Thr212

Site Information
TNtyYLRtFGHNtMD SwissProt Entrez-Gene
Blast this site against: NCBI SwissProt PDB
Site Group ID: 455545

In vivo Characterization
Methods used to characterize site in vivo:	[32P] bio-synthetic labeling ( 28 , 29 ) , immunoprecipitation ( 2 , 4 ) , mass spectrometry ( 2 , 3 , 5 , 7 , 9 , 11 , 12 , 13 , 14 , 16 , 17 , 19 , 21 , 22 , 23 , 24 , 25 , 29 ) , mutation of modification site ( 4 , 28 , 29 ) , peptide sequencing ( 29 ) , phospho-antibody ( 1 , 2 , 4 , 6 , 15 , 18 , 20 , 26 , 27 , 28 ) , western blotting ( 1 , 2 , 4 , 6 , 15 , 18 , 20 , 28 )
Disease tissue studied:	luminal A breast cancer ( 3 ) , luminal B breast cancer ( 3 ) , breast cancer, triple negative ( 3 ) , gastric cancer ( 12 , 13 , 14 ) , gastric carcinoma ( 12 , 13 , 14 ) , melanoma skin cancer ( 5 )
Relevant cell line - cell type - tissue:	'stem, embryonic' ( 15 ) , 293 (epithelial) ( 2 , 4 , 6 , 18 , 26 , 28 , 29 ) , BAEC (endothelial) ( 6 ) , brain ( 1 ) , breast ( 3 ) , erythrocyte-blood ( 20 ) , fibroblast-skin ( 4 ) , Flp-In T-Rex-293 (epithelial) [PRKD1 (human), genetic knockin] ( 9 ) , Flp-In T-Rex-293 (epithelial) ( 9 ) , HeLa (cervical) ( 19 , 23 , 24 ) , Jurkat (T lymphocyte) ( 7 , 11 , 16 , 17 , 21 , 22 , 25 ) , MDCK (epithelial) ( 6 ) , MEF (fibroblast) ( 6 ) , mIMCD-3 (epithelial) ( 6 ) , MKN-45 (gastric) ( 12 , 13 , 14 ) , MMDD1 (renal) ( 6 ) , oocyte [CPEB (mouse)] ( 27 ) , rectal gland tubules ( 28 , 29 ) , WM239A (melanocyte) ( 5 )

Upstream Regulation
Regulatory protein:	CAB39 (human) ( 18 ) , OSR1 (human) ( 15 ) , WNK1 (human) ( 2 ) , WNK3 (human) ( 2 )
Putative in vivo kinases:	OSR1 (human) ( 18 , 26 )
Kinases, in vitro:	OSR1 (human) ( 18 , 26 ) , STLK3 (human) ( 26 )
Treatments:	A-769662 ( 20 ) , A79662 ( 6 ) , BI2536 ( 19 ) , calyculin_A ( 28 ) , colforsin ( 28 ) , compound_C ( 6 ) , hypotonic_buffer ( 18 , 28 ) , intraventricular hemorrhage ( 1 ) , low_chloride ( 4 , 15 ) , osmotic_stress ( 27 ) , siRNA ( 18 ) , sorbitol ( 15 , 26 ) , sucrose ( 20 )

Downstream Regulation
Effects of modification on NKCC1:	activity, induced ( 26 , 28 , 29 ) , enzymatic activity, induced ( 15 )

