Basic Research

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
JASN March 2017, 28 (3) 837-851; DOI: https://doi.org/10.1681/ASN.2016040414

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.

The function of diverse signaling proteins is controlled by phosphorylation or dephosphorylation.1 Src is a nonreceptor tyrosine kinase2 that affects multiple tyrosine signaling pathways, including cytoskeletal dynamics.3 Src can suppress cell adhesion and disrupt the actin cytoskeleton by mediating tyrosine phosphorylation and activation of p190RhoGAP, thereby inactivating RhoA.4,5 Protein kinase A (PKA) or Ca2+/calmodulin dependent kinase II (CaMKII) mediate the serine/threonine phosphorylation of many actin modulating proteins including RhoA6 and Rac1.7 However, little is known about how signals from competing upstream tyrosine versus serine/threonine kinases are integrated to control actin remodeling, which is critically important in podocyte health and disease.8–10

Many vital cellular functions such as motility, regulation of cell shape, intracellular organization, and membrane trafficking depend on the dynamic modulation of the actin cytoskeleton.11–15 Rho family proteins play a central role in the control of pathways that regulate actin cytoskeletal dynamics in podocytes.9,10,16 RhoA promotes stress fiber formation, which is adaptive in podocytes, whereas Rac1 and Cdc42 promote lamellipodia and filopodia formation, respectively, which are correlated with podocyte injury.9,10,16 Rho proteins can cooperate with or antagonize each other through the activity of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs).11,13–15 Additionally, the Rho guanine nucleotide dissociation inhibitors control their homeostasis and localization.17 The importance of Rho protein function in podocytes is further underscored by an abundance of recent work showing human mutations in Rho regulating proteins, such as the Rac1 regulators Arhgdia18,19 and Arhgap24,20 as causes of progressive proteinuric kidney disease. Furthermore, Rac1-induced reactive oxygen species (ROS) negatively regulate RhoA activity by inhibiting the low molecular weight protein tyrosine phosphatase.21 However, little is known about how competing divergent upstream signals through tyrosine versus serine/threonine kinase activity are integrated for the coordinate regulation of Rho protein crosstalk, which is critically important for podocyte structure and function.9,10,22,23

Synaptopodin is a proline-rich actin binding protein that is expressed in highly dynamic cell compartments, such as the foot processes (FP) of pericyte-like podocytes in the kidney and neuronal dendritic spines in the brain.24 The brain of synaptopodin-deficient mice lacking Synpo-short and Synpo-long shows impaired synaptic plasticity.25,26 Synaptopodin exists in three isoforms, neuronal Synpo-short, renal Synpo-long, and Synpo-T.27 In podocytes, gene silencing of synaptopodin or degradation by cathepsin L (CatL) causes the loss of stress fibers and reduction of RhoA abundance and activity.27–29 Mechanistically, synaptopodin promotes stress fiber formation by blocking the c-Cbl–mediated ubiquitination and proteasomal degradation of Nck130 and the Smurf1-mediated ubiquitination and proteasomal degradation of RhoA.28,31 In addition, synaptopodin can suppress filopodia by disrupting Cdc42:IRSp53:Mena signaling complexes.32 The degradation of synaptopodin by CatL is antagonistically regulated by PKA/CaMKII and calcineurin.29 Serine/threonine phosphorylation of synaptopodin by PKA or CaMKII promotes 14–3-3 binding, which protects synaptopodin from cleavage by CatL.29 Dephosphorylation of synaptopodin by calcineurin abrogates the interaction of 14–3-3 with synaptopodin.29 This renders CatL cleavage sites on synaptopodin accessible, thereby promoting the degradation of synaptopodin by CatL. This can be blocked by the calcineurin inhibitor cyclosporine A (CsA) or the cathepsin inhibitor E64.29 These series of experiments demonstrate that synaptopodin has a nodal role in the regulation of RhoA and Cdc42 in podocytes and raises the question of how it may also be involved in the regulation of Rac1.

The transient receptor potential canonical (TRPC) ion channels TRPC5 and TRPC6 are antagonistic regulators of synaptopodin abundance.33,34 Of note, TRPC5-mediated Ca2+ influx mediates synaptopodin degradation and Rac1 activation.33,34 In these studies, TRPC5 channels were activated by protamine sulfate (PS),33 however, previous studies have shown that EGF receptor (EGFR) signaling is also responsible for TRPC5 activation through the vesicular insertion of TRPC5 channels into the plasma membrane.35 These studies led to the question of whether EGFR/TRPC5/Rac1 signaling may be implicated in synaptopodin-mediated cytoskeletal regulation, and how this may intersect with known EGFR/Src tyrosine phosphorylation events.1 EGF/EGFR signaling is of particular interest given important recent work showing that deletion of EGFR in a podocyte-specific manner attenuates diabetic nephropathy,36,37 and that EGFR promotes glomerular injury in rapidly progressive GN.38 Here we show that synaptopodin plays a central role as a coincidence detector of competing EGFR/Src-mediated tyrosine signals versus serine/threonine signals to orchestrate Rho protein–mediated actin dynamics in podocytes.

