Synaptopodin Is a Coincidence Detector of Tyrosine Versus Serine/Threonine Phosphorylation for the Modulation of Rho Protein Crosstalk in Podocytes

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Synaptopodin Is a Coincidence Detector of Tyrosine versus Serine/Threonine Phosphorylation for the Modulation of Rho Protein Crosstalk in Podocytes

† ‡ ‡ | Lisa Buvall,* Hanna Wallentin, Jonas Sieber, § Svetlana Andreeva, Hoon Young Choi, ‡ Peter Mundel,¶ and Anna Greka §

*Departments of Physiology, Institute of Neuroscience and Physiology, and †Clinical and Molecular Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; ‡Department of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, Massachusetts; §Kidney Disease Initiative, The Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts; |Department of Internal Medicine, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea; ¶and Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts

ABSTRACT Tyrosine and serine/threonine signal-transduction pathways inﬂuence many aspects of cell behavior, including the spatial and temporal regulation of the actin cytoskeleton. However, little is known about how input from diverse tyrosine and serine/threonine kinases is integrated to control Rho protein crosstalk and actin remodeling, which are critically important in podocyte health and disease. Here we unveil the proteolytically-regulated, actin organizing protein synaptopodin as a coincidence detector of tyrosine versus serine/threonine phosphorylation. We show that serine/threonine and tyrosine kinases duel for synaptopodin stability versus degradation. EGFR/Src-mediated tyrosine phosphorylation of synaptopodin in podocytes promotes binding to the serine/threonine phosphatase calcineurin. This leads to the loss of 14–3-3 binding, resulting in synaptopodin degradation, Vav2 activation, enhanced Rac1 signaling, and ultimate loss of stress ﬁbers. Our studies reveal how synaptopodin, a single proteolytically-controlled protein, integrates antagonistic tyrosine versus serine/threonine phosphorylation events for the dynamic control of the actin cytoskeleton in podocytes.

J Am Soc Nephrol 28: 837–851, 2017. doi: 10.1681/ASN.2016040414

The function of diverse signaling proteins is con- Many vital cellular functions such as motility, trolled by phosphorylation or dephosphorylation.1 regulation of cell shape, intracellular organization, Src is a nonreceptor tyrosine kinase2 that affects and membrane trafﬁcking depend on the dynamic multiple tyrosine signaling pathways, including cy- modulation of the actin cytoskeleton.11–15 Rho toskeletal dynamics.3 Src can suppress cell adhesion and disrupt the actin cytoskeleton by mediating ty- Received April 9, 2016. Accepted July 29, 2016. rosine phosphorylation and activation of p190Rho- GAP,thereby inactivating RhoA.4,5 Protein kinase A Published online ahead of print. Publication date available at www.jasn.org. (PKA) or Ca2+/calmodulin dependent kinase II (CaMKII) mediate the serine/threonine phosphor- Present address: Dr. Peter Mundel, Third Rock Ventures, 29 Newbury Street, Boston, Massachusetts ylation of many actin modulating proteins including RhoA6 and Rac1.7 However, little is known Correspondence: Dr.AnnaGreka,Glom-NExTCenter,Renal Division, Brigham and Women’s Hospital, Harvard Medical about how signals from competing upstream tyro- School, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, sine versus serine/threonine kinases are integrated Massachusetts 02115. Email: [email protected] or agreka@ to control actin remodeling, which is critically im- broadinstitute.org – portant in podocyte health and disease.8 10 Copyright © 2017 by the American Society of Nephrology

J Am Soc Nephrol 28: 837–851, 2017 ISSN : 1046-6673/2803-837 837 BASIC RESEARCH www.jasn.org family proteins play a central role in the control of pathways activation.33,34 In these studies, TRPC5 channels were activated that regulate actin cytoskeletal dynamics in podocytes.9,10,16 by protamine sulfate (PS),33 however, previous studies have RhoA promotes stress fiber formation, which is adaptive in shown that EGF receptor (EGFR) signaling is also responsible podocytes, whereas Rac1 and Cdc42 promote lamellipodia for TRPC5 activation through the vesicular insertion of TRPC5 and filopodia formation, respectively, which are correlated channels into the plasma membrane.35 These studies led to the with podocyte injury.9,10,16 Rho proteins can cooperate with question of whether EGFR/TRPC5/Rac1 signaling may be im- or antagonize each other through the activity of guanine nu- plicated in synaptopodin-mediated cytoskeletal regulation, cleotide exchange factors (GEFs) and GTPase activating pro- and how this may intersect with known EGFR/Src tyrosine teins (GAPs).11,13–15 Additionally, the Rho guanine nucleotide phosphorylation events.1 EGF/EGFR signaling is of particular dissociation inhibitors control their homeostasis and localiza- interest given important recent work showing that deletion of tion.17 The importance of Rho protein function in podocytes EGFR in a podocyte-specific manner attenuates diabetic ne- is further underscored by an abundance of recent work show- phropathy,36,37 and that EGFR promotes glomerular injury in ing human mutations in Rho regulating proteins, such as the rapidly progressive GN.38 Here we show that synaptopodin Rac1 regulators Arhgdia18,19 and Arhgap24,20 as causes of pro- plays a central role as a coincidence detector of competing gressive proteinuric kidney disease. Furthermore, Rac1- EGFR/Src-mediated tyrosine signals versus serine/threonine induced reactive oxygen species (ROS) negatively regulate signals to orchestrate Rho protein–mediated actin dynamics RhoA activity by inhibiting the low molecular weight protein in podocytes. tyrosine phosphatase.21 However, little is known about how competing divergent upstream signals through tyrosine versus serine/threonine kinase activity are integrated for the coordi- RESULTS nate regulation of Rho protein crosstalk, which is critically important for podocyte structure and function.9,10,22,23 Src-Induced Tyrosine Phosphorylation of Synaptopodin Synaptopodin is a proline-rich actin binding protein that is Promotes Calcineurin Binding expressed in highly dynamic cell compartments, such as the Synaptopodin is a target of serine/threonine phosphoryla- foot processes (FP) of pericyte-like podocytes in the kidney tion by PKA and CaMKII29 but it also contains putative and neuronal dendritic spines in the brain.24 The brain of phospho-acceptor sites for Src. To determine whether synaptopodin-deficient mice lacking Synpo-short and Synpo- synaptopodin is a target of Src, we tested for tyrosine phos- long shows impaired synaptic plasticity.25,26 Synaptopodin exists phorylation of synaptopodin purified from HEK293 cells in three isoforms, neuronal Synpo-short, renal Synpo-long, and coexpressing FRB and Src-iFKBP for inducible activation Synpo-T.27 In podocytes, gene silencing of synaptopodin or of Src by rapamycin.39,40 This approach was on the basis of degradation by cathepsin L (CatL) causes the loss of stress recent studies by Karginov et al.,whodevisedanelegant fibers and reduction of RhoA abundance and activity.27–29 approach for the allosteric regulation of the catalytic activity Mechanistically, synaptopodin promotes stress fiber forma- of protein kinases in living cells including Src.39,40 The in- tion by blocking the c-Cbl–mediated ubiquitination and pro- ducible Src-iFKBP consists of a highly conserved portion of teasomal degradation of Nck130 and the Smurf1-mediated the kinase catalytic domain with a small protein insert ubiquitination and proteasomal degradation of RhoA.28,31 (iFKBP), which inactivates catalytic activity without altering In addition, synaptopodin can suppress filopodia by disrupting other protein functions. Binding to rapamycin and FRB re- Cdc42:IRSp53:Mena signaling complexes.32 The degradation of stores catalytic activity by increasing the rigidity of Src- synaptopodin by CatL is antagonistically regulated by PKA/ iFKBP.39,40 Src-iFKBPisadvantageousovertheclassic CaMKII and calcineurin.29 Serine/threonine phosphorylation constitutively active Src-Y527F because it permits the induc- of synaptopodin by PKA or CaMKII promotes 14–3-3 binding, ible activation of Src in a time- and dose-dependent fashion which protects synaptopodin from cleavage by CatL.29 Dephos- in living cells.39,40 The time- and dose-dependent regulation phorylation of synaptopodin by calcineurin abrogates the inter- of Src activity was critical for the studies described below, action of 14–3-3 with synaptopodin.29 This renders CatL because it allowed us to overcome the problem that over- cleavage sites on synaptopodin accessible, thereby promoting expression of constitutively active Src-Y527F led to rapid cell the degradation of synaptopodin by CatL. This can be blocked death and detachment of podocytes, thereby precluding the by the calcineurin inhibitor cyclosporine A (CsA) or the cathep- study of Src induction in the regulation of podocyte actin sin inhibitor E64.29 These series of experiments demonstrate dynamics. that synaptopodin has a nodal role in the regulation of RhoA To determine whether synaptopodin is tyrosine phosphor- and Cdc42 in podocytes and raises the question of how it may ylated by Src, we used a phospho-proteomic approach. At 0 or also be involved in the regulation of Rac1. 15 minutes after activation of Src-iFKBP, we could not detect The transient receptor potential canonical (TRPC) ion any tyrosine-phosphorylated residues in synaptopodin. In channels TRPC5 and TRPC6 are antagonistic regulators of contrast, after 60 minutes of Src activation, three tyrosine- synaptopodin abundance.33,34 Of note, TRPC5-mediated Ca2+ phosphorylated residues (Y29, Y222, and Y344) were found influx mediates synaptopodin degradation and Rac1 (Figure 1A). The inducible Src-iFKBP kinase employed in this

