Supplemental material

Supplemental figures

Supplemental Fig. S1: Characterization of murine glomerular preparation. (A) Glomerular lysates (Glom) and cortex kidney lysates (remnants of the glomerular preparation) were subjected to immunoblotting and membranes were probed with antibodies against Nephrin and Podocin as glomerular markers as well as NaKATPase (alpha subunit) and the Tamm-Horsefall-glycoprotein (THP). (B) Glomeruli isolated by magnetic beads were lysed in 8M Urea and subjected to three consecutive sonication steps. The urea unsoluble pellet was sonicated and dissolved in 2% SDS buffer. Same volumes of lysates were analyzed for content of Nephrin and Podocin.

Supplemental Fig. S2: Characterization of phosphorylation datasets. (A) Comparison of distribution of serines, threonines and tyrosin phosphorylation sites. (B) Bar graph depiciting the number of phosphorylation sites per protein.

Supplemental Fig. S3: Results of conservational analysis of detected phosphorylation sites A. Automated batch-alignment was performed using the cPhos software and phosphosites were evaluated in terms of conservation of phosphorylation sites and phosphorylation residues. The results show the conservation score of phosphorylation sites and surrounding motifs. (B, C) Examples for high or low conserved phosphorylation sites and motifs in the phosphoproteomic dataset. 9 vertebrate species of the respective homologene group are listed. The central residue (marked with yellow) shows the phosphorylation site. (B) Src pY424 is a highly conserved site (site conservation score = 1.0, motif conservation score = 0.92) (C) Akap1 pS55 is a low conserved phosphorylation sites (site conservation score = 0.65, motif conservation score = 0.66). Residues are colour-coded according to their electrostatic and structural properties (red= negatively charged residues [D;E]; blue: positively charge residues [R;K]; green: prolines)

Supplemental Fig. S4: Modeling and free energy simulation of PHB domain interaction. (A) Modeling of the effect of phosphorylation on Stomatin PHB domain conformation. The crystal structure PDB ID 4FVF was chosen as a reference for this simulation. (B) Construction of homologous model of Podocin based on the stomatin PHB domain dimers and analysis of the effect of phosphorylation on PHB domain conformation. (C) Thermodynamic cycle of Podocin PHB domain phosphorylation and dimerization. The free energy G denotes the effect of phosphorylation on the dimer and can be simulated based on the given equation. (D) Free energy simulation of dimers was performed in a 93.31×62.21×46.66 Å3 sized periodic box of water to analyze the effect of phosphorylation on the dimerization. (E) Monomer was extracted from dimer structure and was dissolved in a 77.758×46.655×46.655 Å3 sized periodic box of water for the free energy simulation of phosphorylation.

Fig. S5: Further characterization of Podocin phosphorylation at T234. Phosphoproteomic analysis of Podocin PHB-domain phosphorylation at T234 in the presence of an inactive (T410A) or constitutively active (T410E) protein kinase C zeta (rPKCz). A representative reconstructed ion chromatogram of the MS1 precursor (“ion current”) is shown.

Supplemental tables

Supplemental Table S1: Tyrosine phosphorylation sites on glomerular proteins

This table depicts high-confidence tyrosin phosphorylation sites on glomerular proteins. In the modified sequence column the original sequence of the phosphopeptide is depicted. “*” denotes serine, threonine and tyrosin phosphorylation whereas “#” denotes (methionin-) oxidation.