Results

Src-Induced Tyrosine Phosphorylation of Synaptopodin Promotes Calcineurin Binding

Synaptopodin is a target of serine/threonine phosphorylation by PKA and CaMKII29 but it also contains putative phospho-acceptor sites for Src. To determine whether synaptopodin is a target of Src, we tested for tyrosine phosphorylation of synaptopodin purified from HEK293 cells coexpressing FRB and Src-iFKBP for inducible activation of Src by rapamycin.39,40 This approach was on the basis of recent studies by Karginov et al., who devised an elegant approach for the allosteric regulation of the catalytic activity of protein kinases in living cells including Src.39,40 The inducible Src-iFKBP consists of a highly conserved portion of the kinase catalytic domain with a small protein insert (iFKBP), which inactivates catalytic activity without altering other protein functions. Binding to rapamycin and FRB restores catalytic activity by increasing the rigidity of Src-iFKBP.39,40 Src-iFKBP is advantageous over the classic constitutively active Src-Y527F because it permits the inducible activation of Src in a time- and dose-dependent fashion in living cells.39,40 The time- and dose-dependent regulation of Src activity was critical for the studies described below, because it allowed us to overcome the problem that overexpression of constitutively active Src-Y527F led to rapid cell death and detachment of podocytes, thereby precluding the study of Src induction in the regulation of podocyte actin dynamics.

To determine whether synaptopodin is tyrosine phosphorylated by Src, we used a phospho-proteomic approach. At 0 or 15 minutes after activation of Src-iFKBP, we could not detect any tyrosine-phosphorylated residues in synaptopodin. In contrast, after 60 minutes of Src activation, three tyrosine-phosphorylated residues (Y29, Y222, and Y344) were found (Figure 1A). The inducible Src-iFKBP kinase employed in this study requires up to 1 hour for full activation with 200 nM rapamycin39; this is also the time point at which we detected robust phosphorylation of synaptopodin. Thus, the kinetics for synaptopodin phosphorylation (between 15 and 60 minutes) most likely reflect the time needed for full activation of Src-FKBP.

Figure 1.
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.

Y222 is conserved in evolution (Figure 1B, arrow) and five amino acids away from the 14–3-3 binding site (T216; Figure 1B, 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 1D). Consistent with the heterologous Co-IP studies (Figure 1C), the activation of Src increased the binding of endogenous calcineurin to endogenous synaptopodin (Figure 1D).

Next, given the loss of 14–3-3 binding after dephosphorylation of synaptopodin by calcineurin,29 we sought to understand 14–3-3 binding in the presence of activated Src. To this end, Co-IP studies showed that activation of Src caused a reduction of 14–3-3 binding to synaptopodin (Figure 2). In contrast, activation of Src did not affect the interaction of 14–3-3 with Src-resistant Synpo-222A (Figure 2). Taken together, these studies showed that Src-induced tyrosine phosphorylation of Y222 increases calcineurin binding to synaptopodin (Figure 1, C and D) to promote serine/threonine dephosphorylation of synaptopodin and reduction in 14–3-3 binding29 (Figure 2).

Figure 2.
Figure 2.

Activation of Src impairs 14–3-3 binding to synaptopodin. Exogenous coimmunoprecipitation studies in HEK293 cells reveal that activation of Src-iFKBP (Src*) reduces the interaction of FLAG-14–3-3β with Myc-synaptopodin when compared with inactive Src-iFKBP (Src). In contrast, the interaction of Src-resistant Synpo-222A with 14–3-3 is not altered by Src activation. Molecular mass markers are in kDa.

Calcineurin Inhibition Protects from Src-Induced Synaptopodin Degradation and Stress Fiber Loss

Synaptopodin promotes stress fiber formation through activation of Nck1-dependent RhoA signaling.28,30,31 Pharmacologic inhibition of PKA or CaMKII reduces synaptopodin protein abundance and disrupts stress fibers, which can be rescued by the calcineurin inhibitor CsA.29 The observed increase in calcineurin binding and reduction of 14–3-3 binding to Src-phosphorylated synaptopodin prompted us to test whether Src reduces synaptopodin protein abundance by promoting the degradation of synaptopodin. In keeping with this hypothesis, activation of Src by rapamcyin in Src-iFKB and FRB-expressing podocytes (Supplemental Figure 4A) reduced synaptopodin protein abundance, which was blocked by CsA or the cathepsin inhibitor E64 (Figure 3A). Functionally, the preservation of synaptopodin protein abundance was associated with an inhibition of Src-induced loss of stress fibers (Figure 3B). In contrast, CsA did not restore stress fibers in synaptopodin-depleted podocytes in the presence of activated Src (Supplemental Figure 4B). The most likely explanation for the observed effects is the activation of Src, rather than on- or off-target effects of rapamycin or infection with lentiviral constructs. In control experiments, well developed stress fibers were present in noninfected podocytes before and after rapamycin treatment, and in Src-iFKB/FRB coinfected podocytes (Supplemental Figure 5A). Moreover, the protein abundance of synaptopodin, active pSrc (417), and inactive pSrc (527) remained unchanged after treating control podocytes (in the absence of Src-iFKBP and FRB) with rapamcyin (200 nM) for 4 hours (Supplemental Figure 5, B and C). However, rapamycin treatment of isolated glomeruli has been shown to attenuate p70S6K phosphorylation,41 and a study in human podocytes has shown efficient block of p70S6K phosphorylation by 24 and 120 hours of treatment with rapamycin,42 raising the possibility that cellular effects of rapamycin not examined in this study could potentially also modulate stress fibers.

Figure 3.
Figure 3.