838 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 837–851, 2017 www.jasn.org BASIC RESEARCH

Figure 1. Src-induced tyrosine phosphorylation of synaptopodin increases calcineurin binding to synaptopodin. (A) MSMS phospho- tyrosine spectra show Src-induced tyrosine phosphorylation sites in synaptopodin. Three specific phospho-tyrosine sites were detected as shown by individual peptide spectra. Phospho-tyrosine residues are shown in gray. pY29: peak y3-y5 + b10-b12; pY222: peak y16 + b2; and pY344: peak y6-y8,b7 + b9. (B) Y222 in synaptopodin is an evolutionarily-conserved phospho-acceptor site for Src. T216 (asterisk) and Y222 (arrow) are in close proximity. (C) Exogenous coimmunoprecipitation studies in HEK293 cells show that activation of Src-iFKBP (Src*) increases the interaction of Myc-synaptopodin (Synpo-WT) with FLAG-calcineurin A (FLAG-CnA) when compared with inactive Src-iFKBP (Src). Alanine substitution of Y222 (Synpo-Y222A) mitigates the interaction with FLAG-CnA at baseline (Src) and no change is seen after activation of Src-iFKBP (Src*). No interaction is found with the negative control Myc-Raver. In the FLAG eluate, FLAG-CnA is marked by an arrow, the other bands represent heavy (*) and light chain (**) of the anti-FLAG antibody used to precipitate FLAG-CnA. (D) Endogenous coimmunoprecipitation in podocytes confirms increased interaction between calcineurin and synaptopodin upon activation of Src (Src*). IP with anti-GFP antibody serves as a negative control. Molecular mass markers are in kDa. study requires up to 1 hour for full activation with 200 nM minutes) most likely reflect the time needed for full activation rapamycin39; this is also the time point at which we detected of Src-FKBP. robust phosphorylation of synaptopodin. Thus, the kinetics Y222 is conserved in evolution (Figure 1B, arrow) and five for synaptopodin phosphorylation (between 15 and 60 amino acids away from the 14–3-3 binding site (T216; Figure 1B,

J Am Soc Nephrol 28: 837–851, 2017 Synaptopodin as a Coincidence Detector 839 BASIC RESEARCH www.jasn.org asterisk), which can be dephosphorylated by the synaptopodin- binding serine/threonine phosphatase calcineurin.29 The close proximity of T216 and Y222 prompted us to ask whether phosphorylation of Y222 by Src can modulate the binding of calcineurin to synaptopodin. In heterologous coimmunoprecipitation (Co-IP) studies in HEK293 cells cotransfected with EGFP-Src-iFKB (Supplemental Figure 1), Cherry-FRB (Supplemental Figure 1), FLAG-calcineurin, and Myc- synaptopodin, we observed increased binding of calcineurin to synaptopodin after activation of Src. Alanine substitution of Y222 mitigated the binding of synaptopodin to calcineurin and no increase in binding was seen after activation of Src- iFKBP (Figure 1C). In contrast, alanine substitution of Y29 or Y344 did not affect the interaction of synaptopodin with calcineurin (Supplemental Figure 2). Of note, treatment with Src inhibitor 1 (SrcI-1) lowered the basal interaction between calcineurin and synaptopodin (Supplemental Figure 3). The interaction between synaptopodin and calcineurin was further examined by endogenous Co-IP studies in podocyte extracts in the presence of the cathepsin inhibitor E64 (to prevent synaptopodin degradation29). In keeping with our published work,29 synaptopodin interacted with calcineurin at baseline Figure 2. Activation of Src impairs 14–3-3 binding to synapto- (Figure 1D). Consistent with the heterologous Co-IP studies podin. Exogenous coimmunoprecipitation studies in HEK293 (Figure 1C), the activation of Src increased the binding cells reveal that activation of Src-iFKBP (Src*) reduces the in- of endogenous calcineurin to endogenous synaptopodin teraction of FLAG-14–3-3b with Myc-synaptopodin when com- (Figure 1D). pared with inactive Src-iFKBP (Src). In contrast, the interaction of – Next, given the loss of 14–3-3 binding after dephosphoryla- Src-resistant Synpo-222A with 14 3-3 is not altered by Src activation. Molecular mass markers are in kDa. tion of synaptopodin by calcineurin,29 we sought to understand 14–3-3 binding in the presence of activated Src. To this end, Co- IPstudies showed that activation of Src caused a reduction of 14– (Figure 3B). In contrast, CsA did not restore stress fibers in 3-3 binding to synaptopodin (Figure 2). In contrast, activation of synaptopodin-depleted podocytes in the presence of activated Src did not affect the interaction of 14–3-3 with Src-resistant Src (Supplemental Figure 4B). The most likely explanation for Synpo-222A (Figure 2). Taken together, these studies showed the observed effects is the activation of Src, rather than on- or that Src-induced tyrosine phosphorylation of Y222 increases off-target effects of rapamycin or infection with lentiviral con- calcineurin binding to synaptopodin (Figure 1, C and D) to structs. In control experiments, well developed stress fibers were promote serine/threonine dephosphorylation of synaptopodin present in noninfected podocytes before and after rapamycin and reduction in 14–3-3 binding29 (Figure 2). treatment, and in Src-iFKB/FRB coinfected podocytes (Supple- mental Figure 5A). Moreover, the protein abundance of synap- Calcineurin Inhibition Protects from Src-Induced topodin, active pSrc (417), and inactive pSrc (527) remained Synaptopodin Degradation and Stress Fiber Loss unchanged after treating control podocytes (in the absence of Synaptopodin promotes stress fiber formation through acti- Src-iFKBP and FRB) with rapamcyin (200 nM) for 4 hours vation of Nck1-dependent RhoA signaling.28,30,31 Pharmaco- (Supplemental Figure 5, B and C). However, rapamycin treat- logic inhibition of PKA or CaMKII reduces synaptopodin ment of isolated glomeruli has been shown to attenuate p70S6K protein abundance and disrupts stress fibers, which can be phosphorylation,41 and a study in human podocytes has shown rescued by the calcineurin inhibitor CsA.29 The observed in- efficient block of p70S6K phosphorylation by 24 and 120 hours crease in calcineurin binding and reduction of 14–3-3 binding of treatment with rapamycin,42 raising the possibility that cellu- to Src-phosphorylated synaptopodin prompted us to test lar effects of rapamycin not examined in this study could poten- whether Src reduces synaptopodin protein abundance by pro- tially also modulate stress fibers. moting the degradation of synaptopodin. In keeping with this hypothesis, activation of Src by rapamcyin in Src-iFKB and Src-Induced Stress Fiber Loss is Blocked by FRB-expressing podocytes (Supplemental Figure 4A) reduced Degradation-Resistant Synaptopodin synaptopodin protein abundance, which was blocked by CsA In a complementary genetic approach, we analyzed the effects or the cathepsin inhibitor E64 (Figure 3A). Functionally, the of Src-resistant Synpo-Y222A, calcineurin-resistant Synpo- preservation of synaptopodin protein abundance was associ- ED,29 or CatL-resistant Synpo-CM1+229 on Src-induced deg- ated with an inhibition of Src-induced loss of stress fibers radation of synaptopodin and loss of stress fibers. Lentiviral

