SUMOylation determines turnover and localization of nephrin at the plasma membrane

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http://www.kidney-international.org & 2014 International Society of Nephrology

SUMOylation determines turnover and localization of nephrin at the plasma membrane Irini Tossidou1,2, Erik Himmelseher1,2, Beina Teng1, Hermann Haller1 and Mario Schiffer1 1

Hannover Medical School, Division of Nephrology, Hannover, Germany

Podocyte effacement and the reformation of foot processes and slit diaphragms can be induced within minutes experimentally. Therefore, it seems likely that the slit diaphragm proteins underlie orchestrated recycling mechanisms under the control of posttranslational modifiers. One of these modifiers, SUMO (small ubiquitin-like modifier), is an ubiquitin-like protein with a 20% corresponding identity to ubiquitin. Modification by SUMOs to proteins on lysine residues can block the ubiquitination of the same site leading to the stabilization of the target protein. Here we found in vitro and in vivo that nephrin is a substrate modified by SUMO proteins thereby increasing its steady-state level and expression at the plasma membrane. A conversion of lysines to arginines at positions 1114 and 1224 of the intracellular tail of murine nephrin led to decreased stability of nephrin, decreased expression at the plasma membrane, and decreased PI3K/AKT signaling. Furthermore, treatment of podocytes with the SUMOylation inhibitor ginkgolic acid led to reduced membrane expression of nephrin. Similarly, the conversion of lysine to arginine at position 1100 of human nephrin caused decreased stability and expression at the plasma membrane. As SUMOylation is a reversible process, our results suggest that SUMOylation participates in the tight orchestration of nephrin turnover at the slit diaphragm. Kidney International advance online publication, 18 June 2014; doi:10.1038/ki.2014.198 KEYWORDS: endocytosis; glomerular disease; glomerular filtration barrier

Correspondence: Irini Tossidou or Mario Schiffer, Division of Nephrology, IFB-TX Hannover, Hannover Medical School, Carl-Neuberg-Street 1, 30625 Hannover, Germany. E-mail: [email protected] or [email protected] 2

These authors contributed equally to this work.

Received 5 February 2013; revised 15 April 2014; accepted 17 April 2014 Kidney International

Podocyte damage including ultrastructural changes and decreased expression of components of the slit diaphragm such as nephrin, podocin, and CD2AP is the basis of many glomerular diseases. Under physiological conditions, it is likely that the slit diaphragm undergoes permanent renewal processes to indemnify its stability in response to changes in filtration pressure. This would require constant reorganization of the podocyte foot processes and the renewal of slit diaphragm components. Several proteins were found to be involved in the formation and reorganization of foot processes. These mechanisms are of fundamental importance during podocyte development and repair.1,2 Thus far, the mechanisms underlying the turnover of slit diaphragm proteins are largely unknown. One of the major components of the slit diaphragm is nephrin. Nephrin is a transmembrane adhesion protein of the Ig superfamily, encoded by NPHS1. Humans and mice lacking nephrin are born without intact slit diaphragms and develop massive proteinuria in utero.3–5 Given its structure and localization, it is not surprising that nephrin, similar to membrane receptors, has signal transduction properties that underlie the same mechanisms of desensitization. We and others showed that nephrin endocytosis is triggered by several molecules, including b-arrestin, protein-kinase C alpha, and CIN85 (Cbl-interacting protein of 85 kDa), the paralog of CD2AP.6–8 The mechanisms by which nephrin molecules are targeted for endocytosis or stabilized at the slit diaphragm are largely unknown. The posttranslational modification of proteins via SUMOylation is a major regulatory pathway of protein function that has an important role in a wide range of cellular processes such as protein stability, localization, and activity.9–11 Vertebrates express four B100 amino-acid SUMO proteins: SUMO-1, -2, -3, and -4. Of those, SUMO-1–3 are ubiquitously expressed, whereas the recently reported SUMO-4 seems to be expressed predominantly in the kidney, lymph node, and spleen. SUMO-2 and -3 are nearly identical, whereas SUMO1 has only 56% identity with SUMO-2 and -3. SUMOs are similar to ubiquitin in their three-dimensional structure, and the steps involved in the SUMO pathway resemble those of the ubiquitin pathway.9,12,13 In contrast to ubiquitination, SUMOs attach to lysines that are often found within a small consensus motif cKxE (where c is a large hydrophobic amino acid and x can be any amino acid). However, some acceptor sites have been identified that do not contain this 1

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motif.12 SUMO modification occurs through an enzymatic pathway consisting of an E1 activation enzyme (SAE-2/1), an E2 conjugating enzyme (Ubc9), and a number of E3 ligases.14 Ubc9 is capable of directly modifying substrates through interaction with the SUMO conjugation motif cKxE.15 Until now, only a few plasma membrane proteins have been identified as SUMO substrates, and molecular details on regulatory aspects are still scarce. One receptor identified as SUMO substrate, where mechanistic insights have been obtained, is the TGF-b receptor type-I. SUMOylation of the TGF-b type-I receptor depends on response to TGF-b and enhances the receptor function by facilitating the recruitment and phosphorylation of Smad3.16,17 Other SUMOylated plasma membrane proteins are the K2P1 potassium-leak channel and the metabotropic glutamate receptor-8 (mGluR8); however, for these two molecules, the consequences of SUMOylation on protein function are still unclear.18,19 As nephrin is a central point for protein interaction and signal transduction at the slit diaphragm, posttranslational modification of the intracellular part could have a key function for slit diaphragm integrity. So far, glycosylation, phosphorylation, and ubiquitination have been shown to modify nephrin posttranslationally.1,2,7,20,21 Here we provide evidence that SUMO proteins are conjugated to nephrin and thereby determine its stability and localization at the plasma membrane. RESULTS SUMO proteins are associated with nephrin in vivo