CsA, E64, Synpo-Y222A, Synpo-ED, or Synpo-CM1+2 protect from Src-induced stress fiber loss. (A) Synaptopodin protein abundance in podocytes is reduced by activated Src-iFKBP (Src*) compared with cells with inactive Src-iFKBP (Src). Src*-induced reduction of synaptopodin protein abundance is blocked by the calcineurin inhibitor CsA or the cathepsin inhibitor E64. GAPDH serves as a loading control. (B) Phalloidin labeling reveals loss of stress fibers after activation of Src (Src*) but not in cells with inactive Src-iFKBP (Src). CsA and E64 protect from Src*-induced stress fiber loss; scale bar 20 μm. (C) GFP-tagged (130 kDa) Src-resistant (Y222A), calcineurin-resistant (ED), or CatL-resistant (CM1+2) but not wild-type (WT) synaptopodin are protected from Src*-induced degradation. Endogenous synaptopodin is seen below the 116 kDa marker. GAPDH serves as a loading control. (D) Overexpression of Synpo-Y222A, Synpo-ED, or Synpo-CM1+2 but not Synpo-WT protects from Src*-induced loss of stress fibers; scale bar 20 μm. (E) Quantitative analysis confirms protection from Src*-induced loss of stress fibers by CsA, E64, Synpo-Y222A, Synpo-ED, or SynpoCM1+2. Values are represented as % stress fiber containing cells ±SEM; n=3; ANOVA; P<0.001. Molecular mass markers are in kDa.

Src-Induced Stress Fiber Loss is Blocked by Degradation-Resistant Synaptopodin

In a complementary genetic approach, we analyzed the effects of Src-resistant Synpo-Y222A, calcineurin-resistant Synpo-ED,29 or CatL-resistant Synpo-CM1+229 on Src-induced degradation of synaptopodin and loss of stress fibers. Lentiviral overexpression of Synpo-Y222A, Synpo-ED, or Synpo-CM1+2 but not of wild-type synaptopodin protected against Src-induced degradation (Figure 3C). Functionally, Synpo-Y222A, Synpo ED, or Synpo CM1+2 but not wild-type synaptopodin conferred resistance against Src-induced loss of stress fibers (Figure 3D). The quantitative analysis of the data in Figure 3, B and D, showed a significant preservation of stress fibers by CsA, E64, Synpo-Y222A, Synpo-ED, or Synpo-CM1+2 (n=50 cells × 3 independent experiments =150 cells; inactive Src: 92%±1.0% SEM stress fiber containing cells; active Src (Src*): 11.3%±1.8%; Src* + CsA: 67.0%±2.0%; Src* + E64: 64.6%±1.8%; Src* + wild-type synaptopodin: 18.3%±2.5%; Src* + Synpo-Y222A: 88.1%±1.1%; Src* + Synpo-ED: 69.2%±3.8%; Src* + CM1+2: 66.5%±1.6%; 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 synaptopodin-depleted cells (n=3; 2.28±0.37 SEM) compared with synaptopodin-replete control cells (0.29±0.09; P<0.01; t test; Figure 4C). Rac1 can increase the production of ROS21,43,44; therefore, we analyzed ROS levels in synaptopodin-depleted cells using flow cytometry. We detected significantly increased ROS levels in synaptopodin-depleted cells (n=3; 5.2%±2.0% SEM) when compared with control shRNA-expressing cells (n=3; 0%±0.7%; P<0.05; t test; Figure 4D).

Figure 4.
Figure 4.

Synaptopodin inhibits Rac1 signaling by blocking Vav2 activation. (A) Western blot analysis shows synaptopodin depletion in synaptopodin knockdown (Synpo shRNA) podocytes. GAPDH serves as a loading control (B). Synaptopodin depletion (synpo shRNA) increases protein abundance of GTP-bound active Rac1 when compared with control shRNA (con) expressing podocytes. (C) Quantitative analysis confirms significant increase of Rac1 activation in synpo shRNA cells. Values are presented as GTP-bound Rac1/Total amount Rac1 ±SEM; n=3; t test; P<0.01. (D) Increased ROS production in synaptopodin-depleted podocytes; data are presented as % of control shRNA cells ±SEM; n=3; t test; P<0.05. (E) Coimmunoprecipitation from HEK293 cells shows interaction between Myc-Vav2 and FLAG-Synpo-short. Myc-α-actinin-4 (Act4) serves as positive control and Myc-Raver (con) as negative control. (F) Endogenous coimmunoprecipitation confirms the interaction between Vav2 and 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 synaptopodin-depleted (synpo shRNA) podocytes when compared with control cells (con shRNA). Total Vav2 levels are not changed. (H) Quantitative analysis of activated Vav2. Values are presented as pVav2/total Vav2 ±SEM; n=3; t test; P<0.05. Molecular mass markers are in kDa.