840 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 837–851, 2017 www.jasn.org BASIC RESEARCH

=150 cells; inactive Src: 92%61.0% SEM stress ﬁber containing cells; active Src (Src*): 11.3%61.8%; Src* + CsA: 67.0%6 2.0%; Src* + E64: 64.6%61.8%; Src* + wild-type synaptopodin: 18.3%62.5%; Src* + Synpo-Y222A: 88.1%61.1%; Src* + Synpo-ED: 69.2%63.8%; Src* + CM1+2: 66.5%61.6%; P,0.001; ANOVA; Figure 3E). The simplest interpretation of these results is that Src-induced degradation of synaptopodin and the resulting loss of stress ﬁbers can be blocked by preserving serine/ threonine phosphorylation of synaptopodin.

Synaptopodin Inhibits Rac1 Signaling Synaptopodin is a positive regulator of RhoA28,30,31 and a negative regulator of Cdc42 signaling.32 Activation of TRPC5 leads to the degradation of synaptopodin and concomitant activation of Rac1.33,34 This prompted us to test whether synaptopodin inhibits Rac1 activation. In podocytes depleted of synaptopodin (Figure 4A, Supplemental Figure 9), Rac1 activation pulldown assays showed increased Rac1 activity (GTP-Rac1) compared with synaptopodin-replete control cells (Figure 4B). The quantitative analysis confirmed a significant increase in Rac1 activity in syn- Figure 3. CsA, E64, Synpo-Y222A, Synpo-ED, or Synpo-CM1+2 protect from Src- aptopodin-depleted cells (n=3; 2.2860.37 induced stress fiber loss. (A) Synaptopodin protein abundance in podocytes is reduced SEM) compared with synaptopodin-re- by activated Src-iFKBP (Src*) compared with cells with inactive Src-iFKBP (Src). Src*- plete control cells (0.2960.09; P,0.01; induced reduction of synaptopodin protein abundance is blocked by the calcineurin t test; Figure 4C). Rac1 can increase the inhibitor CsA or the cathepsin inhibitor E64. GAPDH serves as a loading control. (B) production of ROS21,43,44; therefore, we ana- Phalloidin labeling reveals loss of stress fibers after activation of Src (Src*) but not in lyzed ROS levels in synaptopodin-depleted fi cells with inactive Src-iFKBP (Src). CsA and E64 protect from Src*-induced stress ber cells using flow cytometry. We detected sig- loss; scale bar 20 mm. (C) GFP-tagged (130 kDa) Src-resistant (Y222A), calcineurin- nificantly increased ROS levels in synapto- resistant (ED), or CatL-resistant (CM1+2) but not wild-type (WT) synaptopodin are podin-depleted cells (n=3; 5.2%62.0% protected from Src*-induced degradation. Endogenous synaptopodin is seen below the 116 kDa marker. GAPDH serves as a loading control. (D) Overexpression of Synpo- SEM) when compared with control n 6 Y222A, Synpo-ED, or Synpo-CM1+2 but not Synpo-WT protects from Src*-induced shRNA-expressing cells ( =3; 0% 0.7%; loss of stress fibers; scale bar 20 mm. (E) Quantitative analysis confirms protection from P,0.05; t test; Figure 4D). Src*-induced loss of stress fibers by CsA, E64, Synpo-Y222A, Synpo-ED, or SynpoCM1 +2. Values are represented as % stress fiber containing cells 6SEM; n=3; ANOVA; Synaptopodin Inhibits the Activation P,0.001. Molecular mass markers are in kDa. of the Rac1 GEF Vav2 To examine how synaptopodin can suppress Rac1 activity, we tested whether overexpression of Synpo-Y222A, Synpo-ED, or Synpo-CM1 synaptopodin blocks a Rac1 activating GEF. Synaptopodin +2 but not of wild-type synaptopodin protected against is a proline-rich protein capable of binding to various SH3- Src-induced degradation (Figure 3C). Functionally, Synpo- containing proteins, including CD2AP,45 IRSp53,32 and Nck1/230; Y222A, Synpo ED, or Synpo CM1+2 but not wild-type syn- therefore, we focused on SH3 domain–containing GEFs, and aptopodin conferred resistance against Src-induced loss of further refined our search to those SH3-domain GEFs acti- stress fibers (Figure 3D). The quantitative analysis of the vated downstream of Src. Vav2 is an SH3 domain–containing data in Figure 3, B and D, showed a significant preservation GEF for Rac1,46 which can be activated by Src.47,48 We there- of stress fibers by CsA, E64, Synpo-Y222A, Synpo-ED, or fore asked whether synaptopodin could interact with Vav2. In Synpo-CM1+2 (n=50 cells 3 3 independent experiments heterologous Co-IP experiments in cotransfected HEK293

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Y172-phosphorylated Vav2. Synaptopodin- depleted cells showed an increase in pVav2 (Y172) protein abundance compared with synaptopodin-replete control cells (Figure 4G). The quantitative analysis showed a signiﬁcant increase in pVav2 levels in synaptopodin-depleted cells (n=3; 1.076 0.33 SEM) when compared with control cells (n=3; 0.1060.05; P,0.05; t test; Figure 4H). The simplest interpretation of these results is that synaptopodin inhibits Vav2 activation, thereby preventing Rac1 activity and downstream signaling.