To evaluate whether nephrin is modified by SUMO in vivo, we performed immunofluorescence stainings of frozen kidney cortex sections from 8-week-old C57/Bl6 mice. Confocal microscopy revealed a colocalization of SUMO-1, SUMO-2/3, and Ubc9 with nephrin. SUMO-1 and SUMO-2/3 show a strong nuclear pattern indicated by colocalization with the nuclear podocyte marker WT-1. Ubc9 reveals only a slight colocalization with WT-1 but a stronger colocalization with nephrin (Figure 1a). No colocalization could be observed with the basement membrane marker Nidogen (Supplementary Figure S1A online). Immunoprecipitation experiments from isolated glomeruli show an association of SUMO-1 and SUMO-2/3 with nephrin (Figure 1b). Immunoprecipitation experiments with lysates from cultured podocytes confirm our results from the isolated glomeruli and show an association of SUMO-1 and SUMO2/3 with nephrin. (Figure 1c and Supplementary Figure S1B online). In contrast, immunoprecipitation experiments with SUMO-4 showed no association with nephrin (data not shown). The presence of SUMO-1 and SUMO-2 increases the stability of nephrin and determines the localization

It has been reported by a number of groups that SUMO modification may regulate the stability of proteins.9,10,22 To analyze the stability of nephrin, we performed cycloheximide assays.23,24 We examined the steady-state levels of 2

I Tossidou et al.: SUMOylation of nephrin

flag-mNephrin by comparing HEK-293T cells cotransfected with pEGFPN1 empty vector, pEGFP-SUMO-1, or pEGFPSUMO-2 treated with cycloheximide, a protein biosynthesis inhibitor, for up to 10 h. As depicted in Figure 2a, the steadystate level of murine nephrin is increased in the presence of pEGFP-SUMO-1 and pEGFP-SUMO-2. As we demonstrated that the presence of SUMO-1 and SUMO-2/3 enhanced the stability of nephrin, we wanted to examine whether the inhibition of nephrin SUMOylation could decrease nephrin stability. We transfected HEK-293T cells with mNephrin-flag and cotreated the cells with cycloheximide and ginkgolic acid, an anacardic acid that blocks the formation of the E1-SUMO intermediate.25 The steady-state level of nephrin is decreased in the presence of ginkgolic acid. This result led us to the hypothesis that ginkgolic acid inhibits endogenous SUMOylation of nephrin and thereby decreases its stability. On the other hand, treatment with lactacystin, a proteasomal inhibitor, increases the stability of nephrin by the inhibition of proteasomal degradation. (Figure 2b). As the expression of nephrin at the plasma membrane is necessary for its proper function and formation of the slit diaphragm, we examined whether the presence of SUMO-1 and SUMO-2 enhanced the expression of nephrin at the plasma membrane. To accomplish this, we cotransfected HEK-293T cells with flagmNephrin and pEGFPN1 empty vector and either pEGFPSUMO-1 or pEGFP-SUMO-2 and treated the cells with ginkgolic acid or lactacystin. By using a protein fractionation kit, we prepared a separation of membrane, cytosol, and nuclear fractions. Expression of nephrin is increased in the membrane fraction in the presence of SUMO-1 and SUMO-2 compared with pEGFPN1 alone. Analysis of total cell extracts revealed equal loading (Supplementary Figure S2 online). Treatment with ginkgolic acid decreases nephrin in the membrane fraction, whereas lactacystin treatment increases expression (Figure 2c). Interestingly, we detected two distinct protein bands for nephrin. This migrating behavior of nephrin has been described earlier. The upper band was present in the plasma membrane and the lower band showed an intracellular localization.26 We could also primarily detect the upper band in the membrane fraction, which was earlier described as a specific posttranslationally modified form of nephrin.26 Calreticulin and TGN38, proteins of the membranes of the endoplasmatic reticulum or Golgi compartment, were shown for specificity of fractionation. Immunoprecipitation of cotransfected HEK-293T cells with flag-mNephrin and pEGFPN1 empty vector, pEGFP-SUMO-1, or pEGFP-SUMO-2 show a specific interaction of nephrin with SUMO-1 and SUMO-2/3, which is strongly diminished or almost absent after treatment with ginkgolic acid (Figure 2d). As we demonstrated that the presence of SUMO-1 and SUMO-2 determine localization and expression of nephrin at the plasma membrane, we wanted to verify these results by a biotinylation assay. Biotinylation of the cell surface and streptavidin precipitation reveal a decreased expression of nephrin in the absence of SUMO-1 and after treatment with ginkgolic acid (Figure 2e). Kidney International

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Figure 1 | SUMO proteins are associated with nephrin in vivo. Fluorescence labeling demonstrates nephrin (upper panel) or WT-1 (lower panel) (green) and SUMO-1 and -2/3 and Ubc9 (red) in renal cortex sections of 8-week-old mice (a). Colocalization of the indicated proteins resulting in yellow fluorescence is depicted with white arrows in the merged pictures. Whole lysates from isolated glomeruli (b) or cultured podocytes (c) were precipitated (IP) with SUMO-1 and -2/3 antibodies. The probes were blotted and analyzed for nephrin content. For IgG control, glomerular and podocyte lysates were incubated with normal rabbit IgG instead of specific antibody. Asterisks indicate detection of unspecific IgG heavy chains of the antibodies.

Identification of the potential SUMOylation sites in the cytoplasmatic tail of nephrin

To map the potential sites on the cytoplasmic tail of nephrin that could be modified by SUMOylation, we analyzed potential SUMOylation motifs using ‘SUMOplot prediction’ algorithm.27 We identified one nonconsensus SUMOylation sequence motif, EKTE, which is conserved in human, mouse, and rat nephrin. This predicted lysine could also be found in zebrafish and in invertebrates, including the yellow fever mosquito (Figure 3a). In the cytoplasmic tail region of murine nephrin, we found an additional consensus SUMOylation motif VKYE. Analysis of predicted ubiquitination sites by the program BDM-PUB (http://bdmpub.biocuckoo.org/), from Dr Yu Xue, University of Science & Technology of China, revealed one site in the intracellular part of nephrin. For murine nephrin, the location is on lysine position K1114 and for human nephrin on position K1100 (Supplementary Figure S3 online). To explore whether these sites are modified by SUMOylation, we exchanged the specific lysine to arginine.10,16,23 Next, we generated a mutant of human nephrin where the lysine on Kidney International

position 1100 was converted to arginine. For murine nephrin, we generated two single mutants on positions 1114 and 1224 and additionally a double mutant and used these constructs for in vitro SUMOylation experiments. We incubated purified protein of murine WT-nephrin, single mutants, and double mutants of nephrin with purified SUMO-1 and SUMO-2 according to the manufacturer’s protocol. We detected a weak SUMOylation of the single mutant flag-mNephrinK1224R but no SUMOylation of the single mutant flag-mNephrinK1114R or the double mutant flag-mNephrink1114R/K1224R. In contrast, we detected a strong SUMOylation signal when we used WTmNephrin, especially in the presence of SUMO-1 (Figure 3b). Analysis of purified nephrin proteins revealed equal loading (Supplementary Figure S4B online). Nephrin protein stability is decreased in the absence of SUMO motifs

