lysed in buffer that containedphosphatase inhibitors (see the Materials and Methods section).Glomerular lysates were immunoprecipitated for nephrin and blottedfor phosphotyrosine and nephrin. Similar to the results in culturedcells, we identified tyrosine phosphorylation of nephrin (Figure 7A).Antinephrin immunoblot on glomerular lysates revealed twobands of 180 and 170 kD, consistent with previous reports (26,27).This doublet has been attributed to differential glycosylation.Tyrosine-phosphorylated nephrin always migrated at 180 kD. Whenglomerular lysates were treated with alkaline phosphatase, the180 kD phosphorylated nephrin band disappeared (Figure 7C).However, when the same samples were blotted for nephrin, therewas no visible decrease in the intensity of the 180 kD band(Figure 7C), suggesting either that dephosphorylation does notchange electrophoretic mobility or that only a small fractionof 180-kD nephrin is tyrosine phosphorylated. We also confirmedthat rat glomeruli express Fyn and Src (Figure 7B). The phospho-specificantibody directed at the positive regulatory tyrosine of Src(Y418) recognized a phosphorylated band of 60 kD even in normalrat glomeruli (Figure 7B). Because the region surrounding Y418is highly conserved in all of the related Src-family kinases,the phospho-specific Src antibody is likely to cross-react withthe other members of the family. Thus, at least some Src-familykinases are in an active form even in normal rat glomeruli.Taken together, these results indicate that the cytoplasmicdomain of nephrin could be tyrosine phosphorylated in vitroand in vivo and that Src-family kinases are the likely kinasesresponsible for this phosphorylation.
Figure 7. Nephrin is tyrosine phosphorylated in rat glomeruli. (A) Passive Heymann nephritis (PHN) was induced as in the Materials and Methods section. Glomerular lysates were prepared on day 14, immunoprecipitated for nephrin, and blotted for phosphotyrosine (Py20) and nephrin. Immunoblot of glomerular lysates for nephrin always showed two bands (180 and 170 kD) as reported by others (12,21). These double bands are attributed to differential glycosylation. Tyrosine phosphorylated nephrin always migrated at 180 kD. (Top) Immunoblot. (Bottom) Densitometry. *P < 0.005 versus normal rats; n = 6. (B) On day 14 of PHN, glomerular lysates were analyzed by immunoblotting for Src, phospho(activated)-Src, and Fyn. (C) Glomerular lysates were prepared in buffer without phosphatase inhibitors and incubated with 20 U of calf intestinal alkaline phosphatase for 30 min at 37°C before immunoprecipitation (lane 1).
We next studied how tyrosine phosphorylation of nephrin is affectedin the PHN model of membranous nephropathy. In PHN, it is knownthat complement C5b-9–mediated GEC injury leads to morphologicchanges of GEC and proteinuria. Glomerular lysates from normalrats and rats with PHN were immunoprecipitated with antinephrinantiserum and blotted for phosphotyrosine. We anticipated thatin glomeruli of rats with PHN, tyrosine phosphorylation of nephrinwould be decreased, leading to diminished interaction of nephrinand podocin. However, to our surprise, tyrosine phosphorylationwas markedly increased (4.7 ± 1.0-fold) in glomerulifrom rats with PHN, as compared with normal rats (n = 6; P <0.005; Figure 7A). The amount of active (phosphorylated) Src(-familykinases) was also increased in glomeruli from rats with PHN(Figure 7B). Tyrosine phosphorylation of nephrin was not increasedon day 5, when proteinuria was not observed yet (data not shown).These results suggest that loss of glomerular permselectivitycan be associated with both hypophosphorylation or hyperphosphorylationof nephrin, as occurs respectively in Fyn-deficient mice andin the PHN rat model (see the Discussion section).