Gene Silencing of Vav2 or Rac1 Restores Stress Fibers in Synaptopodin-Depleted Cells Synaptopodin promotes stress fiber formation and RhoA activity by blocking the ubiquitination of Nck1 by c-Cbl30 and by blocking the ubiquitination of RhoA by Smurf1.28,31 Another mechanism for the induction of stress fibers involves the inhibition of Rac1 signaling, which releases Figure 4. Synaptopodin inhibits Rac1 signaling by blocking Vav2 activation. (A) 11,21,49 Western blot analysis shows synaptopodin depletion in synaptopodin knockdown Rac1-mediated inhibition of RhoA. (Synpo shRNA) podocytes. GAPDH serves as a loading control (B). Synaptopodin In addition to the previously reported loss 28,30 fi depletion (synpo shRNA) increases protein abundance of GTP-bound active Rac1 when of RhoA activity, we now nd in- compared with control shRNA (con) expressing podocytes. (C) Quantitative analysis creased Rac1 activity in synaptopodin- confirms significant increase of Rac1 activation in synpo shRNA cells. Values are depleted cells (Figure 4C). Therefore, we presented as GTP-bound Rac1/Total amount Rac1 6SEM; n=3; t test; P,0.01. (D) examined whether inhibition of Rac1 signal- Increased ROS production in synaptopodin-depleted podocytes; data are presented ing could restore stress fibers in synaptopodin- as % of control shRNA cells 6SEM; n=3; t test; P,0.05. (E) Coimmunoprecipitation depleted cells. We suppressed Rac1 signaling from HEK293 cells shows interaction between Myc-Vav2 and FLAG-Synpo-short. Myc- in synaptopodin-depleted podocytes by co- a-actinin-4 (Act4) serves as positive control and Myc-Raver (con) as negative control. expression of Vav2, Rac1, or nonsilencing (F) Endogenous coimmunoprecipitation confirms the interaction between Vav2 and control shRNAs and validated the effi- synaptopodin in podocytes. IP with anti-GFP antibody serves as a negative control. (G) Western blot reveals increased protein abundance of activated Vav2 (pVav2) in syn- ciency of protein depletion by Western aptopodin-depleted (synpo shRNA) podocytes when compared with control cells (con blot (Supplemental Figure 5D). To assess the shRNA). Total Vav2 levels are not changed. (H) Quantitative analysis of activated Vav2. crosstalk between Rac1 and RhoA GTPases Values are presented as pVav2/total Vav2 6SEM; n=3; t test; P,0.05. Molecular mass in the absence of synaptopodin, we ana- markers are in kDa. lyzed changes in total/active Rac1 and RhoA protein abundance in synaptopodin- depleted podocytes before and after silencing cells, we found that Myc-Vav2 could bind to FLAG-Synpo-short of Vav2 or Rac1 (Figure 5A). We found that gene silencing of (Figure 4E). The interaction of synaptopodin with a-actinin-427 Vav2 or Rac1 abrogated the activation of Rac1 and restored served as positive control. No interaction was found with activation of RhoA in synaptopodin-depleted podocytes Myc-raver, serving as negative control (Figure 4E), thereby (Figure 5A). The quantitative analysis confirmed a signifi- confirming the specificity of the interaction. The interaction cant increase in Rac1 activity in synaptopodin-depleted cells between synaptopodin and Vav2 was further examined by (n=3; 1.4060.11 SEM) compared with synaptopodin/Vav2 (n=3; endogenous Co-IP studies; in protein extracts from cultured 0.0160.01; P,0.001; ANOVA) or synaptopodin/Rac1 (n=3; podocytes, anti-Vav2 antibody precipitated Vav2 and copreci- 0.0060.00; P,0.001; ANOVA) codepleted cells (Figure 5B). pitated synaptopodin (Figure 4F). Conversely, anti-synaptopodin Conversely, RhoA activity was significantly reduced in antibody precipitated synaptopodin and coprecipitated Vav2 synaptopodin-depleted cells (n=3; 0.0960.06 SEM) compared (Figure 4F). To test whether the observed interaction between with synaptopodin/Vav2 (n=3; 1.0660.18; P,0.05; ANOVA) synaptopodin and Vav2 is functionally relevant, we examined or synaptopodin/Rac1 (n=3; 1.0360.19; P,0.05; ANOVA) the protein abundance of active Vav2 by Western blot analysis of codepleted cells (Figure 5B).

842 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 837–851, 2017 www.jasn.org BASIC RESEARCH

RhoA signaling28,31 (Figure 5, A and B). Depletion of synaptopodin shifts Rho protein balance toward Rac1 activation and loss of stress ﬁbers, which can be reversed by depletion of Vav2 or Rac1.