Next, we wanted to analyze whether the presence or absence of these SUMOylation motifs affects the protein stability of nephrin. We examined the dynamics of protein turnover 3

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by comparing protein lysates of HEK-293T cells transfected with WT-flag-mNephrin and flag-mNephrinK1114R, flag-mNephrinK1224R, or flag-mNephrinK1114R/K1224R treated

with cycloheximide over 10 h. As depicted in Figures 4a–c, the expression levels of the single and double mutants are significantly decreased compared with WT-Nephrin.

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and quantification revealed a half-life for murine nephrin of about 270 min, which is significantly enhanced in the presence of SUMO-1 and SUMO-2. The lysine mutants displayed a significantly lower half-life (Figure 4d).

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Figure 3 | Identification of the SUMO modification sites in murine and human nephrin. (a) Schematic representation of the evolutionary conserved putative SUMO site in the intracellular part of human and murine nephrin, where lysine on position 1110 in human nephrin and on position 1114 in murine nephrin is the putative acceptor amino acid. Murine nephrin exhibits also a second putative SUMO site on lysine position 1224. This lysine lies within a consensus SUMOylation sequence. (b) Purified flag-mNephrin, flag-mNephrinK1114R, flag-mNephrinK1224R and flag-mNephrinK1114R/K1224R were incubated for 30 min with purified SUMO-1 or SUMO-2 and immunoblotted for SUMO-1 and SUMO-2. Asterisks depict free SUMO-1 and SUMO-2. Purified nephrin protein was immunoblotted for control and detected using anti-flag antibodies.

Figure 2 | SUMO-1 and SUMO-2 enhance the stability of nephrin and determine localization. (a) HEK-293T cells were transiently transfected with mNephrin-flag and SUMO-1-GFP (upper panel), SUMO-2-GFP (lower panel), or with the empty GFPN1. The cells were incubated for 0, 2, 4, 6, 8, and 10 h with 2 mM cycloheximide. Whole-cell lysates were immunoblotted for the detection of flag and GFP. Densitometry summarizes means of nephrin/GAPDH expression (black bars transfected cells with flag-mNephrin and GFPN1, gray bars transfected with mNephrin-flag and SUMO-1-GFP or SUMO-2-GFP) obtained from three independent experiments. *Po0.02 compared GFPN1/SUMO-1 or GFPN1/SUMO-2 by Student’s t-test. (b) HEK-293T cells were transiently transfected with mNephrin-flag. The cells were incubated for 0, 2, 4, 6, 8, and 10 h with 2 mM cycloheximide and simultaneously with 2 mM ginkgolic acid (upper panel) or 2 mM lactacystin (lower panel). Whole-cell lysates were immunoblotted for the detection of flag and GAPDH. Densitometry summarizes the means of nephrin þ DMSO/GAPDH expression (black bars) and nephrin þ ginkgolic acid/GAPDH or nephrin þ lactacystin / GAPDH (gray bar) obtained from three independent experiments. *Po0.01 compared DMSO / ginkgolic acid or DMSO/lactacystin by student’s t-test. HEK-293T cells were transfected with flag-mNephrin and empty vector, SUMO-1-GFP, or SUMO-2-GFP, and treated with DMSO only, ginkgolic acid, or lactacystin. (c) Subcellular fractionation into membrane cytosolic and nuclear fractions depict the expression of mNephrin-flag in the presence of SUMO-1 (left panel) or SUMO-2 (right panel). As control for purity and loading, the fractions were analyzed for Gia3 (membrane fraction), GAPDH (cytosolic fraction), Histone N1 (nuclear fraction), calreticulin (ER-containing fraction), TGN38 Golgi-containing fraction, and GFP. (d) HEK-293T cells were transiently transfected with mNephrin-flag and empty vector, SUMO-1-GFP, or SUMO-2-GFP, and treated with DMSO only or ginkgolic acid. Nephrin was immunoprecipitated with flag-coupled beads. Immunoblot was analyzed for GFP, control lysates were analyzed for flag- and GFP- expression. (e) HEK-293T cells were transiently transfected with mNephrin-flag and empty vector or SUMO-1-GFP and treated with DMSO only, ginkgolic acid, or lactacystin. After surface biotinylation and precipitation with streptavidin beads, immunoblot was analyzed for flag and control lysates for flag and GFP expression. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Kidney International

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Figure 4 | SUMO motif–mutated nephrin displays decreased protein stability. HEK-293T cells were transiently transfected with flagmNephrin and flag-mNephrinK1114R (a), or flag-mNephrinK1224R (b) or mNephrinK1114R/K1224R (c). The cells were incubated for 0, 2, 4, 6, 8, and 10 h with 2 mM cycloheximide. Whole-cell lysates were immunoblotted for flag and GAPDH. Densitometry summarizes the means of WT-nephrin/mutated nephrin expression (black bars transfected cells with mNephrin-flag, gray bars transfected with mutated mNephrin-flag) obtained from three independent experiments. *Po0.02 compared WT-nephrin/mutated nephrin by Student’s t-test. (d) Analysis of half-life times were performed by Excel.