Synaptopodin Inhibits the Activation of the Rac1 GEF Vav2

To examine how synaptopodin can suppress Rac1 activity, we tested whether synaptopodin blocks a Rac1 activating GEF. Synaptopodin is a proline-rich protein capable of binding to various SH3-containing proteins, including CD2AP,45 IRSp53,32 and Nck1/230; therefore, we focused on SH3 domain–containing GEFs, and further refined our search to those SH3-domain GEFs activated downstream of Src. Vav2 is an SH3 domain–containing GEF for Rac1,46 which can be activated by Src.47,48 We therefore asked whether synaptopodin could interact with Vav2. In heterologous Co-IP experiments in cotransfected HEK293 cells, we found that Myc-Vav2 could bind to FLAG-Synpo-short (Figure 4E). The interaction of synaptopodin with α-actinin-427 served as positive control. No interaction was found with Myc-raver, serving as negative control (Figure 4E), thereby confirming the specificity of the interaction. The interaction between synaptopodin and Vav2 was further examined by endogenous Co-IP studies; in protein extracts from cultured podocytes, anti-Vav2 antibody precipitated Vav2 and coprecipitated synaptopodin (Figure 4F). Conversely, anti-synaptopodin antibody precipitated synaptopodin and coprecipitated Vav2 (Figure 4F). To test whether the observed interaction between synaptopodin and Vav2 is functionally relevant, we examined the protein abundance of active Vav2 by Western blot analysis of 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; 1.07±0.33 SEM) when compared with control cells (n=3; 0.10±0.05; 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 Rac1-mediated inhibition of RhoA.11,21,49 In addition to the previously reported loss of RhoA activity,28,30 we now find increased Rac1 activity in synaptopodin-depleted cells (Figure 4C). Therefore, we examined whether inhibition of Rac1 signaling could restore stress fibers in synaptopodin-depleted cells. We suppressed Rac1 signaling in synaptopodin-depleted podocytes by coexpression of Vav2, Rac1, or nonsilencing control shRNAs and validated the efficiency of protein depletion by Western blot (Supplemental Figure 5D). To assess the crosstalk between Rac1 and RhoA GTPases in the absence of synaptopodin, we analyzed changes in total/active Rac1 and RhoA protein abundance in synaptopodin-depleted podocytes before and after silencing of Vav2 or Rac1 (Figure 5A). We found that gene silencing of Vav2 or Rac1 abrogated the activation of Rac1 and restored activation of RhoA in synaptopodin-depleted podocytes (Figure 5A). The quantitative analysis confirmed a significant increase in Rac1 activity in synaptopodin-depleted cells (n=3; 1.40±0.11 SEM) compared with synaptopodin/Vav2 (n=3; 0.01±0.01; P<0.001; ANOVA) or synaptopodin/Rac1 (n=3; 0.00±0.00; P<0.001; ANOVA) codepleted cells (Figure 5B). Conversely, RhoA activity was significantly reduced in synaptopodin-depleted cells (n=3; 0.09±0.06 SEM) compared with synaptopodin/Vav2 (n=3; 1.06±0.18; P<0.05; ANOVA) or synaptopodin/Rac1 (n=3; 1.03±0.19; P<0.05; ANOVA) codepleted cells (Figure 5B).

Figure 5.
Figure 5.

Gene silencing of Vav2 or Rac1 restores Rho protein crosstalk and stress fibers in synaptopodin-depleted podocytes. (A) Western blot analysis showing changes in total and active Rac1 and RhoA protein abundance in synaptopodin-depleted podocytes before and after silencing of Vav2 or Rac1. (B) Quantitative analysis confirms 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 ±SEM or RhoA/Total amount RhoA ±SEM; n=3; ANOVA; ***P<0.001; *P<0.05. (C) Gene silencing of Vav2 or Rac1 restores stress fibers in synaptopodin-depleted podocytes; scale bar 20 μm. (D) Quantitative analysis confirms rescue of stress fiber formation by Vav2 or Rac1 shRNAs. Values are presented as % stress fiber containing cells compared with control shRNA expressing cells ±SEM; n=3; ANOVA; ****P<0.001. Molecular mass markers are in kDa.

We then visualized the actin cytoskeleton by phalloidin staining and found the restoration of stress fibers in synaptopodin-depleted cells codepleted of Vav2 or Rac1 (Figure 5C). The quantitative analysis showed a near complete rescue of stress fiber containing cells after codepletion of Vav2 or Rac1 (n=50 cells × 3 independent experiments =150 cells; control shRNA: 95.5%±1.5% SEM stress fiber containing cells; Synpo shRNA: 4.3%±1.6%; Synpo shRNA + Vav2 shRNA: 84.7%±1.4%; Synpo shRNA + Rac1 shRNA: 86.9%±0.9%; P<0.001; ANOVA; Figure 5D). Similar to gene silencing of Rac1, pharmacologic inhibition of Rac1 with NSC23766 restored stress fibers in synaptopodin-depleted cells (Supplemental Figure 6). Taken together, synaptopodin preserves stress fibers by simultaneously blocking Rac1 (Figure 5, A and B) and promoting 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 hypothesized that PS signals through EGFR, Src, and PI3 kinase activation,1 leading to the degradation of synaptopodin and loss of stress fibers. We observed that PS increased the abundance of phosphorylated pEGFR (Figure 6A). PS also increased protein abundance of active pSrc (Y416) and decreased protein abundance of inactive pSrc (Y527) (Figure 6B). In addition to the previously described increase in Rac1 activity,33 PS also decreased total and active RhoA levels (Figure 6C). In a detailed analysis, we observed that synaptopodin abundance was preserved in cells treated with the EGFR inhibitor AG1478, SrcI-1, the PI3 kinase inhibitor wortmannin, the calcineurin inhibitor CsA, or the CatL inhibitor E64 (Figure 6D). Of note, podocytes do not express lymphocyte-specific protein tyrosine kinase (Lck; Supplemental Figure 7), thereby excluding an effect of SrcI-1 on Lck in our studies. In a complementary genetic approach, we analyzed the effects of Src-resistant Synpo-Y222A, calcineurin-resistant Synpo-ED,29 or CatL-resistant Synpo-CM1+229 on PS-induced degradation of synaptopodin and loss of stress fibers. Synpo-Y222A, Synpo-ED, or Synpo-CM1+2 but not wild-type synaptopodin were resistant to PS-induced degradation (Figure 6E).

Figure 6.
Figure 6.