PS-Induced EGFR-Dependent Activation of Src Triggers Reduction of RhoA Activity and Degradation of Synaptopodin Perfusion of rat or mouse kidneys with the polycation PS causes TRPC5-dependent33 podocyte actin remodeling and FP effacement.50–52 Synaptopodin-deficient mice display impaired recovery from PS-induced FP effacement.27 In vitro, exposure of podocytes to PS leads to loss of stress fibers,33,53 degradation of synaptopodin, and activation of Rac1 in a TRPC5-dependent fashion,33 thereby phenocopying the observed effect of Src activation (Figure 3) and the knockdown of synaptopodin (Figure 4A).28,30 PS has been shown to increase EGFR kinase activity,54,55 by exposing a population of cryptic EGFRs.56 Therefore, we hypothe- sized that PS signals through EGFR, Src, and PI3 kinase activation,1 leading to the degradation of synaptopodin and loss of Figure 5. Gene silencing of Vav2 or Rac1 restores Rho protein crosstalk and stress stress fibers. We observed that PS increased fi bers in synaptopodin-depleted podocytes. (A) Western blot analysis showing changes the abundance of phosphorylated pEGFR in total and active Rac1 and RhoA protein abundance in synaptopodin-depleted po- (Figure 6A). PS also increased protein docytes before and after silencing of Vav2 or Rac1. (B) Quantitative analysis confirms abundance of active pSrc (Y416) and de- significant changes in Rac1 and RhoA activation in synpo shRNA cells before and after silencing of Vav2 or Rac1. Values are presented as GTP-bound Rac1/Total amount Rac1 creased protein abundance of inactive pSrc 6SEM or RhoA/Total amount RhoA 6SEM; n=3; ANOVA; ***P,0.001; *P,0.05. (C) (Y527) (Figure 6B). In addition to the pre- Gene silencing of Vav2 or Rac1 restores stress fibers in synaptopodin-depleted po- viously described increase in Rac1 activity,33 docytes; scale bar 20 mm. (D) Quantitative analysis confirms rescue of stress fiber PS also decreased total and active RhoA formation by Vav2 or Rac1 shRNAs. Values are presented as % stress fiber containing levels (Figure 6C). In a detailed analysis, cells compared with control shRNA expressing cells 6SEM; n=3; ANOVA; we observed that synaptopodin abundance ****P,0.001. Molecular mass markers are in kDa. was preserved in cells treated with the EGFR inhibitor AG1478, SrcI-1, the PI3 kinase inhibitor wortmannin, the calci- We then visualized the actin cytoskeleton by phalloidin neurin inhibitor CsA, or the CatL inhibitor E64 (Figure 6D). staining and found the restoration of stress fibers in synapto- Of note, podocytes do not express lymphocyte-specific protein podin-depleted cells codepleted of Vav2 or Rac1 (Figure 5C). tyrosine kinase (Lck; Supplemental Figure 7), thereby The quantitative analysis showed a near complete rescue of excluding an effect of SrcI-1 on Lck in our studies. In a com- stress fiber containing cells after codepletion of Vav2 or Rac1 plementary genetic approach, we analyzed the effects of Src- (n=50 cells 3 3 independent experiments =150 cells; control resistant Synpo-Y222A, calcineurin-resistant Synpo-ED,29 or shRNA: 95.5%61.5% SEM stress fiber containing cells; Synpo CatL-resistant Synpo-CM1+229 on PS-induced degradation shRNA: 4.3%61.6%; Synpo shRNA + Vav2 shRNA: 84.7%6 of synaptopodin and loss of stress fibers. Synpo-Y222A, 1.4%; Synpo shRNA + Rac1 shRNA: 86.9%60.9%; P,0.001; Synpo-ED, or Synpo-CM1+2 but not wild-type synaptopodin ANOVA; Figure 5D). Similar to gene silencing of Rac1, phar- were resistant to PS-induced degradation (Figure 6E). macologic inhibition of Rac1 with NSC23766 restored stress In keeping with the observed stabilization of synaptopodin fibers in synaptopodin-depleted cells (Supplemental Figure 6). protein abundance (Figure 6E), application of AG1478, SrcI-1, Taken together, synaptopodin preserves stress fibers by simul- wortmannin, CsA, or E64 also protected from PS-induced loss taneously blocking Rac1 (Figure 5, A and B) and promoting of stress fibers (Figure 7A). Overexpression of Synpo-Y222A,

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(n=50 cells 3 3 independent experiments =150 cells; control cells 95.5%60.7% SEM stress fiber containing cells; PS treated cells: 1.7%61.0%; PS + AG1478: 69.2%6 1.3%; PS + Src-1 inhibitor: 79.1%61.0%; PS + wortmannin: 73.3%60.8%; PS + CsA: 49.2%61.0%; PS + E64: 54.1%61.0%; PS + wild-type synaptopodin: 14.0%6 1.3%; PS + Synpo-Y222A: 77.0%62.4%; PS + Synpo-ED: 54.7%63.0%; PS + Synpo-CM1+2: 53.0%62.6%; P,0.001; ANOVA). Finally, to test whether the observed effect could be recapitulated with a phys- iologic ligand rather than PS, we treated podocytes with EGF.38 We found that EGF caused activation of Src (Figure 8A) and degradation of synaptopodin (Figure 8, B and C). Importantly, EGF also caused endogenous tyrosine phosphorylation of synaptopodin, similar to PS (Figure 8C). As expected, the consequence of these signaling events was EGF-mediated loss of podocyte stress fibers (Figure 8D), thereby phenocopying the effects of PS on the actin cytoskeleton (Figure 7). These data further support the conclusion that PS promotes Src signaling through EGFR activation to trigger the degradation of synaptopodin, which results in Rac1 activation and RhoA inactivation, thereby causing the loss of stress fibers in podocytes.

DISCUSSION Figure 6. PS-induced degradation of synaptopodin is blocked by inhibition of EGFR, Src, calcineurin, or CatL. (A) Immunoprecipitation of endogenous EGFR shows in- The experiments described here allowed us creased abundance of tyrosine phosphorylated EGFR (Y1068, Y845) in PS-treated to explore several important questions. podocytes. Total EGFR serves as loading control. (B) PS increases the abundance of First, we show that the actin organizing active (pSrc Y416) and decreases the abundance of inactive (pSrc Y527) endogenous protein synaptopodin is not only phos- Src. Total Src serves as a loading control. (C) PS decreases the abundance of active phorylated by the serine/threonine kinases RhoA. (D) Inhibition of PS-induced degradation of synaptopodin by EGFR blocker PKA and CaMKII,29 but also by the tyro- AG1478 (AG), SrcI-1, PI3K inhibitor wortmannin (Wort), calcineurin inhibitor CsA, or sine kinase Src. Second, our results show cathepsin inhibitor E64. GAPDH serves as a loading control. (E) GFP-Synpo-Y222A, GFP-Synpo-ED, or GFP-Synpo-CM1+2 but not wild-type GFP-Synpo-short (WT) are that serine/threonine and tyrosine phos- resistant against PS-induced degradation. Note that the bands below 130 kDa cor- phorylation duel for synaptopodin stability respond to overexpressed synaptopodin and the bands below 116 kDa to endogenous versus degradation. Serine/threonine synaptopodin. GAPDH serves as a loading control. Molecular mass markers are in kDa. phosphorylation by PKA/CaMKII stabi- lizes synaptopodin.29 In contrast, tyrosine phosphorylation of synaptopodin by Src Synpo ED, or Synpo CM1+2 but not wild-type synaptopodin increases the binding of synaptopodin to calcineurin, thereby had no effect on stress fibers at baseline (Supplemental Figure promoting the dephosphorylation of serine/threonine resi- 8) but conferred protection from PS-induced loss of stress dues required for 14–3-3 binding.29 Third, we demonstrate fibers (Figure 7B). The quantitative analysis (Figure 7C) that synaptopodin can suppress Rac1 signaling by blocking the showed a near complete rescue of stress fibers by AG1478, Vav2-mediated activation of Rac1. These results unveil synapto- SrcI-1, or wortmannin, and to a lesser degree by CsA or E64 podin as a coincidence detector for competing serine/threonine