Gene UniProt symbol Protein Name ID Residue Phosphopeptide sequence Abhd14a Abhydrolase domain-containing Q922Q6 Y59 IFY*REVLPIQQACR protein 14A Abi3 ABI gene family member 3 Q8BYZ1 Y318 VVT*LY*PY*T*RQK Abi3 ABI gene family member 3 Q8BYZ1 Y320 VVT*LY*PY*T*RQK Ank3 ankyrin-3 G5E8K5 Y1461 KRY*SYLTEPSMSPQS*PCER Ankrd61 Ankyrin repeat domain-containing Q9CQM6 Y315 NGES*PIY*M#Y*LQR protein 61 Ankrd61 Ankyrin repeat domain-containing Q9CQM6 Y317 NGES*PIY*M#Y*LQR protein 61 Arhgap35 Rho GTPase-activating protein 35 Q91YM2 Y1105 NEEENIY*SVPHDSTQGK Arntl2 Aryl hydrocarbon receptor nuclear Q2VPD4 Y258 KFHT*VHCT*GY*LR translocator-like protein 2 Bcar1 Breast cancer anti-estrogen Q61140 Y310 GLLSSSHHSVY*DVPPSVSK resistance protein 1 Bckdha 2-oxoisovalerate dehydrogenase Q3U3J1 Y346 IGHHS*TSDDSSAY*RSVDEVNYWDKQDHPIS subunit alpha, mitochondrial R Brca2 Breast cancer type 2 P97929 Y581 FIY*S*VS*DDAS*LQGK susceptibility protein homolog Bzrap1 Peripheral-type benzodiazepine Q7TNF8 Y1766 T*S*RPM#VAAFDY*NPR receptor-associated protein 1 Carkd ATP-dependent (S)-NAD(P)H- K3W4M4 Y103 IGIVGGCQEY*TGAPYFAGISALK hydrate dehydratase Ccdc105 Coiled-coil domain-containing Q9D4K7 Y372 FNQEM#Y*VTRGIIK protein 105 Ccdc147 coiled-coil domain containing 147 B2RW38 Y245 QAEIKAM#QQY*MHKSK Ccdc158 Coiled-coil domain-containing Q8CDI6 Y106 FY*LRQSVIDLQTK protein 158 Ccdc39 Coiled-coil domain-containing Q9D5Y1 Y642 Y*EILT*VVM#LPPEGEEEK protein 39 Cdk2 Cyclin-dependent kinase 2 P97377 Y15 IGEGTY*GVVYK Cntnap1 Contactin-associated protein 1 O54991 Y105 YMLLY*GDR Ctnnd1 Catenin delta-1 E9Q8Z8 Y96 LNGPQDHNHLLY*STIPR Ctnnd1 Catenin delta-1 E9Q8Z8 Y280 FHPEPY*GLEDDQRSMGYDDLDYGMM#SDY GTAR Ctnnd1 Catenin delta-1 E9Q8Z8 Y865 SQSSHSY*DDSTLPLIDR Cyth2 Cytohesin-2 D3YU95 Y391 DEWIKS*IQAAVS*VDPFY*EM#LAAR Dock3 Dedicator of cytokinesis protein 3 F8VPQ1 Y1203 LMERLLDY*RDCM#K Dpy19l3 Protein dpy-19 homolog 3 Q71B07 Y612 TQAVY*QIY*AKR Dpy19l3 Protein dpy-19 homolog 3 Q71B07 Y615 TQAVY*QIY*AKR Drg2 Developmentally-regulated GTP- Q9QXB9 Y332 Y*ALVWGT*S*TKYS*PQR binding protein 2 Dyrk1a Dual specificity tyrosine- Q61214 Y321 IYQY*IQSR phosphorylation-regulated kinase 1A Dyrk4 Dual specificity tyrosine- Q8BI55 Y379 VYTY*IQSR phosphorylation-regulated kinase 4 Eef1a1 Elongation factor 1-alpha 1 P10126 Y418 SGDAAIVDMVPGKPMCVESFSDY*PPLGR Epb4.1 Protein 4.1 A2A841 Y665 ERLDGENIY*IR Epb41l3 Band 4.1-like protein 3 Q9WV92 Y479 DSVSAAEVGTGQY*ATTK Erbb2ip Protein LAP2 B7ZNX6 Y1097 RTEGDY*LSYR