PS-induced degradation of synaptopodin is blocked by inhibition of EGFR, Src, calcineurin, or CatL. (A) Immunoprecipitation of endogenous EGFR shows increased abundance of tyrosine phosphorylated EGFR (Y1068, Y845) in PS-treated podocytes. Total EGFR serves as loading control. (B) PS increases the abundance of active (pSrc Y416) and decreases the abundance of inactive (pSrc Y527) endogenous Src. Total Src serves as a loading control. (C) PS decreases the abundance of active RhoA. (D) Inhibition of PS-induced degradation of synaptopodin by EGFR blocker AG1478 (AG), SrcI-1, PI3K inhibitor wortmannin (Wort), calcineurin inhibitor CsA, or 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 resistant against PS-induced degradation. Note that the bands below 130 kDa correspond to overexpressed synaptopodin and the bands below 116 kDa to endogenous synaptopodin. GAPDH serves as a loading control. Molecular mass markers are in kDa.

In keeping with the observed stabilization of synaptopodin protein abundance (Figure 6E), application of AG1478, SrcI-1, wortmannin, CsA, or E64 also protected from PS-induced loss of stress fibers (Figure 7A). Overexpression of Synpo-Y222A, Synpo ED, or Synpo CM1+2 but not wild-type synaptopodin had no effect on stress fibers at baseline (Supplemental Figure 8) but conferred protection from PS-induced loss of stress fibers (Figure 7B). The quantitative analysis (Figure 7C) showed a near complete rescue of stress fibers by AG1478, SrcI-1, or wortmannin, and to a lesser degree by CsA or E64 (n=50 cells × 3 independent experiments =150 cells; control cells 95.5%±0.7% SEM stress fiber containing cells; PS treated cells: 1.7%±1.0%; PS + AG1478: 69.2%±1.3%; PS + Src-1 inhibitor: 79.1%±1.0%; PS + wortmannin: 73.3%±0.8%; PS + CsA: 49.2%±1.0%; PS + E64: 54.1%±1.0%; PS + wild-type synaptopodin: 14.0%±1.3%; PS + Synpo-Y222A: 77.0%±2.4%; PS + Synpo-ED: 54.7%±3.0%; PS + Synpo-CM1+2: 53.0%±2.6%; P<0.001; ANOVA).

Figure 7.
Figure 7.

PS-induced stress fiber loss is blocked by inhibition of EGFR, Src, calcineurin, or CatL. (A) Phalloidin staining shows rescue of PS-induced loss of stress fibers by AG1478, SrcI-1, wortmannin, CsA, or E64. (B) Overexpression of GFP-Synpo-Y222A, GFP-Synpo-ED, or GFP-Synpo-CM1+2 but not wild-type GFP-Synpo-short protects against PS-induced stress fiber loss in podocytes; scale bar 20 μm. (C) Quantitative analysis confirms protection by CsA, E64, Synpo-Y222A, Synpo-ED, or SynpoCM1+2. Values are presented as % stress fiber containing cells ±SEM; n=3; ANOVA; ****P<0.001. Molecular mass markers are in kDa.

Finally, to test whether the observed effect could be recapitulated with a physiologic 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.

Figure 8.
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 μm. (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.

Discussion

The experiments described here allowed us to explore several important questions. First, we show that the actin organizing protein synaptopodin is not only phosphorylated by the serine/threonine kinases PKA and CaMKII,29 but also by the tyrosine kinase Src. Second, our results show that serine/threonine and tyrosine phosphorylation duel for synaptopodin stability versus degradation. Serine/threonine phosphorylation by PKA/CaMKII stabilizes synaptopodin.29 In contrast, tyrosine phosphorylation of synaptopodin by Src increases the binding of synaptopodin to calcineurin, thereby promoting the dephosphorylation of serine/threonine residues required for 14–3-3 binding.29 Third, we demonstrate that synaptopodin can suppress Rac1 signaling by blocking the Vav2-mediated activation of Rac1. These results unveil synaptopodin as a coincidence detector for competing serine/threonine and tyrosine signals to regulate Rho protein crosstalk in podocytes (Figure 8D).

Calcineurin has a central role in the regulation of diverse calcium-dependent signaling events.57 In addition to the transcription factor NFAT,58 various cytoskeletal proteins such as microtubule-associated protein 2,59,60 tubulin,60 tau factor,60 and slingshot61 are regulated by calcineurin-mediated serine/threonine dephosphorylation. Our results illuminate a novel mechanism of coordinate tyrosine kinase (Src) and serine/threonine phosphatase (calcineurin) activities on the same protein substrate: Src phosphorylation enables the binding of calcineurin to synaptopodin. Importantly, synaptopodin does not contain the known calcineurin-binding motifs PxIxIT62,63 or LxVP,64 suggesting that calcineurin binding is indeed dependent on Src activity. We speculate that synaptopodin contains a novel calcineurin-binding site, whose binding affinity is increased by Src-mediated phosphorylation at Y222. 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 t1/231 and high susceptibility to proteolytic degradation.24,29