Cellular localization of the nephrin lysine mutants

To further examine the localization of the lysine nephrin mutants in podocytes, we cotransfected pEGFP-WT-mNephrin, pEGFP-mNephrinK1114R, pEGFP-mNephrinK1224R, and pEGFP-mNephrinK1114R/K1224R with a pDsred2-ER construct that encodes for calreticulin, a protein that is specifically expressed in the endoplasmic reticulum. It is known that the transport pathway of nephrin goes via the ER.26 Fluorescence analysis revealed that the lysine mutants displayed a complete ER colocalization, except for pEGFP-mNephrinK1224R, which was also slightly detected at the plasma membrane. pEGFPWT-mNephrin displayed both a plasma membrane and an ER localization (Figure 5a). Furthermore, lactacystin treatment revealed a strong cytosolic and membrane localization of nephrin, whereas treatment with tunicamycin, an inhibitor of N-glycosylation, and ginkgolic acid displayed a complete ER localization (Supplementary Figure S5 online). The depicted results of nephrin expression are representative of the majority of transfected cells verified in three independent experiments. To examine intracellular localization of nephrin lysine mutants in more detail and complement our fluorescence analysis, we performed subcellular fractionation of 6

transfected HEK-293T cells. The lysine mutants flag-mNephrinK1114R and flag-mNephrinK1114R/K1224R could not be detected in the plasma membrane fraction. In contrast, flag-mNephrinK1224R is slightly expressed at the plasma membrane compared with flag-WT-mNephrin, which is clearly detectable in that compartment. Interestingly, the lower band of the lysine mutant predominantly was detected in the cytosolic fraction, indicating a dependency of SUMO protein modification for localization of nephrin at the plasma membrane (Figure 5b). Analysis of total cell extracts revealed equal loading (Supplementary Figure S4B online). As it was demonstrated earlier that nephrin is involved in PI3K-dependent AKT activation in podocytes,28 we wanted to examine AKT phosphorylation in the presence of the lysine mutants of nephrin. HEK-293T cells were transfected with flag-WT-mNephrin, flag-mNephrinK1114R, flag-mNephrinK1224R, and flag-mNephrinK1114R/K1224R constructs. Before lysis, the cells were incubated for 30 min with the PI3K-inhibitor LY294002. Western blot analysis showed a significantly reduced AKT phosphorylation of the lysine mutants compared with WT-Nephrin. Compared with NephrinK1114R and NephrinK1114R/K1224R, NephrinK1224R revealed a stronger AKT Kidney International

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Figure 5 | Reduced expression of SUMO motif–mutated nephrin at the plasma membrane. Podocytes were transfected with plasmids expressing GFP-tagged wild-type or mutant nephrin constructs as indicated (green) and pDS2Red-ER-vector, which labels the endoplasmatic reticulum (red). The transfected cells were analyzed by fluorescence microscopy. The nucleus is stained with 40 ,6-diamidino-2-phenylindole (blue). (b) HEK-293T cells were transiently transfected with mNephrin-flag and flag-mNephrinK1114R, flag-mNephrinK1224R, or mNephrinK1114R/K1224R. Subcellular fractionation into membrane cytosolic and nuclear fractions was performed and analyzed for nephrin content. As a control, fractions were analyzed for Gia3 (membrane fraction), GAPDH (cytosolic fraction) and Histone N1 (nuclear fraction), calreticulin (ER-containing fraction), and TGN38 (Golgi-containing fraction) expression. (c) HEK-293T cells were transiently transfected with mNephrin-flag and flagmNephrinK1114R or flag-mNephrinK1224R or mNephrinK1114R/K1224R and incubated with LY294002 (PI3K-Inhibitor) or as control with DMSO for 30 min. Whole-cell lysates were immunoblotted for phospho-AKTS473, AKT, and flag.

phosphorylation (Figure 5c). These data indicate that nephrin-dependent AKT phosphorylation depends on the SUMOylation state of nephrin. SUMO proteins are associated with human nephrin

To evaluate whether human nephrin is modified by SUMO, we performed immunoprecipitation experiments with human podocyte lysates, which revealed an association of SUMO-1 and SUMO-2/3 with nephrin, similar to the results that we had obtained in murine podocytes (Figure 6a). To examine the influence of SUMO proteins on steady-state levels of mychNephrin, HEK-293T cells were cotransfected with pEGFP empty vector, pEGFP-SUMO-1, or pEGFP-SUMO-2, and treated with cycloheximide for up to 10 h. Similar to our Kidney International

results obtained for the murine nephrin construct, we found that the steady-state level of human myc-Nephrin is increased in the presence of pEGFP-SUMO-1 and pEGFP-SUMO-2 (Figure 6b). As human nephrin has only one predicted SUMO motif, we also examined the protein stability and steady-state levels of human nephrin by comparing HEK-293T cells transfected with human myc-Nephrin and myc-NephrinK1100R, treated with cycloheximide for up to 10 h. As depicted in Figure 6c, the protein stability of the human nephrin mutant was significantly decreased compared with WT nephrin. Analysis and quantification reveal a half-life for human nephrin at 500 min, which is significantly enhanced in the presence of SUMO-1 and SUMO-2. The lysine mutant displayed a significantly lower half-life (Figure 6d). 7

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Lysate

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Figure 6 | SUMOylation of human nephrin in vitro. (a) Cultured human podocytes were immunoprecipitated (IP) with SUMO-1 and -2/3 antibodies. The probes were analyzed for nephrin content. For IgG control, glomerular lysates were incubated with normal rabbit IgG instead of specific antibody. Asterisk indicates unspecific IgGs. (b) HEK-293T cells were transiently transfected with hNephrin-myc and SUMO-1-GFP (left panel) or SUMO-2-GFP (right panel) or with the empty GFPN1. The cells were incubated for 0, 2, 4, 6, 8, and 10 h with 2 mM cycloheximide. Whole-cell lysates were immunoblotted for myc and GFP. Densitometry summarizes means of hNephrin-myc/GAPDH expression (black bars transfected cells with hNephrin-myc and GFPN1, gray bars transfected with hNephrin-myc and SUMO-1-GFP or SUMO-2-GFP) obtained from three independent experiments. *Po0.01 compared GFPN1/SUMO-1 or GFPN1/SUMO-2 by Student’s t-test. (c) HEK-293T cells were transiently transfected with hNephrin-myc and hNephrinK1100R-myc. The cells were incubated for 0, 2, 4, 6, 8, and 10 h with 2 mmol/L cycloheximide. Wholecell lysates were immunoblotted for myc and GAPDH. Densitometry summarizes means of hNephrin/GAPDH and hNephrinK1100R/GAPDH expression (black bars transfected cells with hNephrin-myc and GFPN1, gray bars transfected with hNephrin-myc and hNephrinK1100R-myc obtained from three independent experiments). *Po0.03 compared GFPN1/SUMO-1 or GFPN1/SUMO-2 by Student’s t-test. (d) Analysis of half-life times were performed by Excel. (e) HEK-293T cells were transiently transfected with hNephrin-myc and hNephrinK1100R-myc. Subcellular fractionation into membrane cytosolic and nuclear fractions depict expression of human nephrin. As control for purity and loading, the fractions were analyzed for Gia3 (membrane fraction), GAPDH (cytosolic fraction), Histone H1 (nuclear fraction), calreticulin (ER-containing fraction), and TGN38 (Golgi-containing fraction).