Tyrosine phosphorylation of nephrin has been reported by fourother groups (9–12). Verma et al. (11) demonstrated thatthe Src-family kinase Fyn directly binds to and phosphorylatesnephrin. Simons et al. (10) reported that rat nephrin was tyrosinephosphorylated when rats received an injection of an antibodythat causes morphologic changes of podocytes. Similarly, Lahdenperaet al. (9) recently reported that clustering of nephrin by antibodiescaused strong tyrosine phosphorylation of nephrin in the cytoplasmicdomain. However, the direct consequence of nephrin phosphorylationwas not addressed in these studies. Nephrin and podocin bothare critical for glomerular permselectivity (2). Direct interactionof nephrin and podocin was demonstrated and is believed to beimportant for their functions (6,7). Thus, the current resultsdemonstrating that tyrosine phosphorylation of nephrin by Fynaugments nephrin–podocin interaction suggests that tyrosinephosphorylation of nephrin has a direct implication in normalGEC function. Podocin does not contain any domain known to bindphosphotyrosine (e.g., SH2 domain, PTB domain). Thus, most likely,tyrosine phosphorylation causes conformational changes of nephrin,which facilitate interaction with podocin. Another possibilityis that phosphorylated nephrin recruits a phosphotyrosine-bindingadapter molecule, which in turn recruits podocin. Of interest,it was recently demonstrated that the cytoplasmic domain ofnephrin binds to the p85 regulatory subunit of PI3K and togetherwith CD2AP and podocin activates the AKT signaling pathway (12)It is possible that Fyn-mediated tyrosine phosphorylation ofnephrin not only augments its interaction with podocin but alsofacilitates recruitment of additional signaling molecules suchas PI3K. In addition, it was recently reported that podocinfacilitates recruitment of nephrin to the lipid raft (8). Itwill be of interest to study whether tyrosine phosphorylationof nephrin by Src-family kinases would affect this process.However, because Src-family kinases are primarily localizedin the lipid raft (13), it is more likely that tyrosine phosphorylationwould modulate nephrin–podocin interaction after nephrinis recruited to the lipid raft. Precise location where Fyn ismodulating nephrin–podocin interaction will require furtherstudies.
Src-family kinases are known to regulate signaling of adhesionmolecules. c-Src has a pivotal role in integrin-mediated signaltransduction via focal adhesion kinase and also in cadherin-mediatedsignal transduction via catenins (15,28). Because nephrin actsas an adhesion molecule between adjacent foot processes of GEC,by analogy to integrins and cadherins, it is conceivable thatthe cytoplasmic domain of nephrin could serve as a scaffoldfor protein complexes that contain Src-family kinases and transmitsignals into the cells. A recent review by Benzing (29) highlightedthe role of the slit diaphragm proteins in intracellular signalingpathways and the potential role of tyrosine phosphorylationof nephrin and Neph1 in these pathways. Our data indicatingthat Fyn augments AP-1 promoter activation by nephrin and podocinis in line with these newly emerging concepts.
Fyn-knockout mice demonstrate morphologic changes of podocytesand/or proteinuria (11,16). Because nephrin can be phosphorylatedby Fyn, it is tempting to hypothesize that Fyn-mediated tyrosinephosphorylation of nephrin is important for the maintenanceof normal morphology and permselectivity of GEC. Increased nephrin–podocininteraction by Fyn in vitro and tyrosine phosphorylation ofnephrin in normal rat glomeruli would be in agreement with thishypothesis. Curiously, in the PHN rat model of membranous nephropathy,nephrin tyrosine phosphorylation was increased by almost fivefold,as compared with normal rats. This seems to contradict the hypothesisthat tyrosine phosphorylation by Fyn is important for normalGEC function. There are several possible interpretations forthese results. It is possible that the primary target of injuryin PHN is not nephrin; thus, hyperphosphorylation of nephrinis either an epiphenomenon or a compensatory mechanism afterGEC injury. Alternatively, a delicate balance of nephrin phosphorylationand dephosphorylation might be important for maintenance ofnormal podocyte function. Although facilitation of nephrin–podocininteraction by the action of Fyn may be necessary for normalpodocyte function, transient interruption of this associationmay also be required in vivo. Thus, hyperphosphorylation ofnephrin as observed in PHN could lead to inappropriate regulationof nephrin–podocin interaction. In addition, we have notedthat protein expression of nephrin was consistently decreasedwhen it was expressed with Fyn (Figures 2E, 4D, and 5B). Lahdenperaet al. (9) also noted that the nephrin mutant that lacks mostof the cytoplasmic domain (thus cannot be phosphorylated) hada higher protein expression, as compared with the wild-typenephrin. Thus, it is also possible that hyperphosphorylationof nephrin could lead to a decreased protein stability of nephrin.