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indeed dependent on Src activity. We speculate that synaptopodin contains a novel calcineurin-binding site, whose binding affinity is increased by Src-mediated phos- phorylationatY222.Alternatively,the interaction may also involve an adaptor protein. Although this question does not directly influence the conclusions of our study, future studies will be designed to address it. Dynamic control and balance of Rho protein signaling is crucial for podocyte survival.10 The regulation of the actin cytoskeleton in podocytes, in particular, is critical for their function as the gatekeepers of the kidney filter barrier.10 Synaptopodin is a unique proline rich protein known to play a critical role in the regulation of actin dynamics in podocytes and neurons.24 However, the precise mechanisms by which it achieves this remained elusive. We now unveil coincident phosphorylation of synaptopodin by serine/threonine and tyrosine kinases as a signaling mechanism that can balance the crosstalk of Rho proteins. We speculate that this coincidence detection paradigm represents an efficient way for the podocyte to rapidly shift Rho protein balance by changing the ratio of tyrosine to serine/threonine phosphorylation of synaptopodin, a protein with a short 31 t1/2 and high susceptibility to proteolytic Figure 7. PS-induced stress fiber loss is blocked by inhibition of EGFR, Src, calcineurin, degradation.24,29 or CatL. (A) Phalloidin staining shows rescue of PS-induced loss of stress fibers by Receptor tyrosine kinase receptors AG1478, SrcI-1, wortmannin, CsA, or E64. (B) Overexpression of GFP-Synpo-Y222A, (RTKs) are key regulators of several cellular GFP-Synpo-ED, or GFP-Synpo-CM1+2 but not wild-type GFP-Synpo-short protects processes.1 EGFR is a well characterized against PS-induced stress fiber loss in podocytes; scale bar 20 mm. (C) Quantitative RTK known to regulate many cellular analysis confirms protection by CsA, E64, Synpo-Y222A, Synpo-ED, or SynpoCM1+2. Values are presented as % stress fiber containing cells 6SEM; n=3; ANOVA; processes such as proliferation, differenti- ****P,0.001. Molecular mass markers are in kDa. ation, cytoskeletal regulation, and transcription.1,65 Src has long been implicated in signal transduction pathways down- and tyrosine signals to regulate Rho protein crosstalk in podocytes stream of EGFR-induced tyrosine phosphorylation.66,67 PS is (Figure 8D). involved in EGFR activation54–56 and EGFR-dependent Calcineurin has a central role in the regulation of diverse activation of the Ca2+-permeable TRPC5 channel.34,35 In- calcium-dependent signaling events.57 In addition to the tran- triguingly, TRPC5 activation leads to the degradation of syn- scription factor NFAT,58 various cytoskeletal proteins such as aptopodin downstream of PS.33 Our data unify these previous microtubule-associated protein 2,59,60 tubulin,60 tau factor,60 findings into a cohesive signaling pathway in which synapto- and slingshot61 are regulated by calcineurin-mediated serine/ podin is the substrate of Src-mediated tyrosine phosphoryla- threonine dephosphorylation. Our results illuminate a novel tion triggered by PS-induced EGFR activation (Figure 8D). mechanism of coordinate tyrosine kinase (Src) and serine/ According to this pathway, PI3 kinase (PI3K) inhibition may threonine phosphatase (calcineurin) activities on the same antagonize PS-induced suppression of synaptopodin through protein substrate: Src phosphorylation enables the binding at least two synergistic mechanisms: (1) PS-induced synapto- of calcineurin to synaptopodin. Importantly, synaptopodin podin degradation requires TRPC5-mediated activation of does not contain the known calcineurin-binding motifs calcineurin29,33 and EGF-induced membrane insertion of PxIxIT62,63 or LxVP,64 suggesting that calcineurin binding is TRPC5 requires PI3 kinase activity.35 (2) EGF-induced PI3

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Figure 8. EGF induces Src activation, synaptopodin phosphorylation and degradation, and loss of stress fibers. (A) EGF increases abundance of active (pSrc Y416) and decreases abundance of inactive (pSrc Y527) endogenous Src. Total Src serves as a loading control. (B) EGF induces degradation of synaptopodin. GAPDH serves as a loading control. (C) A phospho-tyrosine antibody (p-Tyr) detects enhanced tyrosine phosphorylation of immunoprecipitated synaptopodin as a consequence of EGF treatment in the presence of E64 (to prevent synaptopodin degradation). PS and PS+E64 serve as controls. A western blot (below) with synaptopodin antibody confirms the identity of the protein on the p-Tyr blot as synaptopodin. (D) Phalloidin staining reveals EGF-induced loss of stress fibers; scale bar 50 mm. (E) Synaptopodin as a coincidence detector: a model. PS triggers EGFR signaling, and downstream activation of Src and PI3K resulting in Vav2-mediated Rac1 activation (Rac1-GTP). Rac1-GTP promotes PIP(5)K-dependent membrane insertion and activation of TRPC5, thereby increasing Ca2+ influx and activation of calcineurin (CN). Src increases CN binding to synaptopodin, which disrupts PKA/CaMKII-dependent 14–3-3 binding, resulting in CatL-mediated degradation of synaptopodin. Loss of synaptopodin increases Vav2 activation, resulting in Rac1-mediated increase in ROS production, thereby promoting RhoA inactivation. Loss of RhoA activation is compounded by increased ubiquitination of the RhoA activator Nck1 by c-Cbl (Nck1-Ub), and by increased ubiquitination of RhoA-GDP (RhoA-GDP-Ub) by Smurf1 due to synaptopodin depletion. Molecular mass markers are in kDa.