To examine the expression profiles of the human nephrin lysine mutant in more detail, we performed subcellular fractionation of transfected HEK-293T cells. The lysine 8

mutant myc-hNephrinK1100R was only slightly detected in the plasma membrane fraction compared with normal human nephrin, indicating a clear dependency of human Kidney International

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I Tossidou et al.: SUMOylation of nephrin

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Figure 7 | Treatment of mice with ginkgolic acid leads to de-SUMOylation of nephrin in vivo and glomerular proteinuria. Fluorescence labeling demonstrates (a) nephrin (green) and Ubc9 (red), (b) nephrin and SUMO-1, and (c) nephrin and SUMO-2/3 in renal cortex sections of 8-week-old mice treated with 500 mg of ginkgolic acid or DMSO as control. Colocalization of the indicated proteins resulted in yellow fluorescence and is depicted with white arrows in the merged pictures. (a, right panel) Coomassie gel of urine samples indicates proteinuria in mice 24 h after treatment with ginkgolic acid. Whole lysates from isolated glomeruli were immunoprecipitated (IP) with SUMO-1 (b, right panel) and -2/3 (c, right panel) antibodies. The probes were blotted and analyzed for nephrin content. Asterisks indicate detection of unspecific IgG heavy chains of the antibodies. (d) Podocytes transduced with an adenoviral pEGFP-Nephrin expression vector were treated with DMSO only for control or with 2 mM ginkgolic acid for 10 h. Expression pattern was analyzed according to normal cellular distribution and membrane association of GFP-Nephrin (expression pattern A, control) or perinuclear expression (expression pattern B, ginkgolic acid–treated cells). Hundred cells from each of three independent experiments were analyzed blindly for cellular distribution and plasma membrane localization of GFP-Nephrin. Analysis shows the percentage of cells with GFP-Nephrin in expression pattern A or B after DMSO or ginkgolic acid treatments.

nephrin protein stability and subcellular localization on the SUMOylation state (Figure 6e). Analysis of total cell extracts revealed equal loading (Supplementary Figure S4C online). Treatment of mice with ginkgolic acid leads to proteinuria and de-SUMOylation of nephrin in vivo

To evaluate the in vivo significance for nephrin SUMOylation, we treated 8-week-old male mice with ginkgolic acid (500 mg/animal) dissolved in DMSO or with the same volume of DMSO only as a control for 24 h. Before and after that, spot urine was collected and the animals were killed. Immunofluorescence staining of frozen kidney sections from both groups revealed a decreased nephrin expression and colocalization with Ubc9, SUMO-1, and SUMO-2/3 in the ginkgolic acid–treated mice compared with the DMSOtreated group (Figures 7a–c, left panels). Furthermore, mice treated with ginkgolic acid for 24 h exhibited a significant proteinuria, as confirmed by Coomassie gel (Figure 7a, right panel). Immunoprecipitation experiments from isolated glomeruli indicated a decreased association of SUMO-1 and SUMO-2/3 with nephrin in glomerular protein lysates Kidney International

from ginkgolic acid–treated mice (Figures 7b and c, right panels). To confirm these results further in cultured podocytes, we generated an adenoviral GFP-Nephrin expression vector and transduced podocytes with a wild-type GFP-Nephrin. The cells were treated with ginkgolic acid or with DMSO for 10 h. Next, cells were analyzed for cellular distribution of GFP. More than 80% of the cells treated with DMSO only showed a homogeneous GFP distribution throughout the podocyte cell body including plasma membrane localization of nephrin (expression pattern A) compared with cells treated with ginkgolic acid, with less than 10% of cells showing nephrin at the plasma membrane (Figure 7d). In contrast, the GFP expression in the ginkgolic acid–treated group was in a perinuclear pattern (expression pattern B), suggestive for the Golgi/ER area. These data indicate a direct effect of SUMOylation on nephrin expression in vivo, as well as in cultured podocytes. DISCUSSION

The central role of nephrin in maintaining glomerular filtration barrier function was clearly defined in several publications in 9

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the past decade.3–5 Several groups have suggested that along with genetic diseases also alterations in normal nephrin expression are involved in acquired glomerular diseases.26,29 SUMOylation of proteins can alter their function, activity, or localization.9,11,12 Until now, only a few membrane proteins or receptors are known to be regulated by SUMOylation. Among these are K þ channels, nonselective cation channels, kainite receptors, glutamate receptor-8, and the TGF-b receptor.16–19,30,31 A well-described posttranslational modification of nephrin is glycosylation. The group of Karl Tryggvasson proposed that N-glycosylation of nephrin is crucial for its proper folding and plasma membrane localization.21 Furthermore, they concluded that inhibition of this process could be an important factor in the pathogenesis and disease onset of some acquired glomerular diseases.21 However, the detailed processes regarding how nephrin is transported and stabilized at the plasma membrane remain unclear. In this report, we present evidence that nephrin is a target protein for SUMOylation by SUMO-1 and SUMO-2/3. We found that nephrin is SUMOylated in vivo and in vitro. Western blot assays using the protein biosynthesis inhibitor cycloheximide revealed a significantly increased protein stability of nephrin in the presence of SUMO-1 and SUMO-2 and decreased stability after treatment with the SUMOylation inhibitor ginkgolic acid. Interestingly, nephrin shows a significantly shorter half-life in the absence of the SUMO proteins. These data represent supporting evidence that nephrin undergoes a tightly regulated turnover process. To analyze whether SUMO proteins can enhance the expression of nephrin in the plasma membrane compartment, which is crucial for stabilization and maintenance of the slit diaphragm, we performed subcellular fractionation and biotinylation assays. The results revealed an enhanced expression of nephrin in the plasma membrane in the presence of SUMO-1 and SUMO-2 and a decreased expression after treatment with ginkgolic acid, a SUMOylation inhibitor that disrupts the formation of the E1-SUMO intermediate. Interestingly, on SDS gel electrophoresis, nephrin is migrating in two isoforms: the upper band can be detected in the plasma membrane, whereas the lower band is primarily present in the cytosolic fraction. The two different protein forms have been observed by other groups previously, and they already speculated that this minor difference in molecular migration behavior between the two nephrin isoforms is most likely due to specific posttranslational modifications.26 After treatment with ginkgolic acid, the expression of the upper band is strongly diminished but not absent. Therefore, we speculate that SUMOylation is only one of multiple modifications that could act on nephrin simultaneously. On the basis of these results, we hypothesized that non-SUMOylated nephrin can leave the plasma membrane fraction for intracellular parts and is either degraded or recycled to the plasma membrane. To further analyze the potential influence of SUMOylation on nephrin, we searched for putative lysines within the intracellular part of nephrin that could be modified by 10

