National Heart, Lung, and Blood Institute Epithelial Systems Biology Laboratory

Deep Phosphoproteomics of PKA-Null mpkCCD Cells: Short-Term Vasopressin Treatment

This database provides phosphopeptide ratios of desmopressin (dDAVP)-treated and untreated PKA-null mpkCCD collecting duct cells, determined using Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and an Orbitrap Fusion Lumos Tribrid mass spectrometer. PKA-null cells were developed from mpkCCD cells by deleting both PKA catalytic subunit genes via CRISPR-Cas9 (PMID: 28973931). 

Brief Method: For short-term dDAVP stimulation, three independent clones of PKA-null mpkCCD cells were maintained in the absence of dDAVP for 2 h (wash-out phase) on the day of experiment, and were then treated with either 0.1 nM dDAVP or vehicle for 30 min on the basolateral side only. The proteins were extracted, mixed (total = 2mg), reduced, alkylated and digested with Trypsin/LysC. Both Fe-NTA and TiO2 columns (Thermo Fisher) were used for parallel enrichment of phosphopeptides. Protein mass spectrometry of each sample was performed using both HCD and EThcD fragmentation.

Data Analysis: Proteome Discoverer 1.4 (version 1.4.0.288) using two search engines: Mascot and SequestHT [mouse Swiss-Prot (April 24, 2017)]. Data were filtered to eliminate low abundance phosphopeptides based on MS1 AUC. The identification and quantification of mono-phosphopeptide have been confirmed by manual inspection of MS spectra. Phosphosite was designated with * (site assignment probability , >80%). The basic aminio acids (R or K) at position -3 relative to the phosphorylated amino acid were highlighted with bold red. The acidic amino acids (D or E) at position +2 and +3 were highlighted with bold blue. Pfp, probability of false positive ratio difference.

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Reference: Datta A, Yang CR, Limbutara K, Chou CL, Rinschen MM, Raghuram V, Knepper MA. PKA-independent vasopressin signaling in renal collecting duct. FASEB J. 2020;34:6129-6146. (PMID: 32219907).

The website was created by Arnab Datta, Anika Kao, Chin-Rang Yang and Mark A. Knepper. Please contact Mark A. Knepper for comments or questions: knep@helix.nih.gov.

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Gene Symbol Accession Site(s) Annotation Centralized Sequence Log2  (dDAVP/ Ctrl)    -log10(Pfp)
Rin3 P59729 S411 Ras and Rab interactor 3 GISRTAS*LNLPPQ 0.86 3.52
Arhgef12 Q8R4H2 S41 Rho guanine nucleotide exchange factor 12 KVERSSS*HDFDPT 0.79 3.28
Camsap2 Q8C1B1 S439 Calmodulin-regulated spectrin-associated protein 2 GIIRSVS*NEGLTL 0.75 3.16
Gys1 Q9Z1E4 S711 Glycogen [starch] synthase, muscle GSKRSNS*VDTGPS 0.70 2.98
Crebbp P45481 S120 CREB-binding protein LGAMGKS*PLNQGD 0.59 2.63
Misp Q9D279 S364 Mitotic interactor and substrate of PLK1 GLQRSLS*SDCILS 0.59 2.67
Strn3 Q9ERG2 S202 Striatin-3 RSQRVRS*LLGLSN 0.58 2.95
Ccser2 Q3UHI0 S222 Serine-rich coiled-coil domain-containing protein 2 KMVRSQS*FSHSIQ 0.58 2.66
Krt19 P19001 S59 Keratin, type I cytoskeletal 19 VTSSSGS*YGGVRG 0.52 2.47
Crtc1 Q68ED7 S151 CREB-regulated transcription coactivator 1 SWRRTNS*DSALHQ 0.49 2.24
Wdr70 Q3TWF6 S641 WD repeat-containing protein 70 MFAQVES*DDEESK 0.49 2.75
Dennd4c A6H8H2 S1321 DENN domain-containing protein 4C YKDRSTS*LSALVR 0.48 1.98
Serbp1 Q9CY58 S203 Plasminogen activator inhibitor 1 RNA-binding protein SGSDRSS*FSHYSG 0.48 2.18
Psmd11 Q8BG32 S14 26S proteasome non-ATPase regulatory subunit 11 EFQRAQS*LLSTDR 0.48 1.97
Osbp Q3B7Z2 S377 Oxysterol-binding protein 1 GHKRTGS*NISGAS 0.39 2.48
Arhgef2 Q60875 S163 Rho guanine nucleotide exchange factor 2 GHFNDES*PLGLRQ 0.39 2.11
Arhgef2 Q60875 S151 Rho guanine nucleotide exchange factor 2 SLAKSVS*TTNIAG 0.39 2.31
Lrba Q9ESE1 T1011 Lipopolysaccharide-responsive and beige-like anchor protein SNTELQT*HDMSSD 0.37 2.44
Ralgapa1 Q6GYP7 S772 Ral GTPase-activating protein subunit alpha-1 TRVRHFS*QSEDTG 0.33 2.62
Lrba Q9ESE1 S1000 Lipopolysaccharide-responsive and beige-like anchor protein SIGRASS*IDSASN 0.30 2.75
Stk39 Q9Z1W9 S383 STE20/SPS1-related proline-alanine-rich protein kinase RRVPGSS*GHLHKT 0.29 2.60
Lipe P54310 S559 Hormone-sensitive lipase SMRRSVS*EAALAQ 0.25 3.34
Rplp2 P99027 S79 60S acidic ribosomal protein P2 VSAAPGS*AAPAAG -0.18 1.33
Flna Q8BTM8 S968 Filamin-A SVGVSPS*LDLSKI -0.20 2.98
Ybx3 Q9JKB3 S52 Y-box-binding protein 3 AALLAGS*PGGDAA -0.20 2.75
Prkci Q62074 T411 Protein kinase C iota type PGDTTST*FCGTPN -0.34 2.39
Prkci Q62074 T415 Protein kinase C iota type TSTFCGT*PNYIAP -0.35 2.32
Rrp7a Q9D1C9 S99 Ribosomal RNA-processing protein 7 homolog A KPDLAES*PTEPKS -0.35 2.12
Sipa1l1 Q8C0T5 S1626 Signal-induced proliferation-associated 1-like protein 1 HGEFSAS*DSSLTD -0.45 1.99
Epb41l1 Q9Z2H5 S378 Band 4.1-like protein 1 TFFRLVS*PEPPPK -0.45 2.39
Rplp2 P99027 S86 60S acidic ribosomal protein P2 AAPAAGS*APAAAE -0.59 3.65
Tjp1 P39447 S280 Tight junction protein ZO-1 LSDSIHS*ANASER -0.65 2.25
Pdap1 Q3UHX2 S60 28 kDa heat- and acid-stable phosphoprotein EKKSLDS*DESEDE -0.83 1.25
Fn1 P11276 S285 Fibronectin ASAGSGS*FTDVRT -0.84 3.67
Thumpd1 Q99J36 S88 THUMP domain-containing protein 1 DQQPSGS*EGEDDD -0.94 4.60
Ncl P09405 S145 Nucleolin NAKKEDS*DEDEDE -1.64 16.00
Gtf2f1 Q3THK3 S311 General transcription factor IIF subunit 1 SESSEES*EEEKPP -2.46 15.60
Gene Symbol Accession Site(s) Annotation Centralized Sequence Log2  (dDAVP/ Ctrl)    -log10(Pfp)


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