Fam180a Protein FAM180A Q8BR21 Y135 EDFERIT*LTLAYT*AY*R Fat1 FAT tumor suppressor homolog 1 F2Z4A3 Y3062 SNAEIT*Y*T*LFGSGAEK precursor Fgd1 FYVE, RhoGEF and PH domain- P52734 Y93 ALRFS*Y*HLEGSQPR containing protein 1 Gab1 GRB2-associated-binding protein Q9QYY0 Y660 SSGSGSSM#ADERVDY*VVVDQQK 1 Gm4955 G3UZV2 Y20 MVNY*YKQIVLLSGLEYM#NDY*NFR Protein Gm4955 Gm6434 F7CXS7 Y67 PY*VNY*RAPAGIDT*PLT*VK Protein Gm6434 Gm6434 F7CXS7 Y70 PY*VNY*RAPAGIDT*PLT*VK Protein Gm6434 Gm8298 uncharacterized protein J3QPI0 Y235 EY*KHGPFLT*R LOC666803 precursor Gpx1 Glutathione peroxidase 1 P11352 Y147 Y*IIWSPVCR Grm7 Metabotropic glutamate receptor G5E8D5 Y492 NGDAPGRYDIFQYQTT*NT*TNPGY*R 7 Gstt4 Glutathione S-transferase theta-4 Q9D4P7 Y6 GLELY*M#DLLS*APCR Hes1 Transcription factor HES-1 P35428 Y110 AQM#TAALSTDPSVLGKY*R Hipk3 Homeodomain-interacting protein A2AQH3 Y359 TVCSTY*LQSR kinase 3 Idh3b isocitrate dehydrogenase 3, beta Q91VA7 Y120 VAIIGKIYT*PM#EY*K subunit Ifitm3 Interferon-induced Q9CQW9 Y27 IKEEY*EVAEMGAPHGSASVR transmembrane protein 3 Ipo13 Importin-13 Q8K0C1 Y735 M#Y*ST*VPQAS*ALDLTR Kdr Vascular endothelial grow th factor P35918 Y1212 FHY*DNTAGISHYLQNSK receptor 2 Kiaa1731 Centrosomal protein KIAA1731 Q8BQ48 Y586 LLEY*QT*VLK homolog Kif23 kinesin family member 23 E9Q5G3 Y380 TCM#EVLRENQT*Y*GT*NK Lyn Tyrosine-protein kinase Lyn P25911 Y194 SLDNGGYY*ISPR Mapk1 Mitogen-activated protein kinase P63085 Y185 VADPDHDHTGFLTEY*VATR 1 Mapk12 Mitogen-activated protein kinase O08911 Y185 QADSEM#TGY*VVTR 12 Mapk3 Mitogen-activated protein kinase D3Z3G6 Y205 IADPEHDHTGFLTEY*VATR 3 Mbp myelin basic protein F6RT34 Y99 PGLCHMY*KDS*HTR Mmrn1 Multimerin-1 B2RPV6 Y1205 YPPVTTFS*GY*LLY*R Mmrn1 Multimerin-1 B2RPV6 Y1208 YPPVTTFS*GY*LLY*R Mrps22 28S ribosomal protein S22, Q9CXW2 Y102 T*FRPAIQPLKPPT*Y*K mitochondrial Mrps35 28S ribosomal protein S35, Q8BJZ4 Y289 EELLGTKEVEDY*QK mitochondrial Myh10 Myosin-10 Q3UH59 Y680 IVGLDQVT*GMT*ET*AFGSAY*K Myo10 Unconventionnal myosin-X F8VQB6 Y1482 LY*TKLLNEAT*RWSSAIQNVT*DT*K Myo1a Unconventional myosin-Ia O88329 Y585 NLYS*KNPNY*IRCIK Ncf2 Neutrophil cytosol factor 2 O70145 Y351 ELKLS*VPM#PY*M#LK Nedd9 Enhancer of filamentation 1 O35177 Y344 ANPEERDGVY*DVPLHNPADAK Olfr318 olfactory receptor 318 Q5NCD7 Y123 Y*IAICQPLHY*PVLM#S*RK Olfr318 olfactory receptor 318 Q5NCD7 Y132 Y*IAICQPLHY*PVLM#S*RK Papss2 Bifunctional 3'-phosphoadenosine O88428 Y21 STNVVY*QAHHVSR 5'-phosphosulfate synthase 2 Pdk1 [Pyruvate dehydrogenase Q8BFP9 Y163 NRHNDVIPT*M#AQGVT*EY*K [lipoamide]] kinase isozyme 1, mitochondrial Polr2a DNA -directed RNA polymerase II P08775 Y413 RGNS*QY*PGAKY*IIR subunit RPB1 Polr2a DNA -directed RNA polymerase II P08775 Y418 RGNS*QY*PGAKY*IIR subunit RPB1 Prpf4b Serine/threonine-protein kinase Q61136 Y849 LCDFGSASHVADNDITPY*LVSR PRP4 homolog Ptk2 Focal adhesion kinase 1 P34152 Y614 YMEDSTY*YK Ptp4a2 Protein tyrosine phosphatase O70274 Y11 PAPVEISY*ENM#RFLITHNPT*NAT*LNK type IVA 2

Pxn Paxillin F8VQ28 Y118 AGEEEHVY*SFPNK Sat1 Diamine acetyltransferase 1 A2BES2 Y27 LIKELAKY*EY*M#EDQVILT*EK Sat1 Diamine acetyltransferase 1 A2BES2 Y29 LIKELAKY*EY*M#EDQVILT*EK Shh Sonic hedgehog protein Q62226 Y45 LT*PLAY*KQFIPNVAEKT*LGAS*GR Slc22a1 Solute carrier family 22 member 1 O08966 Y545 AKENTIY*LQVQTGK Slc6a20a Sodium- and chloride-dependent Q8VDB9 Y204 VVY*FTAS*M#PY*CVLIIYLVR transporter XTRP3A Sorbs1 Sorbin and SH3 domain- Q62417 Y325 SIY*EYQPGK containing protein 1 Sorcs1 VPS10 domain-containing E9QQ02 Y495 T*FIT*Y*NK receptor SorCS1 Spsb1 SPRY domain-containing SOCS Q9D5L7 Y19 T*VDM#RDPT*Y*R box protein 1 Src Neuronal proto-oncogene P05480 Y424 LIEDNEY*TAR tyrosine-protein kinase Src Stat3 Signal transducer and activator of P42227 Y705 YCRPESQEHPEADPGSAAPY*LK transcription 3 Stk38 Serine/threonine-protein kinase Q91VJ4 Y115 DT*GHVY*AM#KILR 38 Susd1 sushi domain containing 1 E9Q3H4 Y685 LY*YGEY*YNAPLKTGNEY*CILLR precursor Susd1 sushi domain containing 1 E9Q3H4 Y696 LY*YGEY*YNAPLKTGNEY*CILLR precursor Tex2 Testis-expressed sequence 2 Q6ZPJ0 Y234 QESDTVS*Y*KPPDSK protein Tjp2 Tight junction protein ZO-2 Q9Z0U1 Y1095 IEIAQKHPDIY*AVPIK Tln1 Talin-1 P26039 Y26 TMQFEPSTMVY*DACR Tln1 Talin-1 P26039 Y70 ALDY*YM#LR Tmc8 Transmembrane channel-like B0QZP7 Y45 M#LPY*AM#ADKRFIR protein 8 Tnk2 Activated CDC42 kinase 1 G3X9X7 Y874 VS*ST*HY*Y*LLPER Tnk2 Activated CDC42 kinase 1 G3X9X7 Y875 VS*ST*HY*Y*LLPER Tns1 tensin 1 E9Q0S6 Y1480 HAAYGGY*STPEDR Tns1 tensin 1 E9Q0S6 Y1558 AGSLPNY*ATINGK Trrap transformation/transcription E9PWT1 Y892 AELM#QALWRT*LRNPADSISHVAY*R domain-associated protein Ttll1 Probable tubulin polyglutamylase Q91V51 Y268 WTVNNLRLY*LES*TRGR TTLL1 Ttyh2 Protein tw eety homolog 2 Q3TH73 Y494 Y*ENVPLIGR Vcl Vinculin Q64727 Y822 SFLDSGY*R Vmn1r202 vomeronasal 1 receptor 202 Q8R259 Y218 RVLY*LHS*S*R Vmn2r74 vomeronasal receptor Vmn2r74 E9PW21 Y79 HLIFS*VY*LALEEINK precursor Yes1 Tyrosine-protein kinase Yes Q04736 Y192 GAY*SLSIR Yes1 Tyrosine-protein kinase Yes Q04736; Y424 LIEDNEY*TAR P05480 Zdhhc5 Palmitoyltransferase ZDHHC5 Q8VDZ4 Y533 LLPTGPPHREPS*PVRY*DNLSR 2310030G0 Uncharacterized protein C11orf52 Q9D8L0 Y79 TSNSTSEESDLHY*ADIHVLR 6Rik homolog