I Tossidou et al.: SUMOylation of nephrin

SUMOylation. We detected an evolutionary conserved lysine residue, which lies within a nonconsensus sequence in murine and human nephrin. A second putative lysine, which lies within a SUMO-consensus sequence, could be only found in murine nephrin. Our results demonstrate that mutated human and murine nephrin that cannot be SUMOylated display a decreased expression in the plasma membrane and a decreased protein half-life. Interestingly, SUMO can compete with ubiquitin for lysine sites. Analysis of predicted ubiquitination sites by the BDM-PUB software from Dr Yu Xue, University of Science & Technology of China, revealed that there is only one such site in the intracellular part of nephrin. In murine nephrin, the site is detected on lysine position K1114 and for human nephrin on position K1100 (Supplementary Figure S3 online), which we have identified as specific SUMOylation sites in this work. Further analysis of SUMOylation and ubiquitination could elucidate the detailed trafficking pathway of nephrin, and further studies on this are ongoing in our laboratory. The Tryggvasson group previously published that defective nephrin trafficking could be caused by a missense mutation in the NPHS1 gene.26 When we performed a literature search, we could not find a published mutation matching the site for the described SUMOylated lysine found in human nephrin. Huber et al.28 showed that nephrin associates with phosphoinositide 3-OH and enhances AKT-dependent signaling. We detected a decreased phosphorylation of AKT when SUMOylation is decreased. Interestingly, mutation of the additional lysine residue, which can only be detected in murine nephrin, does not abolish SUMOylation completely and still reveals plasma membrane localization. Therefore, we conclude that the evolutionary conserved lysine in murine and human nephrin has the more dominant physiological relevance. Previously, we and others could show a raftmediated internalization triggered by proteins such as PKCa and b-arrestin.6,8 SUMOylation could be involved in this sensitive physiological process and could be an important factor that stabilizes nephrin at the slit diaphragm. Treatment of mice with the SUMOylation inhibitor ginkgolic acid underlines our in vitro findings. At 24 h after treatment, mice developed strong glomerular proteinuria, and showed a decreased expression of glomerular nephrin. To prove that the ginkgolic acid treatment induces de-SUMOylation of nephrin in vivo, we performed immunoprecipitation experiments with SUMO-1 and SUMO-2/3 from glomerular lysates of DMSO- and ginkgolic acid–treated animals, which revealed a reduced association with nephrin in the ginkgolic acid–treated animals. These in vivo data support the overall mechanism of nephrin SUMOylation and the significance of this posttranslational modification for slit diaphragm integrity and glomerular barrier function. In conclusion, our results show that SUMOylation of nephrin is crucial for normal nephrin localization, stability, and proper function. As SUMOylation is a reversible process, this pathway could be important in the ‘recycling’ of nephrin to the cell surface. A dysregulated SUMOylation or Kidney International

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I Tossidou et al.: SUMOylation of nephrin

? Ubiquitination

(RIPA buffer with 1 mM sodium orthovanadate, 50 mM NaF). To all buffers, 200 mg/l okadaic acid and 20 g/l N-ethylmaleimide (SigmaAldrich) were added. Lysates were centrifuged at 11,000 r.p.m., and aliquots of the supernatants (40 mg protein/lane) were separated by 8% SDS–PAGE. Densitometry analysis was performed using the Quantity One Software (Bio-Rad, Hercules, CA).

SUMOylation

DESUMOylation K1100R K1114R K1224R K1114A K1224A

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Figure 8 | Schematic working model of the proposed SUMOylation process of nephrin in podocytes. Nephrin is SUMOylated and transported to the plasma membrane of the podocytes. De-SUMOylation or ubiquitination could lead to endocytosis of nephrin and either to proteasomal degradation or recycling by reSUMOylation. Mutation on the putative SUMO sites of nephrin leads to rapid proteasomal degradation.

ubiquitination process of murine and human nephrin at the identified residues could lead to the destabilization of the slit diaphragm (Figure 8). Thus, SUMOylation and de-SUMOylation of nephrin might be an important novel mechanism involved in the pathogenesis of proteinuria. MATERIALS AND METHODS Antibodies and reagents Primary antibodies used for western blotting studies were antiNephrin rabbit polyclonal IgG, GAPDH rabbit polyclonal IgG, Histone H1 rabbit polyclonal IgG, TGN38 goat polyclonal IgG, and SUMO-4 rabbit polyclonal IgG, which were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Myc rabbit polyclonal IgG, GFP rabbit polyclonal IgG, flag rabbit polyclonal IgG, SUMO-1 rabbit polyclonal IgG, SUMO-2/3 rabbit polyclonal IgG, phosphoAKTS473 rabbit polyclonal IgG, AKT rabbit polyclonal IgG, and Calreticulin rabbit polyclonal IgG were from Cell Signaling Technologies (Danvers, MA). Gia3 rabbit polyclonal IgG (Millipore, Darmstadt, Germany), Nephrin guinea pig polyclonal IgG were purchased from Progen (Heidelberg, Germany). The secondary antibodies for Western blotting, goat anti-rabbit IgG HRP and goat anti mouse-IgG HRP, were supplied by Santa Cruz Biotechnology (Santa Cruz). Inhibitors were purchased from the following vendors: cycloheximide (for inhibition of translation) from Sigma-Aldrich (St Louis, MO), and ginkgolic acid (for inhibition of SUMOylation) and LY294002 (for inhibition of PI3K) from Calbiochem, Darmstadt, Germany. Western blot analysis For the analysis of whole-cell protein lysates, cultured HEK-293T cells, either treated or untreated cells, were lysed on ice in RIPA buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.5% sodium deoxycholate, 1% nonidet P-40, and 0.1% SDS) containing protease inhibitors (complete mini; Roche Diagnostics, Mannheim, Germany). For the analysis of whole-cell protein lysates from cultured podocytes, treated or untreated cells were lysed on ice in lysis buffer Kidney International