In summary, the current study demonstrates that the cytoplasmicdomain of nephrin is tyrosine phosphorylated in vivo and invitro. This tyrosine phosphorylation influences nephrin–podocininteraction and nephrin-mediated signaling. How tyrosine phosphorylationof nephrin is modulated and contributes to proteinuria in kidneydiseases requires further studies.
Acknowledgments
This study was supported by grants from the Canadian Instituteof Health Research (T.T., S.L.); the Kidney Foundation of Canada(T.T., S.L.); and the Ministry of Education, Science, Cultureand Sports of Japan (H.K.). T.T. and S.L. are recipients ofa scholarship from the Canadian Institute of Health Research.
We are grateful for the assistance of Dr. Benzing in settingup AP-1 luciferase assay.
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Received for publication December 4, 2003.
Accepted for publication September 15, 2004.
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Top
Abstract
Introduction
-actinin-4 (2). These molecules are believed to maintain the normal morphology and permselectivity of podocyte by interacting with the actin cytoskeleton, but their precise mechanisms of action are largely unknown. 
150 amino acids), which contains six tyrosine residues conserved in human, mouse, and rat sequences (Figure 1A). Recently, several groups reported that nephrin is tyrosine phosphorylated in the cytoplasmic domain (9–11). The injection of a monoclonal antibody that causes morphologic changes of podocyte induced marked tyrosine phosphorylation of nephrin (10). Clustering of nephrin by antibodies caused strong tyrosine phosphorylation of nephrin in the cytoplasmic domain (9). Also, it was shown that the Src-family tyrosine kinase Fyn bound and phosphorylated nephrin (11). However, the significance of nephrin tyrosine phosphorylation has yet to be elucidated. It was recently reported that the cytoplasmic domain of nephrin binds to the regulatory p85 subunit of phosphoinositide 3-kinase (PI3K) and together with CD2AP and podocin activates the Akt signaling pathway (12). It was suggested that this binding is dependent on tyrosine phosphorylation of nephrin (12). 
-galactoside, and lipofectamine 2000 reagent were purchased from Invitrogen-Life Technologies (Burlington, ON, Canada). Sodium orthovanadate and other standard chemicals were from Sigma Chemical Co. (St. Louis, MO). Protease inhibitor cocktail, reagents for PCR, and calf intestinal alkaline phosphatase were from Roche Diagnostics (Laval, QC, Canada). Antiphosphotyrosine antibody Py20 was from BD Biosciences (San Jose, CA). Rabbit anti-CD2AP antibody, rabbit anti-Fyn antibody, mouse anti-Myc antibody, and protein A-Sepharose were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Src [pY418] phospho-specific antibody was from Biosource International (Camarillo, CA). Src phosphorylated at Y418 is known to be active. Dual-Luciferase Reporter Assay System was from Promega (Madison, WI). cDNA encoding Src, T lymphocyte Fyn (FynT), kinase-negative FynT, and C-terminal Src kinase (Csk; all in the mammalian expression vector pME18S) were gifts from Dr. Junichi Abe (University of Rochester, Rochester, NY) (17–19). cDNA Tac/zeta/zeta (20) was a gift from Dr. Sylvain Latour (Hôpital Necker Enfants-Malades, Paris, France). Isolation of full-length rat nephrin was reported earlier (21), and the coding region was subcloned into the EcoRV/XbaI sites of pcDNA3.1/HisA (Invitrogen-Life Technologies). Plasmids for AP-1 luciferase, nephrin-F, and F-podocin were gifts from Dr. Thomas Benzing (Freiburg, Germany) (6). 