Supplemental Table S2.

Gene ontology terms enriched and deenriched in phosphoproteins with serine/threonine phosphorylation sites. The five most enriched molecular function and cellular component GO-Term categories are depicted. A phosphoproteomic mouse tissue atlas dataset of serine/threonine phosphorylated proteins by Huttlin et al. was used as a background dataset (Huttlin et al. Cell 2010).

Enrichment Benj. Hoch. Category GO Term factor -lg(P value) FDR Count Molecular guanyl-nucleotide exchange 1.62 5.23 0.00125 22 Function factor activity phospholipid binding 1.47 6.92 0.00011 36 nucleoside-triphosphatase 1.4 5.63 0.00056 59 regulator activity GTPase regulator activity 1.4 5.63 0.00064 32 enzyme regulator activity 1.39 6.8 0.00008 62 Cellular cell-cell adherens junction 1.86 3.5 0.01667 22 Component lamellipodium 1.8 4.83 0.00207 36 adherens junction 1.78 7.22 0.00005 59 focal adhesion 1.77 4.21 0.00468 32 anchoring junction 1.75 7.16 0.00003 62

Deenriched Enrichment Benj. Hoch. Category GO Term factor -lg(P value) FDR Count Molecular gated channel activity 1.86 4.49 0.01667 15 Function Cellular ion channel complex 1.8 3.75 0.00207 7 Component

Supplemental Table S3.

Gene ontology terms enriched in phosphoproteins with tyrosin phosphorylation sites. A phosphoproteomic mouse tissue atlas dataset of tyrosin phosphorylated proteins by Huttlin et al. was used as a background dataset (Huttlin et al. Cell 2010). No phosphorylated proteins were found to be deenriched.

Enrichment Benj. Hoch. Category GO Term Factor -lg(p-value) FDR count Molecular signal transducer activity 2.87 4.2 0.021 14 function molecular transducer 2.87 4.2 0.041 14 activity Cellular focal adhesion 3.8 3.95 0.017 9 compartment cell-substrate adherens 3.8 3.95 0.025 9 junction cell-substrate junction 3.78 4.32 0.021 10

Supplemental Table S4: phosphorylation sites on podocyte specific proteins. This table outlines phosphorylation sites on proteins shown to be significantly higher expressed in podocytes. These candidate proteins are based on a proteomic study by Boerries et al., Kidney International 2013. In the modified sequence column the original sequence of the phosphopeptide is

depicted. “*” denotes serine, threonine and tyrosin phosphorylation whereas “#” denotes (methionin- )oxidation.