Receptor tyrosine kinase receptors (RTKs) are key regulators of several cellular processes.1 EGFR is a well characterized RTK known to regulate many cellular processes such as proliferation, differentiation, cytoskeletal regulation, and transcription.1,65 Src has long been implicated in signal transduction pathways downstream of EGFR-induced tyrosine phosphorylation.66,67 PS is involved in EGFR activation54–56 and EGFR-dependent activation of the Ca2+-permeable TRPC5 channel.34,35 Intriguingly, TRPC5 activation leads to the degradation of synaptopodin downstream of PS.33 Our data unify these previous findings into a cohesive signaling pathway in which synaptopodin is the substrate of Src-mediated tyrosine phosphorylation triggered by PS-induced EGFR activation (Figure 8D). According to this pathway, PI3 kinase (PI3K) inhibition may antagonize PS-induced suppression of synaptopodin through at least two synergistic mechanisms: (1) PS-induced synaptopodin degradation requires TRPC5-mediated activation of calcineurin29,33 and EGF-induced membrane insertion of TRPC5 requires PI3 kinase activity.35 (2) EGF-induced PI3 kinase signaling can increase Vav2-mediated Rac1 activity.1 Rac1 activity, in addition to PI3 kinase activity, is also required for EGF-induced membrane insertion and function of TRPC5.34,35 The observed positive effect of PI3K inhibition on synaptopodin protein abundance lends further support to the conclusion that PS signals through EGFR to trigger the degradation of synaptopodin. These data are of particular interest in the context of important recent work showing that EGFR signaling is an important mediator of injury in glomerular disease,38 and that deletion of EGFR in a podocyte-specific manner attenuates diabetic nephropathy.36,37 The implications of this work may also be far reaching given recent findings that urinary EGF may serve as a robust biomarker for the progression of CKD.68

In conclusion, our data reveal a signaling network, which integrates input from upstream receptor pathways (EGFR), kinase systems (Src versus PKA/CaMKII), and the Rho proteins to regulate essential cellular functions (Figure 8D). As shown in this model, PS induces Src and PI3K activation, downstream of the EGFR, leading to Vav2-mediated activation of Rac1 and ensuing PIP(5)Kα-dependent membrane insertion and activation of TRPC5.34,35 TRPC5-mediated Ca2+ influx increases calcineurin activity,34 thereby abrogating the PKA/CAMKII-mediated serine/threonine phosphorylation and 14–3-3 binding of synaptopodin.29 The resulting degradation of synaptopodin29,33 leads to increased Rac1 activity, ROS production, and inactivation of RhoA. PS also induces tyrosine phosphorylation of Y222 in synaptopodin by Src. This in turn increases the binding of calcineurin to synaptopodin, thereby promoting the loss of 14–3-3 binding and CatL-mediated degradation.29 Degradation of synaptopodin abrogates its capacity to block the c-Cbl–induced proteasomal degradation of Nck130 and the Smurf1-induced proteasomal degradation of RhoA28; it also reduces the capacity of synaptopodin to block the Vav2-mediated activation of Rac1, leading to increased ROS production, and decreased RhoA activity (Figure 8). Thus, the absence of synaptopodin shifts the overall Rho protein balance from RhoA toward Rac1. Our studies reveal the proteolytically regulated, actin organizing protein synaptopodin as a coincidence detector of serine/threonine and tyrosine signaling, capable of translating signals from distinct kinase pathways into coordinated changes in Rho protein–mediated remodeling of the actin cytoskeleton. Our work offers insight into a fundamental cell biologic mechanism, while also recognizing the therapeutic implications of this discovery for the millions of patients with proteinuria,10 the result of a damaged podocyte actin cytoskeleton.

Concise Methods

TiO2 and LC-MS/MS Analysis

TiO2 and LC-MS/MS analysis were done by the Yale Keck Proteomic Center. For in-gel protein digestion, the gel band was washed with 250 μl 50% acetonitrile/50% water for 5 minutes followed by 250 μl of 50 mM ammonium bicarbonate/50% acetonitrile/50% water for 30 minutes. One final wash was done using 10 mM ammonium bicarbonate/50% acetonitrile/50% water for 30 minutes. After washing, the gel pieces were dried in a Speedvac. Trypsin was prepared by mixing 10 µl 0.1mg/ml trypsin (Promega, Madison, WI) with 140 µl 10 mM ammonium bicarbonate and adding 15 µl PhosStop (Roche, Basel, Switzerland) as prepared following the manufacturer’s directions. The gel was then rehydrated with 30 µl of the trypsin solution and digested at 37°C for 16 hours. For titanium dioxide enrichment, the digest was acidified with 0.5% TFA, 50% acetonitrile. TopTips (Glygen Corporation) were prepared by washing three times with 40 µl each of 100% acetonitrile, followed by 0.2 M sodium phosphate pH 7.0, and 0.5% TFA, 50% acetonitrile. Washes were spun through into an Eppendorf tube at 2000 rpm for 1 minute. The acidified digest supernatant was loaded into the TopTip, spun at 1000 rpm for 1 minute, and then at 3000 rpm for 2 minutes. Gel pieces were rinsed with 40 µl 0.5% TFA, 50% acetonitrile, with the supernatant transferred to the TopTip and the spin repeated. The TopTip was then washed with 40 µl 0.5% TFA, 50% acetonitrile, and the spin repeated. The flow through from these washes was saved and analyzed by LC-MS/MS as below. Phosphopeptides were eluted from the TopTip by three times 30 µl 28% ammonium hydroxide. Both the flow through and eluted fractions were dried in a SpeedVac, and redried from 40 µl of water. Samples were dissolved in 3 µl 70% formic acid, vortexed, diluted with 7 µl 0.1% TFA, spun, and transferred to LC-MS/MS vials where 5 µl was injected for LC-MS/MS on the Thermo Scientific LTQ Orbitrap Elite. The LTQ Orbitrap Elite is equipped with a Waters nanoAcquity UPLC system, and uses a Waters Symmetry C18 180 µm × 20 mm trap column and a 1.7 µm, 75 µm × 250 mm nanoAcquity UPLC column (37°C) for peptide separation. Trapping was done at 5 µl/min, 99% Buffer A (100% water, 0.1% formic acid) for 3 minutes. Peptide separation was performed at 300 nl/min with Buffer A: 100% water, 0.1% formic acid; and Buffer B: 100% CH3CN, 0.075% formic acid. A linear gradient (51 minutes) was run with 1% buffer B at initial conditions, 65% B at 50 minutes, and 85% B at 51 minutes. MS was acquired in the Orbitrap part of the instrument (300–2000 m/z) using 1 microscan, and a maximum inject time of 500 ms and up to 10 MS/MS were performed per MS using collision-induced dissociation in the Orbitrap with Multistage Activation. The data were searched using Mascot Distiller and the Mascot search algorithm (version 2.4.0) for uninterpreted MS/MS spectra after using the Mascot Distiller program to generate Mascot-compatible files. Search parameters used were variable methionine oxidation; propionamide modification of cysteine; and phosphorylation of serine, threonine, or tyrosine; a peptide tolerance of +10 ppm; MS/MS fragment tolerance of +0.62 Da; and peptide charges of +2 or +3. Normal and decoy database searches were run.