846 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 837–851, 2017 www.jasn.org BASIC RESEARCH kinase signaling can increase Vav2-mediated Rac1 activity.1 of 50 mM ammonium bicarbonate/50% acetonitrile/50% water for Rac1 activity, in addition to PI3 kinase activity, is also required 30 minutes. One final wash was done using 10 mM ammonium bicar- for EGF-induced membrane insertion and function of bonate/50% acetonitrile/50% water for 30 minutes. After washing, TRPC5.34,35 The observed positive effect of PI3K inhibition the gel pieces were dried in a Speedvac. Trypsin was prepared by on synaptopodin protein abundance lends further support to mixing 10 ml0.1mg/mltrypsin(Promega,Madison,WI)with the conclusion that PS signals through EGFR to trigger the 140 ml 10 mM ammonium bicarbonate and adding 15 ml PhosStop degradation of synaptopodin. These data are of particular in- (Roche, Basel, Switzerland) as prepared following the manufacturer’s terest in the context of important recent work showing that directions. The gel was then rehydrated with 30 ml of the trypsin EGFR signaling is an important mediator of injury in glomer- solution and digested at 37°C for 16 hours. For titanium dioxide en- ular disease,38 andthatdeletionofEGFRinapodocyte- richment, the digest was acidified with 0.5% TFA, 50% acetonitrile. specific manner attenuates diabetic nephropathy.36,37 The TopTips (Glygen Corporation) were prepared by washing three times implications of this work may also be far reaching given recent with 40 ml each of 100% acetonitrile, followed by 0.2 M sodium phos- findings that urinary EGF may serve as a robust biomarker for phate pH 7.0, and 0.5% TFA, 50% acetonitrile. Washes were spun the progression of CKD.68 through into an Eppendorf tube at 2000 rpm for 1 minute. The acid- In conclusion, our data reveal a signaling network, which ified digest supernatant was loaded into the TopTip, spun at 1000 rpm integrates input from upstream receptor pathways (EGFR), for 1 minute, and then at 3000 rpm for 2 minutes. Gel pieces were kinase systems (Src versus PKA/CaMKII), and the Rho proteins rinsed with 40 ml 0.5% TFA, 50% acetonitrile, with the supernatant to regulate essential cellular functions (Figure 8D). As shown transferred to the TopTip and the spin repeated. The TopTip was then in this model, PS induces Src and PI3K activation, down- washed with 40 ml 0.5% TFA, 50% acetonitrile, and the spin repeated. stream of the EGFR, leading to Vav2-mediated activation of The flow through from these washes was saved and analyzed by Rac1 and ensuing PIP(5)Ka-dependent membrane inser- LC-MS/MS as below. Phosphopeptides were eluted from the TopTip tion and activation of TRPC5.34,35 TRPC5-mediated Ca2+ by three times 30 ml 28% ammonium hydroxide. Both the flow influx increases calcineurin activity,34 thereby abrogating the through and eluted fractions were dried in a SpeedVac, and redried PKA/CAMKII-mediated serine/threonine phosphorylation from 40 ml of water. Samples were dissolved in 3 ml 70% formic acid, and 14–3-3 binding of synaptopodin.29 The resulting degra- vortexed, diluted with 7 ml 0.1% TFA, spun, and transferred to dation of synaptopodin29,33 leads to increased Rac1 activity, LC-MS/MS vials where 5 ml was injected for LC-MS/MS on the ROS production, and inactivation of RhoA. PS also induces Thermo ScientificLTQOrbitrapElite.TheLTQOrbitrapEliteis tyrosine phosphorylation of Y222 in synaptopodin by Src. equipped with a Waters nanoAcquity UPLC system, and uses a Waters This in turn increases the binding of calcineurin to synapto- Symmetry C18 180 mm 3 20 mm trap column and a 1.7 mm, 75 mm 3 podin, thereby promoting the loss of 14–3-3 binding and 250 mm nanoAcquity UPLC column (37°C) for peptide separation. CatL-mediated degradation.29 Degradation of synaptopodin Trapping was done at 5 ml/min, 99% Buffer A (100% water, 0.1% abrogates its capacity to block the c-Cbl–induced proteasomal formic acid) for 3 minutes. Peptide separation was performed at 300 degradation of Nck130 and the Smurf1-induced proteasomal nl/min with Buffer A: 100% water, 0.1% formic acid; and Buffer B: 28 degradation of RhoA ; it also reduces the capacity of synap- 100% CH3CN, 0.075% formic acid. A linear gradient (51 minutes) topodin to block the Vav2-mediated activation of Rac1, leading was run with 1% buffer B at initial conditions, 65% B at 50 minutes, to increased ROS production, and decreased RhoA activity and 85% B at 51 minutes. MS was acquired in the Orbitrap part of the (Figure 8). Thus, the absence of synaptopodin shifts the overall instrument (300–2000 m/z) using 1 microscan, and a maximum inject Rho protein balance from RhoA toward Rac1. Our studies time of 500 ms and up to 10 MS/MS were performed per MS using reveal the proteolytically regulated, actin organizing protein collision-induced dissociation in the Orbitrap with Multistage Activa- synaptopodin as a coincidence detector of serine/threonine tion. The data were searched using Mascot Distiller and the Mascot and tyrosine signaling, capable of translating signals from dis- search algorithm (version 2.4.0) for uninterpreted MS/MS spectra tinct kinase pathways into coordinated changes in Rho protein– after using the Mascot Distiller program to generate Mascot-compat- mediated remodeling of the actin cytoskeleton. Our work offers ible files. Search parameters used were variable methionine oxidation; insight into a fundamental cell biologic mechanism, while also propionamide modification of cysteine; and phosphorylation of ser- recognizing the therapeutic implications of this discovery for ine, threonine, or tyrosine; a peptide tolerance of +10 ppm; MS/MS the millions of patients with proteinuria,10 the result of a dam- fragment tolerance of +0.62 Da; and peptide charges of +2 or +3. aged podocyte actin cytoskeleton. Normal and decoy database searches were run.

Plasmid Constructs CONCISE METHODS EGFP-Src-iFKBP and Cherry-FRB constructs, allowing the inducible activation of Src by adding rapamycin,39,40 were obtained from

TiO2 and LC-MS/MS Analysis Dr. Klaus Hahn. Flag-tagged wild-type synaptopodin, calcineurin- TiO2 and LC-MS/MS analysis were done by the Yale Keck Proteomic resistant Synpo-ED, CatL-resistant Synpo-CM1+2, 14–3-3, and con- Center. For in-gel protein digestion, the gel band was washed with stitutive active calcineurin have been described previously.29 Vav2, 250 ml 50% acetonitrile/50% water for 5 minutes followed by 250 ml synaptopodin, a-actinin-4, and raver cDNAs were subcloned into