Residue Gene symbol Protein name UniProtID number Modified sequence 0610010K14Rik Chromatin complexes subunit BAP18 D3Z687 S96 VYEDSGIPLPAES*PKKGPK Medium-chain specific acyl-CoA dehydrogenase, Acadm mitochondrial P45952 T351 RNT*YYASIAK Actr3 Actin-related protein 3 Q99JY9 S418 HNPVFGVM#S* Canx Calnexin P35564 S582 AEEDEILNRS*PR QKSDAEEDGVTGS*QDEEDS Canx Calnexin P35564 S563 KPK QKS*DAEEDGVTGSQDEEDS Canx Calnexin P35564 S553 KPK Cd2ap CD2-associated protein Q9JLQ0 S510 FNGGHS*PTQS*PEK Cd2ap CD2-associated protein Q9JLQ0 S580 AEADDGKRNS*VDELR Cd2ap CD2-associated protein Q9JLQ0 S514 FNGGHS*PTQS*PEK Cd2ap CD2-associated protein Q9JLQ0 S458 S*VDLDAFVAR Bifunctional ATP-dependent dihydroxyacetone AS*YISSAQLDQPDPGAVAAA Dak kinase/FAD-AMP lyase (cyclizing) Q8VC30 S545 AIFR Dlg1 Disks large homolog 1 Q811D0 S619 FGDILHVINAS*DDEWWQAR Dlg1 Disks large homolog 1 Q811D0 S573 EQM#M#NSSVSSGS*GSLR Dlg1 Disks large homolog 1 Q811D0 S575 EQM#M#NSSVSSGSGS*LR Dpysl2 Dihydropyrimidinase-related protein 2 O08553 T509 GLYDGPVCEVSVT*PK TVT*PAS*SAKTSPAKQQAPP Dpysl2 Dihydropyrimidinase-related protein 2 O08553 T514 VR Dync1h1 Cytoplasmic dynein 1 heavy chain 1 Q9JHU4 S4366 TDS*TSDGRPAWMR Dync1h1 Cytoplasmic dynein 1 heavy chain 1 Q9JHU4 S1228 RKDS*AIQQQVANLQMK Epb41l5 Band 4.1-like protein 5 Q8BGS1 S581 EDS*LLTHK Epb41l5 Band 4.1-like protein 5 Q8BGS1 S348 FRYS*GKTEYQTTK SWKGDDS*PVANGAEPAGQ Epn3 Epsin-3 Q91W69 S257 R Heterogeneous nuclear ribonucleoprotein U-like AVEEQGDDQDS*EKSKPAGS Hnrnpul2 protein 2 Q00PI9 S183 DGER Heterogeneous nuclear ribonucleoprotein U-like Hnrnpul2 protein 2 Q00PI9 S159 S*GDETPGSEAPGDK Ilk Integrin-linked protein kinase O55222 S186 NGTLNKHS*GIDFK Ilk Integrin-linked protein kinase O55222 S232 S*RDFNEECPR Iqgap2 Ras GTPase-activating-like protein IQGAP2 Q3UQ44 S764 IQSLLRAS*K Iqgap2 Ras GTPase-activating-like protein IQGAP2 Q3UQ44 S1461 S*IKLDGKAEPK Iqgap2 Ras GTPase-activating-like protein IQGAP2 Q3UQ44 S16 YGS*IVDDER VETVNREPLTMHS*DREDDT Kirrel Kin of IRRE-like protein 1 Q80W68 S584 ASISTATR Kirrel Kin of IRRE-like protein 1 Q80W68 S775 FSYTSQHS*DYGQR VETVNREPLTMHSDREDDTA Kirrel Kin of IRRE-like protein 1 Q80W68 T595 S*IST*ATR VETVNREPLTMHS*DREDDT Kirrel Kin of IRRE-like protein 1 Q80W68 S592 AS*ISTATR VETVNREPLTM#HS*DREDD Kirrel Kin of IRRE-like protein 1 Q80W68 T590 T*ASISTATR Cation-dependent mannose-6-phosphate GVGDDQLGEES*EERDDHLL M6pr receptor P24668 S268 PM Membrane-associated guanylate kinase, WW and Magi2 PDZ domain-containing protein 2 Q9WVQ1 S1013 IIPQEELNS*PTSAPSSEK Mapt Microtubule-associated protein A2A5Y6 S510 SGYSSPGS*PGTPGSR Mapt Microtubule-associated protein A2A5Y6 S507 SGYSS*PGS*PGTPGSR AKTDHGAEIVYKS*PVVS*GD Mapt Microtubule-associated protein A2A5Y6 S708 T*SPR TDHGAEIVYKS*PVVS*GDTS* Mapt Microtubule-associated protein A2A5Y6 S704 PR Mapt Microtubule-associated protein A2A5Y6 S506 SGYS*SPGS*PGTPGSR