Plasmid Constructs

EGFP-Src-iFKBP and Cherry-FRB constructs, allowing the inducible activation of Src by adding rapamycin,39,40 were obtained from Dr. Klaus Hahn. Flag-tagged wild-type synaptopodin, calcineurin-resistant Synpo-ED, CatL-resistant Synpo-CM1+2, 14–3-3, and constitutive active calcineurin have been described previously.29 Vav2, synaptopodin, α-actinin-4, and raver cDNAs were subcloned into pCMV-Myc-C vector (Clontech). Synpo-Y222A, synpo-Y29A, and synpo-Y344A were obtained by using Synpo-short27 in the pCMV-Myc-C vector as a template to perform point mutations with QuikChange Multi Site-Directed Mutagenesis Kit (STRATAGENE) according to the Manufacturer’s protocol. All constructs were verified by DNA sequencing.

Cell Culture and Transient Transfection

Conditionally immortalized murine podocytes were propagated at 33°C in RPMI containing 10% FCS (Invitrogen, Carlsbad, CA), 100 U/ml penicillin (Invitrogen), 100 U/ml streptomycin (Invitrogen), and 10 U/ml mouse recombinant γ-interferon (Cell Sciences) to induce activation of the T-antigen.69 To differentiate the podocytes, cells were trypsinized, replated, and cultured in γ-interferon–free media at 37°C for 10–14 days. Transient transfection of HEK293 cells was done by using FuGene 6 Reagent (Roche) at a 1:3 DNA/FuGene ratio. Podocytes were treated with the following compounds (all from Sigma-Aldrich, St. Louis, MO) for 1 hour: SrcI-1 at 5 μM, CsA at 200 nM, E64 at 4 μM, wortmannin at 100 nM, AG1478 at 500 nM, and EGF at 25 ng/ml. PS was used at 600 μg/ml for 1 hour. The Rac1-kinase inhibitor NSC23766 (Millipore) was used at 50 μM for 1 hour. To activate Src-FKB,39,40 we added 200 nM rapamycin (Sigma-Aldrich) for 4 hours to podocytes and for 1 hour to HEK293 cells.

Lentiviral Gene Silencing and Overexpression

Lentivirus-mediated gene silencing of synaptopodin was done according to recently described protocols.30 To silence Vav2, shRNA hairpins were designed using the RNAi consortiums hairpin design protocol (http://www.broadinstitute.org/rnai/public) of the Harvard–Massachusetts Institute of Technology Broad Institute and cloned into pLKO.1 vector. The p190RhoGAP hairpins were obtained from Open Biosystems, Rac1 hairpins from Sigma-Aldrich. Lentivirus production in HEK293T cells and infection of podocytes was done as previously described.34 Several shRNAs were screened by Western blotting for knockdown efficiency (Supplemental Figure 9) and hairpins were chosen for further experiments on the basis of silencing efficiency. Selected shRNA sequences are shown in Supplemental Table 1. For experiments in which more than one shRNA was used, both shRNAs were pooled. A nonsilencing shRNA served as negative control. For lentivirus-mediated overexpression, the allosteric engineered inducible c-Src construct RapR-Src and FRB were subcloned into the lentiviral VVPW vector. EGFP-tagged synaptopodin constructs (wild-type, Synpo-Y222A, Synpo-ED, and Synpo-CM1+2) were subcloned into the lentiviral vector VVPW and lentiviral particles were produced using HEK 293T cells as recently described in detail.30,34 Podocytes were infected at day nine after differentiation with 4 μg/ml polybrene (Sigma-Aldrich). When two infections were performed, they were performed on two consecutive days, the first one on day nine and the second on day ten. Podocytes were harvested or processed for immunostaining at 96 hours after the first infection.30,34 In keeping with previous results,30 we observed near complete (>95%) infection efficiency of Src-iFKBP and FRB expression in podocytes (Supplemental Figure 4A).