J Am Soc Nephrol 28: 837–851, 2017 Synaptopodin as a Coincidence Detector 847 BASIC RESEARCH www.jasn.org pCMV-Myc-C vector (Clontech). Synpo-Y222A, synpo-Y29A, and enous coimmunoprecipitation from differentiated podocytes, were synpo-Y344A were obtained by using Synpo-short27 in the pCMV- performed as previously described.28,31,32 The following primary an- Myc-C vector as a template to perform point mutations with QuikChange tibodies were used to detect proteins by immunoblotting: anti–EGF Multi Site-Directed Mutagenesis Kit (STRATAGENE) according to the Receptor (no. 4267; Cell Signaling Technology, Danvers, MA), anti– Manufacturer’s protocol. All constructs were verified by DNA sequencing. p-EGF Receptor Y845 (no. 2231; Cell Signaling Technology), anti–p- EGF Receptor Y1068 (no. 3777; Cell Signaling Technology), Cell Culture and Transient Transfection anti–Calcineurin A (no. 2614; Cell Signaling Technology), anti–p- Conditionally immortalized murine podocytes were propagated at 33°C Src Y416 (no. 6943; Cell Signaling Technology), anti–p-Src Y527 in RPMI containing 10% FCS (Invitrogen, Carlsbad, CA), 100 U/ml pen- (no. 2105; Cell Signaling Technology), anti-Src (no. 2123; Cell Sig- icillin (Invitrogen), 100 U/ml streptomycin (Invitrogen), and 10 U/ml naling Technology), anti–p190RhoGAP (no. 610149; BD Biosciences, mouse recombinant g-interferon (Cell Sciences) to induce activation of San Jose, CA), anti-pVAV2 (no. sc-16409-R; Santa Cruz Biotechnology, the T-antigen.69 To differentiate the podocytes, cells were trypsinized, re- Santa Cruz, CA), anti-Rac1 (no. 610650; BD Biosciences) (all used at plated, and cultured in g-interferon–free media at 37°C for 10–14 days. 1:1000), and anti-synaptopodin NT antibody24 (at 1:5000). Anti-Vav2 Transient transfection of HEK293 cells was done by using FuGene 6 Re- (no. 05–1569; Millipore) was used at 1:500, anti–phospho-tyrosine (no. agent (Roche) at a 1:3 DNA/FuGene ratio. Podocytes were treated with the 8954; Cell Signaling Technology) at 1:2000, and anti-Myc (no. 2278; following compounds (all from Sigma-Aldrich, St. Louis, MO) for 1 hour: Cell Signaling Technology) at 1:1000, anti-FLAG (no. F3165; Sigma- SrcI-1 at 5 mM, CsA at 200 nM, E64 at 4 mM, wortmannin at 100 nM, Aldrich) at 1:10,000, and anti-GAPDH (no. CB1001; EMD-Millipore) AG1478 at 500 nM, and EGF at 25 ng/ml. PS was used at 600 mg/ml for at 1:5000. The secondary antibodies were peroxidase-conjugated 1 hour. The Rac1-kinase inhibitor NSC23766 (Millipore) was used at 50 mM confirmation specific mouse anti-rabbit (no. 5127; Cell Signaling Tech- for 1 hour. To activate Src-FKB,39,40 we added 200 nM rapamycin nology), horseradish peroxidase–conjugated goat anti-mouse, goat (Sigma-Aldrich) for 4 hours to podocytes and for 1 hour to HEK293 cells. anti-rabbit (no. W402B, no. W401B; Promega) or goat anti-rat (no. 7077; Cell Signaling Technology); they were used at 1:5000, 1:20,000, Lentiviral Gene Silencing and Overexpression 1:10,000, and 1:1000, respectively. In the immunoprecipitation studies Lentivirus-mediated gene silencing of synaptopodin was done ac- the synaptopodin NT,24 GFP, and Flag antibodies were used at 1:100, cording to recently described protocols.30 To silence Vav2, shRNA whereas the VAV2 antibody was used at 1:50. hairpins were designed using the RNAi consortiums hairpin design protocol (http://www.broadinstitute.org/rnai/public) of the Harvard– Rho Protein Activation Assays Massachusetts Institute of Technology Broad Institute and cloned Activated RhoAwas measured with a commercial Rho activation assay into pLKO.1 vector. The p190RhoGAP hairpins were obtained from kit (Millipore) using a GST-tagged fusion protein corresponding to Open Biosystems, Rac1 hairpins from Sigma-Aldrich. Lentivirus pro- residues 7–89 of mouse Rhotekin Rho-binding domain according to duction in HEK293T cells and infection of podocytes was done as the manufacturer’s instructions and as reported previously.30 After previously described.34 Several shRNAs were screened by Western the pulldown, eluted active RhoA was detected by immunoblotting blotting for knockdown efficiency (Supplemental Figure 9) and hair- using a rabbit monoclonal RhoA antibody (Cell Signaling Technol- pins were chosen for further experiments on the basis of silencing ogy) at 1:1000. Activated Rac1 was measured with a commercial Rac1 efficiency. Selected shRNA sequences are shown in Supplemental activation assay kit (Millipore) using the p21-binding domain of p21- Table1.ForexperimentsinwhichmorethanoneshRNAwas activated protein kinase to bind GTP-bound Rac1, according to the used, both shRNAs were pooled. A nonsilencing shRNA served as manufacturer’s instructions and as reported previously.33 After the negative control. For lentivirus-mediated overexpression, the allo- pulldown, active Rac1 was detected by immunoblotting using a steric engineered inducible c-Src construct RapR-Src and FRB were monoclonal anti-Rac1 antibody (BD Biosciences). Total Rac1 subcloned into the lentiviral VVPW vector. EGFP-tagged synapto- served as loading controls. Rac1 activity was calculated as active podin constructs (wild-type, Synpo-Y222A, Synpo-ED, and Synpo- Rac1/total Rac1 obtained from the quantification of three indepen- CM1+2) were subcloned into the lentiviral vector VVPW and dent experiments. lentiviral particles were produced using HEK 293T cells as recently described in detail.30,34 Podocytes were infected at day nine after ROS Analysis differentiation with 4 mg/ml polybrene (Sigma-Aldrich). When The ROS indicator CM-H2DCFDA (Invitrogen) was used to detect two infections were performed, they were performed on two con- ROS. Podocytes were suspended in PBS buffer containing 1.5 mM secutive days, the first one on day nine and the second on day ten. CM-H2DCFDA on ice in the dark for 30 minutes. Cells were washed Podocytes were harvested or processed for immunostaining at 96 and resuspended in PBS buffer to detect ROS in podocytes in the hours after the first infection.30,34 In keeping with previous results,30 FITC channel using the Flow Cytometer BD LSRFORTESSA. we observed near complete (.95%) infection efficiency of Src- iFKBP and FRB expression in podocytes (Supplemental Figure 4A). Immunocytochemistry Immunocytochemistry was performed after fixing podocytes in 2% Western Blot and Immunoprecipitation paraformaldehyde and 4% sucrose in PBS followed by permeabiliza- SDS-PAGE, Western blotting, coimmunoprecipitation of FLAG- and tion using 0.3% Triton X-100 in PBS for 5 minutes at room GFP- fusion proteins from transfected HEK cells, as well as endog- temperature. After washing with PBS, cells were stained using

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Gee HY, Saisawat P, Ashraf S, Hurd TW, Vega-Warner V, Fang H, Beck L.B. was supported by the Swedish Research Council and The BB, Gribouval O, Zhou W, Diaz KA, Natarajan S, Wiggins RC, Lovric S, Swedish Governmental Agency for Innovation Systems; J.S. by Swiss Chernin G, Schoeb DS, Ovunc B, Frishberg Y, Soliman NA, Fathy HM, Goebel H, Hoefele J, Weber LT, Innis JW, Faul C, Han Z, Washburn J, National Science Foundation fellowship P3SMP3_151739; P.M. by Antignac C, Levy S, Otto EA, Hildebrandt F: ARHGDIA mutations cause National Institutes of Health grants DK057683, DK062472, nephrotic syndrome via defective RHO GTPase signaling. J Clin Invest DK091218; and A.G. by NIH grants DK083511, DK093746, and 123: 3243–3253, 2013 DK095045. 19. Shibata S, Nagase M, Yoshida S, Kawarazaki W, Kurihara H, Tanaka H, fi L.B, H.W., J.S., S.A., and H.Y.C. performed the experiments; Miyoshi J, Takai Y, Fujita T: Modi cation of mineralocorticoid receptor function by Rac1 GTPase: implication in proteinuric kidney disease. Nat L.B, H.W., J.S., S.A., H.Y.C, P.M., and A.G. analyzed the data; P.M and Med 14: 1370–1376, 2008 A.G. designed the experiments and supervised the project; L.B., P.M., 20. Akilesh S, Suleiman H, Yu H, Stander MC, Lavin P, Gbadegesin R, and A.G. wrote the paper. Antignac C, Pollak M, Kopp JB, Winn MP, Shaw AS: Arhgap24 inactivates Rac1 in mouse podocytes, and a mutant form is associated with familial focal segmental glomerulosclerosis. J Clin Invest 121: 4127– DISCLOSURES 4137, 2011 21. Nimnual AS, Taylor LJ, Bar-Sagi D: Redox-dependent downregulation A.G. declares consultation services for Bristol Myers Squibb New York, NY, of Rho by Rac. Nat Cell Biol 5: 236–241, 2003 Merck Kenilworth, NJ, Astellas Northbrook, IL, and Third Rock Ventures 22. Tian X, Ishibe S: Targeting the podocyte cytoskeleton: from patho- Boston, MA. P.M. declares consultation services for Third Rock Ventures. genesis to therapy in proteinuric kidney disease [published online

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