Mapt Microtubule-associated protein A2A5Y6 T539 VAVVRT*PPKS*PSASK Mapt Microtubule-associated protein A2A5Y6 S570 IGS*TENLKHQPGGGK Mapt Microtubule-associated protein A2A5Y6 S664 IGS*LDNITHVPGGGNKK TTPSPKT*PPGSGEPPKSGE Mapt Microtubule-associated protein A2A5Y6 T489 R Mapt Microtubule-associated protein A2A5Y6 S522 S*RTPS*LPTPPTREPK Nes Nestin Q6P5H2 S728 ERQES*LKSPEEEDQQAFR Nes Nestin Q6P5H2 S816 LVEKES*QES*LKSPEEEDQR Nes Nestin Q6P5H2 S813 LVEKES*QES*LKSPEEEDQR Nes Nestin Q6P5H2 S688 S*PEEDQQAFRPLEK Nes Nestin Q6P5H2 S731 ERQESLKS*PEEEDQQAFR Nes Nestin Q6P5H2 S819 LVEKESQESLKS*PEEEDQR Nphs2 Podocin Q91X05 S362 AQGS*INYPSSSKPVEPLNPK Nphs2 Podocin Q91X05 T234 SLT*EILLER Nphs2 Podocin Q91X05 S382 KKDS*PML Palld Palladin Q9ET54 S901 IAS*DEEIQGTK Palld Palladin Q9ET54 S1146 SRDS*GDENEPIQER Pard3 Partitioning defective 3 homolog B7ZNY3 S906 GRGCNES*FRAAIDK Pard3b Partitioning defective 3 homolog B Q9CSB4 S801 SYDGPEEADADGLS*DKSSR Pard3b Partitioning defective 3 homolog B Q9CSB4 S1088 GGS*ADPVDYLTASPR Pard3b Partitioning defective 3 homolog B Q9CSB4 S575 SMS*MEGNIR Pard3b Partitioning defective 3 homolog B Q9CSB4 S780 GRGCNES*FR Pard3b Partitioning defective 3 homolog B Q9CSB4 S990 DGRPLS*PDHLEGLYAK Pard3b Partitioning defective 3 homolog B Q9CSB4 S730 SDS*PGKDFGPTLGLKK Pard3b Partitioning defective 3 homolog B Q9CSB4 S635 NDSSILYPFGTYS*PQDKRK Pard3b Partitioning defective 3 homolog B Q9CSB4 S346 ASSPEGEEPAS*PQQSK Pard3b Partitioning defective 3 homolog B Q9CSB4 S1182 GSS*PDQYPYR Parva Alpha-parvin Q9EPC1 S28 KKDDS*FLGK Pdlim5 PDZ and LIM domain protein 5 Q8CI51 S228 RGS*QGDIKQQNGPPR AIGGIILT*ASHNPGGPNGDF Pgm2 Phosphoglucomutase-1 A2CEK3 T133 GIK Membrane-associated progesterone receptor Pgrmc2 component 2 Q80UU9 S98 KRDFS*LEQLR Membrane-associated progesterone receptor LLKPGEEPSEYT*DEEDTKDH Pgrmc2 component 2 Q80UU9 T205 SK cAMP-dependent protein kinase type II-alpha RVS*VCAETFNPDEEEEDND Prkar2a regulatory subunit Q8K1M3 S97 PR Ptpro Receptor-type tyrosine-protein phosphatase O E9Q612 T869 ECGAGT*FVNFASLER Rhpn1 Rhophilin-1 E9Q7Q7 S31 KGYGS*FVQNQPGQLQSHR Sec22b Vesicle-trafficking protein SEC22b O08547 S137 NLGS*INTELQDVQR Na(+)/H(+) exchange regulatory cofactor NHE- Slc9a3r1 RF1 P70441 S285 SAS*SDTSEELNSQDSPKR Na(+)/H(+) exchange regulatory cofactor NHE- Slc9a3r1 RF1 P70441 S289 SAS*S*DTS*EELNSQDSPK Na(+)/H(+) exchange regulatory cofactor NHE- Slc9a3r1 RF1 P70441 S337 SS*KRAPQMDWSK Na(+)/H(+) exchange regulatory cofactor NHE- Slc9a3r1 RF1 P70441 S275 EALVEPASES*PRPALAR Na(+)/H(+) exchange regulatory cofactor NHE- Slc9a3r1 RF1 P70441 S297 SASSDTSEELNSQDS*PKR Na(+)/H(+) exchange regulatory cofactor NHE- Slc9a3r1 RF1 P70441 T288 SASSDT*SEELNSQDSPK Na(+)/H(+) exchange regulatory cofactor NHE- Slc9a3r2 RF2 Q9JHL1 S298 RDPFQES*GLHLSPTAAEAK Na(+)/H(+) exchange regulatory cofactor NHE- Slc9a3r2 RF2 Q9JHL1 S280 SDLPGS*EKDNEDGSTWK Na(+)/H(+) exchange regulatory cofactor NHE- Slc9a3r2 RF2 Q9JHL1 S303 RDPFQESGLHLS*PTAAEAK