Podocyte culture Culture of conditionally immortalized mouse podocytes was performed as described previously.32 For propagation of podocytes, cells were cultured on type-I collagen (BD Biosciences, Bedford, MA) at 33 1C in the presence of 10 U/ml recombinant mouse g-interferon (Cell Sciences, Canton, MA) to enhance the expression of a thermosensitive T antigen. Podocytes were maintained at 37 1C for 14 days without g-IFN for inducing differentiation. The medium consists of RPMI 1640 (Biochrom, Berlin, Germany) containing 10% fetal bovine serum (FBS) (PAA, Pasching, Austria), 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA), and 10 U/ml recombinant mouse g-interferon. The cells were inoculated every 3 or 4 days into a new medium. For cultivation of conditionally immortalized human podocytes, 10 mg/ml insulin (Sigma-Aldrich) was added to the medium without recombinant mouse g-interferon. Animal treatment Eight-week-old male C57/Bl6 mice obtained from Charles River (Sulzfeld, Germany) were injected intraperitoneally with 500 mg of ginkgolic acid or DMSO only for 24 h. The total injection volume was 100 ml (containing 50 ml of DMSO with or without ginkgolic acid and 50 ml of PBS). Urine samples were collected before and 24 h after treatment. A volume of 2 ml of urine was loaded for Coomassie gel. For immunofluorescence, kidneys were flushed with PBS and immediately frozen in tissue molds containing optimal cutting temperature (OCT) compound. Isolation and processing of glomeruli Glomeruli were isolated from kidneys of 8-week-old C57/Bl6 mice using a sequential sieving technique with mesh sizes of 180, 100, and 71 mm. The fraction collected from the 71-mm sieve contained glomeruli and was resuspended in lysis buffer, disrupted with a dounce homogenizer by hand with 50 up and down strokes, and immunoprecipitation experiments were performed with these lysates. HEK-293T- cell culture Human embryonic kidney 293 cells (from DSMZ, Braunschweig, Germany) were cultured in a medium consisting of Dulbecco’s MEM high Glucose with L-glutamine (PAA) containing 10% FBS (PAA) and 1% antibiotic–antimycotic solution (PAA). Plasmid construction The coding sequence for the mature human SUMO1, SUMO-2, and SUMO3 was amplified by PCR, cleaved by BamHI and Xho1, and cloned into BamHI/Xho1-cleaved pcDNA3 (gift from Dr Rainer Niedenthal, Medical School Hannover, Institute of Physiological Chemistry). The pRc/CMV-HA-Ubc9 was provided by R. Bernards (Netherlands Cancer Institute). Mutant flag-mNephrinK1114R, flagmNephrinK1224R, flag-mNephrinK1114R/K1224R, and hNephrinK1100Rmyc were generated by Site-Directed Mutagenesis using PfuUltra II Fusion HS DNA Polymerase (Agilent, Santa Clara). All constructs were confirmed by sequencing. For the generation of an adenoviral pEGFP-Nephrin expression vector, we cloned murine nephrin in the 11

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pDNOR221 vector (Invitrogen). This entry clone was cloned into the pAd/CMV/V5-DEST vector (Invitrogen). The transformation was performed by using electrocompetent cells. The colonies growing on the plate were resistant against antibiotics and could be purified for the expression procedure. Transfection The day before transfection, HEK-293T cells or podocytes were seeded on plates for time courses or on coverslides for immunofluorescence assays. Cells were transfected using Fugene HD transfection reagent for 48 h at 37 1C (Roche Diagnostics) according to the manufacturer’s protocol. For adenoviral transfection, the adenoviral stock was thawed and the appropriate amount of virus was diluted with fresh complete medium. The culture medium from the cells was removed. The medium containing virus was mixed gently by pipetting and it was added to the cells. The plate was gently swirled to disperse the medium and incubated at 37 1C overnight. The following day, the medium containing virus was removed and replaced with fresh, complete culture medium. The cells were harvested on the desired day and used for expression of the recombinant proteins. Immunoprecipitation HEK-293T cells were transfected as mentioned above. Then, the cells were washed carefully with ice-cold PBS on ice. For lysis, 900 ml of RIPA buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton and 0.25% deoxycholate þ protease inhibitors) was added to cells. The lysate was incubated for 15 min on ice and centrifuged at 14,000 r.p.m. for 15 min at 4 1C. A volume of 50 ml of flag-beads (50% slurry in Triton buffer) (Sigma) was added to the supernatant and rotated overhead at 4 1C for 1 h (up to overnight). Next, the beads were centrifuged at 3,000 r.p.m. for 1 min at 4 1C and washed with RIPA buffer five times. Proteins were eluted by boiling the beads in Laemmli buffer and separated by SDS polyacrylamide gel electrophoresis. For immunoprecipitation from glomerular and podocyte protein lysates, 500 mg of total cell lysate was incubated with anti-SUMO1 or anti-SUMO-2/3 antibody and Agarose-A beads in IP buffer (25 mM Tris-HCL, pH 7.5, 1 mM DTT, 30 mM MgCl2, 40 mM NaCl, 0.5% NP-40, and protease inhibitors) and rotated overnight at 4 1C. The pellets were washed three times in IP buffer and separated by SDS polyacrylamide gel electrophoresis. For isotype-matched control, immunoprecipitation was performed with a normal rabbit IgG. For adenoviral transduction of podocytes, cells were plated in complete growth medium. On the day of transduction, the adenoviral stock was thawed and the appropriate amount of virus was diluted with fresh complete medium. The culture medium from the cells was removed. The medium containing virus was mixed gently by pipetting and added to the cells. The plate was gently swirled to disperse the medium and incubated at 37 1C overnight. The following day, the medium containing virus was removed and replaced with fresh, complete culture medium. The cells were harvested on the desired day and used for expression of the recombinant proteins. Subcellular fractionation Fractionation was performed by the subcellular fractionation kit for cultured cells from Thermo Scientific. According to the manufacturer’s protocol, membrane fraction consists of dissolved plasma, mitochondria, and ER/Golgi membranes, cytosol fraction consists of 12