Western Blot and Immunoprecipitation

SDS-PAGE, Western blotting, coimmunoprecipitation of FLAG- and GFP- fusion proteins from transfected HEK cells, as well as endogenous coimmunoprecipitation from differentiated podocytes, were performed as previously described.28,31,32 The following primary antibodies were used to detect proteins by immunoblotting: anti–EGF Receptor (no. 4267; Cell Signaling Technology, Danvers, MA), anti–p-EGF Receptor Y845 (no. 2231; Cell Signaling Technology), anti–p-EGF Receptor Y1068 (no. 3777; Cell Signaling Technology), anti–Calcineurin A (no. 2614; Cell Signaling Technology), anti–p-Src Y416 (no. 6943; Cell Signaling Technology), anti–p-Src Y527 (no. 2105; Cell Signaling Technology), anti-Src (no. 2123; Cell Signaling Technology), anti–p190RhoGAP (no. 610149; BD Biosciences, San Jose, CA), anti-pVAV2 (no. sc-16409-R; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Rac1 (no. 610650; BD Biosciences) (all used at 1:1000), and anti-synaptopodin NT antibody24 (at 1:5000). Anti-Vav2 (no. 05–1569; Millipore) was used at 1:500, anti–phospho-tyrosine (no. 8954; Cell Signaling Technology) at 1:2000, and anti-Myc (no. 2278; Cell Signaling Technology) at 1:1000, anti-FLAG (no. F3165; Sigma-Aldrich) at 1:10,000, and anti-GAPDH (no. CB1001; EMD-Millipore) at 1:5000. The secondary antibodies were peroxidase-conjugated confirmation specific mouse anti-rabbit (no. 5127; Cell Signaling Technology), horseradish peroxidase–conjugated goat anti-mouse, goat 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, 1:10,000, and 1:1000, respectively. In the immunoprecipitation studies the synaptopodin NT,24 GFP, and Flag antibodies were used at 1:100, whereas the VAV2 antibody was used at 1:50.

Rho Protein Activation Assays

Activated RhoA was measured with a commercial Rho activation assay kit (Millipore) using a GST-tagged fusion protein corresponding to residues 7–89 of mouse Rhotekin Rho-binding domain according to the manufacturer’s instructions and as reported previously.30 After the pulldown, eluted active RhoA was detected by immunoblotting using a rabbit monoclonal RhoA antibody (Cell Signaling Technology) at 1:1000. Activated Rac1 was measured with a commercial Rac1 activation assay kit (Millipore) using the p21-binding domain of p21-activated protein kinase to bind GTP-bound Rac1, according to the manufacturer’s instructions and as reported previously.33 After the pulldown, active Rac1 was detected by immunoblotting using a monoclonal anti-Rac1 antibody (BD Biosciences). Total Rac1 served as loading controls. Rac1 activity was calculated as active Rac1/total Rac1 obtained from the quantification of three independent experiments.

ROS Analysis

The ROS indicator CM-H2DCFDA (Invitrogen) was used to detect ROS. Podocytes were suspended in PBS buffer containing 1.5 μM CM-H2DCFDA on ice in the dark for 30 minutes. Cells were washed and resuspended in PBS buffer to detect ROS in podocytes in the FITC channel using the Flow Cytometer BD LSRFORTESSA.

Immunocytochemistry

Immunocytochemistry was performed after fixing podocytes in 2% paraformaldehyde and 4% sucrose in PBS followed by permeabilization using 0.3% Triton X-100 in PBS for 5 minutes at room temperature. After washing with PBS, cells were stained using rhodamine-labeled phalloidin at 1:750 (Molecular Probes) to visualize F-actin.28 Image analysis was performed in ImageJ or Adobe Photoshop for Mac OS X. Confocal images were acquired with a Zeiss upright confocal microscope. Images from an optical slice of approximately 1–5 µm were acquired at a resolution of 1200 pixels per inch with Zeiss Pascal software.34 Image analysis was performed in ImageJ or Adobe Photoshop CS5.1.

Stress Fiber Quantification

Stress fiber–containing cells were quantified as described previously.31,34 We included at least three independent trials for each experimental condition, where >50 cells were counted in each trial in 5–10 independent images per trial.

RT-PCR Analysis of Src Family Kinase Expression

To determine the mRNA expression levels of the Src family kinases Lck and Src, mRNA was extracted from mouse tissue and cultured podocytes using an RNeasy mini kit (Qiagen, Germantown, MD) according to the manufactures instructions. The cDNA was prepared using dNTPs, oligo-dT, RNase out, and reverse transcription (Superscript) supplied by Life Technologies (Carlsbad, CA). To detect Src, Lck, and GAPDH, mRNA primers described in Supplemental Table 2 were used. The thermal cycling conditions were 94°C for 2 minutes, followed by 35 cycles of 94°C for 15 seconds, 55°C for 15 seconds, and 72°C for 30 seconds.

Statistical Analysis

Statistical significance was evaluated using GraphPad Prism 6.0 software by one-way ANOVA with Dunnett multiple comparison test or t test. P<0.05 was considered significant. Values are reported as mean±SEM.

Disclosures

A.G. declares consultation services for Bristol Myers Squibb New York, NY, Merck Kenilworth, NJ, Astellas Northbrook, IL, and Third Rock Ventures Boston, MA. P.M. declares consultation services for Third Rock Ventures.

Acknowledgments

We thank the Yale Keck Proteomic Center for TiO2 and LC-MS/MS analysis. We also thank Dr. Klaus Hahn, University of North Carolina at Chapel Hill, for providing FRB and Src-iFKB.

L.B. was supported by the Swedish Research Council and The Swedish Governmental Agency for Innovation Systems; J.S. by Swiss National Science Foundation fellowship P3SMP3_151739; P.M. by National Institutes of Health grants DK057683, DK062472, DK091218; and A.G. by NIH grants DK083511, DK093746, and DK095045.

L.B, H.W., J.S., S.A., and H.Y.C. performed the experiments; L.B, H.W., J.S., S.A., H.Y.C, P.M., and A.G. analyzed the data; P.M and A.G. designed the experiments and supervised the project; L.B., P.M., and A.G. wrote the paper.

Footnotes

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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, Anna Greka
JASN Mar 2017, 28 (3) 837-851; DOI: 10.1681/ASN.2016040414
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