KPSLVSDLPWEGASPQSPSF Sntb2 Beta-2-syntrophin Q61235 S213 SGSEDSGS*PK Sntb2 Beta-2-syntrophin Q61235 S75 GLGPPS*PPAPPR Sntb2 Beta-2-syntrophin Q61235 S238 NLS*MPDLENR Snx1 Sorting nexin-1 Q9WV80 S280 AVGTQALS*GAGLLK Snx1 Sorting nexin-1 Q9WV80 S188 RFS*DFLGLYEK Sord Sorbitol dehydrogenase Q64442 S171 RGSVS*LGNK Stx4 Syntaxin-4 P70452 S15 QGDNIS*DDEDEVR Stx4 Syntaxin-4 P70452 S117 AIEPQKEEADENYNS*VNTR DRAS*PAAAEEAVPEWASCL Synpo Synaptopodin E9Q3E2 S689 KS*PR DRAS*PAAAEEAVPEWASCL Synpo Synaptopodin E9Q3E2 S672 K Synpo Synaptopodin E9Q3E2 S1002 MRS*PQPAS*PAR Synpo Synaptopodin E9Q3E2 S512 AWAPPASSMADRS*PQPQR Synpo Synaptopodin E9Q3E2 S1007 MRS*PQPAS*PAR Synpo Synaptopodin E9Q3E2 S1091 RGS*LPTEASCTT Synpo Synaptopodin E9Q3E2 S535 LLGQRS*PVLER Synpo Synaptopodin E9Q3E2 S1023 S*PLPIGPSSCASPR Synpo Synaptopodin E9Q3E2 S1084 GWNGS*LR Synpo Synaptopodin E9Q3E2 S946 SVS*PLRS*ETEARPPSR Synpo Synaptopodin E9Q3E2 S942 SVS*PLRSETEARPPSR Synpo Synaptopodin E9Q3E2 S1018 GAAFS*PIPR Synpo Synaptopodin E9Q3E2 S134 STS*FTENDLK Synpo Synaptopodin E9Q3E2 S1037 S*PQAAPSRPFPYR Synpo Synaptopodin E9Q3E2 S1051 S*PTDSDVSLDSEDSGLK Synpo Synaptopodin E9Q3E2 S833 AAS*PAKPSSLDLVPNLPR VRS*PPSYSTLYPSSDPKPSH Synpo Synaptopodin E9Q3E2 S567 LK Synpo Synaptopodin E9Q3E2 S258 VAS*EEEEVPLVVYLK Synpo2 Synaptopodin-2 E9Q1U2 S895 AQS*PTPSLPASWK VQIPVSHPDPEPVS*DNEDD Tjp1 Tight junction protein ZO-1 B9EHJ3 S125 S*YDEEVHDPR FNHNLLPSETVHKPELSSKT* Tjp1 Tight junction protein ZO-1 B9EHJ3 T1484 PT*SPK Tjp1 Tight junction protein ZO-1 B9EHJ3 S622 SREDLS*AQPVQTKFPAYER Tjp1 Tight junction protein ZO-1 P39447 S912 IDS*PGLKPASQQK Tjp1 Tight junction protein ZO-2 B9EHJ3 S912 IDS*PGLKPASQQVYR Tjp1 Tight junction protein ZO-1 P39447 S175 S*VASSQPAKPTK Tjp1 Tight junction protein ZO-1 B9EHJ3 S1058 FEEPAPLS*YDSR Tjp1 Tight junction protein ZO-1 P39447 S617 S*REDLSAQPVQTK AVPVS*PSAVEEDEDEDGHT Tjp1 Tight junction protein ZO-1 B9EHJ3 S1534 VVATAR Tjp2 Tight junction protein ZO-2 Q9Z0U1 S968 DAS*PPPAFKPEPPK Tjp2 Tight junction protein ZO-2 Q9Z0U1 Y1095 IEIAQKHPDIY*AVPIK Tjp2 Tight junction protein ZO-2 Q9Z0U1 S895 MSYLTAMGADYLS*CDSR Tjp2 Tight junction protein ZO-2 Q9Z0U1 S404 RQQYS*DQDYHSSTEK Tjp2 Tight junction protein ZO-2 Q9Z0U1 S213 S*IDRDYDRDYER Tjp2 Tight junction protein ZO-2 Q9Z0U1 S1136 GSYGS*DPEEEEYR SYHEAYEPDYGGGYS*PSYD Tjp2 Tight junction protein ZO-2 Q9Z0U1 S239 RR Tjp2 Tight junction protein ZO-2 Q9Z0U1 S395 SFS*PEER Tjp2 Tight junction protein ZO-2 Q9Z0U1 S441 S*TGDITAAGVTEASREPR

Tjp2 Tight junction protein ZO-2 Q9Z0U1 S209 GLDRDFVSRDHS*R Tjp2 Tight junction protein ZO-2 Q9Z0U1 S107 KVQVAPLQGS*PPLSHDDR Tmod3 Tropomodulin-3 Q9JHJ0 S71 LLS*YLEK Tpd52 Tumor protein D52 F8WHQ1 S198 NSPTFKS*FEEKVENLK Tpd52 Tumor protein D52 Q62393 S170 KLEDVKNS*PTFK Tpd52l2 Tumor protein D54 A2AUD5 S189 NSATFKS*FEDR Tpd52l2 Tumor protein D54 A2AUD5 S96 S*WHDVQVSTAYVK GRM#S*MKEVDEQMLNVQN Tubb4b Tubulin beta-4B chain P68372 S322 K Cytochrome b-c1 complex subunit 1, Uqcrc1 mitochondrial Q9CZ13 S212 RLS*RTDLTDYLNR AAQAS*LNALNDPIAVEQALQ Utrn Utrophin E9Q6R7 S933 EK Voltage-dependent anion-selective channel Vdac1 protein 1 Q60932 S117 LTFDSSFS*PNTGKK Vim Vimentin P20152 S73 LRSS*VPGVR Vim Vimentin P20152 S56 SLYSSS*PGGAYVTR Vim Vimentin P20152 S55 SLYSS*SPGGAYVTR Vim Vimentin P20152 S72 LRS*SVPGVR QVQS*LTCEVDALKGTNESL Vim Vimentin P20152 S325 ER Vim Vimentin P20152 S51 S*LYSSSPGGAYVTR

Supplemental Table S5: phosphorylation sites on bona fide podocyte specific proteins. This table outlines phosphorylation sites on proteins known to be almost exclusively expressed in podocytes. The references for the respective hypothesis-driven studies can be found in the main text.