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soluble cytoplasmic contents, and nuclear fraction consists of soluble nuclear extracts. Biotinylation assay HEK-293T cells were transfected with the appropriate constructs for 48 h. The cells were washed three times with ice-cold PBS containing 1 mM MgCl2 and 0.1 mM CaCl2 to remove any containing proteins. A volume of 80 ml of 10 mM Sulfo-NHS-SS-Biotin (Pierce) per milliliter PBS was added to the cells and incubated at room temperature for 30 min. After incubation, cells were washed once with ice-cold PBS, and the nonreacted biotinylation reagent was quenched by incubation with 50 mM Tris (pH 8.0) followed by three washes in ice-cold PBS. Cells were lysed in RIPA buffer, centrifuged at 14,000 r.p.m. for 15 min at 4 1C, and the resulting supernatant was incubated with 50 ml of 50% streptavidin agarose (Thermo Scientific) with overnight rotation at 4 1C. After the beads were washed five times with RIPA buffer, bound proteins were eluted with sample buffer by boiling for 5 min. Biotinylated mNephrin-flag was analyzed by immunoblotting. Purification of flag-fusion proteins from transfected HEK-293T cells HEK-293T cells were transfected as mentioned above. The cells were washed carefully with ice-cold PBS on ice. For lysis, 900 ml of RIPA buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, and 0.25% deoxycholate þ protease inhibitors) was added to cells. The lysate was incubated for 15 min on ice and centrifuged at 14,000 r.p.m. for 15 min at 4 1C. A volume of 50 ml of flag-beads (50% slurry in Triton buffer) (Sigma) were added to the supernatant and rotated at 4 1C for 1 h (up to overnight). Next, the beads were centrifuged at 3000 r.p.m. for 1 min at 4 1C and washed with RIPA buffer three times and with ice-cold PBS twice. The beads were resuspended in 50 ml of 500 ng/ml Flag-peptide (Sigma-Aldrich) and incubated for 1 h. The beads were centrifuged at 3000 r.p.m. for 1 min at 4 1C and the eluate was taken for experiments immediately or stored at  80 1C. In vitro SUMOylation For in vitro SUMOylation, the SUMOylation kit from Enzo life Sciences was used according to the manufacturer´s protocol. Purified WT-flag-mNephrin-flag-mNephrinK1114R, flag-mNephrinK1224R, and flag-mNephrinK1114R/K1224R were incubated for 30 min at 30 1C with purified SUMO-1 or SUMO-2 and Ubc9. The whole reaction was used for immunoblotting. Immunofluorescence After dissection, kidneys of 8-week-old C57/Bl6 mice were flushed with PBS and immediately frozen in tissue molds containing optimal cutting temperature (OCT) compound. Sections were blocked with 10% normal donkey serum and stained with the appropriate primary antibody, followed by Cy3-conjugated donkey anti-rabbit or Cy3-conjugated fluorescein isothiocyanate–conjugated donkey anti-guinea pig antibodies (Jackson Immunoresearch). For nephrin localization studies in podocytes, cells were plated in 24-wells on glass slides. The following day, the cells were transfected with 0.5 mg of DNA and differentiated for 3 days. Then they were washed with PBS 3 times and fixed with 4% paraformaldehyde. Fluorescence was enhanced with Aqua PolyMount þ DAPI (40 ,6-diamidino-2-phenylindole). The pictures were taken with a Leica confocal microscope Inverted-2 or with a Leica DM LB Kidney International

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microscope and Leica Application Suite Software (Leica, Bonn, Germany). Postprocessing was done with Photoshop 7.0.

8.

9.

Statistics Data are shown as the mean s.d. and were compared by the Student’s t-test. Data analysis was performed using the Excel statistical software. Significant differences were accepted when Pp0.05. DISCLOSURE

10.

11. 12.

All the authors declared no competing interests.

13.

ACKNOWLEDGMENTS

14.

We thank Germaine Puncha and Sandy Zachura for excellent technical assistance, Tobias Huber (Freiburg, Germany) for providing the SV5-Nephrin constructs, Dr Rainer Niedenthal (Medical School Hannover, Germany) for providing the SUMO constructs, R Bernards (Netherland Cancer institute) for providing the Ubc9 construct, Dr Nina Jones (Guelph, Canada) for providing the human Nephrin-myc construct, and Dr Moin Saleem (Bristol, UK) for providing the human podocytes. This work was supported by grants from Deutsche Forschungsgemeinschaft (SCHI 587/2, 3, 4, 6) to MS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.

15.

16.

17. 18.

19. 20.

SUPPLEMENTARY MATERIAL Figure S1. SUMO proteins are associated with Nephrin in vivo. Figure S2. Control lysates from subcellular fractionations. Figure S3. Predicted ubiquitin-binding sites. Figure S4. Negative control for in vitro SUMOylation. Figure S5. Nephrin expression after treatment with lactacystin, tunicamycin, and ginkgolic acid. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki

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Jones N, Blasutig IM, Eremina V et al. Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes. Nature 2006; 440: 818–823. Verma R, Kovari I, Soofi A et al. Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization. J Clin Invest 2006; 116: 1346–1359. Kestila M, Lenkkeri U, Mannikko M et al. Positionally cloned gene for a novel glomerular protein–nephrin–is mutated in congenital nephrotic syndrome. Mol Cell 1998; 1: 575–582. Putaala H, Soininen R, Kilpelainen P et al. The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death. Hum Mol Genet 2001; 10: 1–8. Ruotsalainen V, Ljungberg P, Wartiovaara J et al. Nephrin is specifically located at the slit diaphragm of glomerular podocytes. Proc Natl Acad Sci USA 1999; 96: 7962–7967. Quack I, Rump LC, Gerke P et al. beta-Arrestin2 mediates nephrin endocytosis and impairs slit diaphragm integrity. Proc Natl Acad Sci USA 2006; 103: 14110–14115. Tossidou I, Teng B, Drobot L et al. CIN85/RukL is a novel binding partner of nephrin and podocin and mediates slit diaphragm turnover in podocytes. J Biol Chem 2010; 285: 25285–25295.

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