Gene symbol Protein name UniProtID Residue number Modified sequence

Pla2r1 Secretory phospholipase A2 receptor Q62028 S1486 GRPICIS*P

Pdlim2 PDZ and LIM domain protein 2 Q8R1G6 S124 SACFS*PVSLSPR

Pdlim2 PDZ and LIM domain protein 2 Q8R1G6 S199 VLLHS*PGRPSS*PR

Pdlim2 PDZ and LIM domain protein 2 Q8R1G6 S210 FSS*LDLEEDSEVFK

Pdlim2 PDZ and LIM domain protein 2 Q8R1G6 T276 ALAT*PPKLHTCEK

Slc43a1 Large neutral amino acids transporter small subunit 3 G3X8X3 S450 DGAS*TKFTRPR

Nphs1 Nephrin Q9QZS7 S1112 LAEEIS*EKT*EAGS*EEDR

Nphs1 Nephrin Q9QZS7 S1119 TEAGS*EEDRIR

Fat1 FAT tumor suppressor homolog 1 precursor F2Z4A3 Y3062 SNAEIT*Y*T*LFGSGAEK

Fat1 FAT tumor suppressor homolog 1 precursor F2Z4A3 T3063 SNAEIT*Y*T*LFGSGAEK

A Glom Kidney kDa Nephrin

130

Podocin 70

55

Na K ATPase 100

THP 100

B 8 M Urea Pellet Sonication 2% SDS 1 2 3 kDa Nephrin

130

Podocin 70

55

Fig. S1 A es 3000

2000

1000 Number of phosphosi t

S T Y B

1200

eins 1000 o t

800

600

400

200 Number of phosphop r 0 1 2 3 4 5 6-10 >10 Number of phosphosites

Fig. S2 A Motif conservation Site conservation high (score: 0.95-1) medium (score: 0.8-0.95) low (score: <0.8)

B Src, pY424 HID: 21120 RefSeq Species Amino acid window NP_033297.2 M. musculus 418 LIEDNEYTARQGA 430 NP_001003837.2 D. rerio 411 LIEDNEYTARQGA 423 NP_990788.2 G. gallus 410 LIEDNEYTARQGA 422 NP_001104274.1 B. taurus 419 LIEDNEYTARQGA 431 XP_865870.1 C. lupus 413 LIEDNEYTARQGA 425 NP_114183.1 R. norvegicus 419 LIEDNEYTARQGA 431 XP_001092180.1 M. mulatta 413 LIEDNEYTARQGA 425 XP_001141228.2 P. troglodytes 413 LIEDNEYTARQGA 425 NP_938033.1 H. sapiens 413 LIEDNEYTARQGA 425

C Akap1, pS55

HID: 31165 RefSeq Species Amino acid window NP_001036006.1 M. musculus 49 IKDRRLSEEACPG 61 NP_001091649.2 D rerio 52 EGSNGMVDKSCPA 64 XP_415913.1 G. gallus 54 NDSPPKTEACVPQ 66 XP_002695633.1 B. taurus 53 AQEVLPVEDSRPG 65 XP_003435322.1 C. lupus 52 IKEPLSMEDPCPA 64 NP_446117.1 R. norvegicus 49 IEDRLPTEEACPG 61 XP_001105339.1 M. mulatta 53 IKEPLPMEDVCPE 65 XP_001172289.1 P. troglodytes 54 IKEPLPMEDVCPK 66 NP_003479.1 H. sapiens 54 IKEPLPVEDVCPK 66

Fig. S3 A B

Stomatin dimer (PDB: 4FVF) Podocin dimer Podocin dimmerer

Phosphoryla on at T234

C G(Dimerization) Podocin (Podocin)2

Gmono (Phosphorylation) Gdimer (Phosphorylation)

Podocin-T234Ph (Podocin-T234Ph)2 Gphos (Dimerization)

G Gphos (Dimerization) - G(Dimerization)

Gdimer (Phosphorylation) - 2 Gmono (Phosphorylation) D E

Fig. S4 SLT*EILLERK (+2): NPHS2, T234

)

6 Podocin +PKCz T410A 0 5.80

1

x

(

4.35

y

t

i

s 2.90

n

e t 1.45

n

I

)

6 Podocin +PKCz T410E 0 5.80

1

x

( 4.35

y

t

i

s 2.90

n

e t 1.45

n

I

31.8 32.0 32.2 32.4 32.6 32.8 Retention Time Fig. S5