References
1	Karimy JK, et al. (2017) Inflammation-dependent cerebrospinal fluid hypersecretion by the choroid plexus epithelium in posthemorrhagic hydrocephalus. Nat Med 23, 997-1003 28692063 Curated Info
2	Zhang J, et al. (2016) Functional kinomics establishes a critical node of volume-sensitive cation-Cl- cotransporter regulation in the mammalian brain. Sci Rep 6, 35986 27782176 Curated Info
3	Mertins P, et al. (2016) Proteogenomics connects somatic mutations to signalling in breast cancer. Nature 534, 55-62 27251275 Curated Info
4	Kahle KT, et al. (2016) Peripheral motor neuropathy is associated with defective kinase regulation of the KCC3 cotransporter. Sci Signal 9, ra77 27485015 Curated Info
5	Stuart SA, et al. (2015) A Phosphoproteomic Comparison of B-RAFV600E and MKK1/2 Inhibitors in Melanoma Cells. Mol Cell Proteomics 14, 1599-615 25850435 Curated Info
6	Fraser SA, et al. (2014) Activation of AMPK reduces the co-transporter activity of NKCC1. Mol Membr Biol 31, 95-102 24702155 Curated Info
7	Mertins P, et al. (2013) Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Methods 10, 634-7 23749302 Curated Info
8	Shiromizu T, et al. (2013) Identification of missing proteins in the neXtProt database and unregistered phosphopeptides in the PhosphoSitePlus database as part of the Chromosome-centric Human Proteome Project. J Proteome Res 12, 2414-21 23312004 Curated Info
9	Franz-Wachtel M, et al. (2012) Global detection of protein kinase D-dependent phosphorylation events in nocodazole-treated human cells. Mol Cell Proteomics 11, 160-70 22496350 Curated Info
10	Beli P, et al. (2012) Proteomic Investigations Reveal a Role for RNA Processing Factor THRAP3 in the DNA Damage Response. Mol Cell 46, 212-25 22424773 Curated Info
11	Mulhern D (2012) CST Curation Set: 13109; Year: 2012; Biosample/Treatment: cell line, Jurkat/calyculin_A & pervanadate; Disease: T cell leukemia; SILAC: -; Specificities of Antibodies Used to Purify Peptides prior to LCMS: p[STY] Curated Info
12	Mulhern D (2012) CST Curation Set: 13402; Year: 2012; Biosample/Treatment: cell line, MKN-45/untreated; Disease: gastric carcinoma; SILAC: -; Specificities of Antibodies Used to Purify Peptides prior to LCMS: p[STY] Curated Info
13	Mulhern D (2012) CST Curation Set: 13405; Year: 2012; Biosample/Treatment: cell line, MKN-45/untreated; Disease: gastric carcinoma; SILAC: -; Specificities of Antibodies Used to Purify Peptides prior to LCMS: p[STY] Curated Info
14	Mulhern D (2012) CST Curation Set: 13458; Year: 2012; Biosample/Treatment: cell line, MKN-45/untreated; Disease: gastric carcinoma; SILAC: -; Specificities of Antibodies Used to Purify Peptides prior to LCMS: p[STY] Curated Info
15	Thastrup JO, et al. (2012) SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation. Biochem J 441, 325-37 22032326 Curated Info
16	Guo A (2011) CST Curation Set: 12453; Year: 2011; Biosample/Treatment: cell line, Jurkat/calyculin_A & pervanadate; Disease: T cell leukemia; SILAC: -; Specificities of Antibodies Used to Purify Peptides prior to LCMS: (K/R)Xp[ST](I/L/V) Curated Info
17	Guo A (2011) CST Curation Set: 12454; Year: 2011; Biosample/Treatment: cell line, Jurkat/calyculin_A & pervanadate; Disease: T cell leukemia; SILAC: -; Specificities of Antibodies Used to Purify Peptides prior to LCMS: (K/R)Xp[ST](I/L/V) Curated Info
18	Filippi BM, et al. (2011) MO25 is a master regulator of SPAK/OSR1 and MST3/MST4/YSK1 protein kinases. EMBO J 30, 1730-41 21423148 Curated Info
19	Kettenbach AN, et al. (2011) Quantitative phosphoproteomics identifies substrates and functional modules of aurora and polo-like kinase activities in mitotic cells. Sci Signal 4, rs5 21712546 Curated Info
20	Sid B, et al. (2010) Stimulation of human and mouse erythrocyte Na(+)-K(+)-2Cl(-) cotransport by osmotic shrinkage does not involve AMP-activated protein kinase, but is associated with STE20/SPS1-related proline/alanine-rich kinase activation. J Physiol 588, 2315-28 20442269 Curated Info
21	Possemato A (2010) CST Curation Set: 9436; Year: 2010; Biosample/Treatment: cell line, Jurkat/calyculin_A & pervanadate; Disease: T cell leukemia; SILAC: -; Specificities of Antibodies Used to Purify Peptides prior to LCMS: p[ST]P Curated Info
22	Zhou J (2009) CST Curation Set: 6052; Year: 2009; Biosample/Treatment: cell line, Jurkat/untreated; Disease: T cell leukemia; SILAC: -; Specificities of Antibodies Used to Purify Peptides prior to LCMS: (F/Y/M)Xp[ST](L/I/M) Curated Info
23	Zhou J (2008) CST Curation Set: 4842; Year: 2008; Biosample/Treatment: cell line, HeLa/UV; Disease: cervical adenocarcinoma; SILAC: -; Specificities of Antibodies Used to Purify Peptides prior to LCMS: p[ST]Q Antibodies Used to Purify Peptides prior to LCMS: Phospho-ATM/ATR Substrate Motif (pS/pTQG) Antibody Cat#: 5074, PTMScan(R) Phospho-ATM/ATR Substrate Motif (pS/pTQG) Immunoaffinity Beads Cat#: 1993 Curated Info
24	Zhou J (2008) CST Curation Set: 4778; Year: 2008; Biosample/Treatment: cell line, HeLa/UV; Disease: cervical adenocarcinoma; SILAC: -; Specificities of Antibodies Used to Purify Peptides prior to LCMS: p[ST]Q Antibodies Used to Purify Peptides prior to LCMS: Phospho-ATM/ATR Substrate Motif (pS/pTQG) Antibody Cat#: 5074, PTMScan(R) Phospho-ATM/ATR Substrate Motif (pS/pTQG) Immunoaffinity Beads Cat#: 1993 Curated Info
25	Possemato A (2007) CST Curation Set: 2796; Year: 2007; Biosample/Treatment: cell line, Jurkat/calyculin_A & pervanadate; Disease: T cell leukemia; SILAC: -; Specificities of Antibodies Used to Purify Peptides prior to LCMS: p[ST]Q Antibodies Used to Purify Peptides prior to LCMS: Phospho-ATM/ATR Substrate Motif (pS/pTQG) Antibody Cat#: 5074, PTMScan(R) Phospho-ATM/ATR Substrate Motif (pS/pTQG) Immunoaffinity Beads Cat#: 1993 Curated Info
26	Vitari AC, et al. (2006) Functional interactions of the SPAK/OSR1 kinases with their upstream activator WNK1 and downstream substrate NKCC1. Biochem J 397, 223-31 16669787 Curated Info
27	Giménez I, Forbush B (2005) Regulatory phosphorylation sites in the NH2 terminus of the renal Na-K-Cl cotransporter (NKCC2). Am J Physiol Renal Physiol 289, F1341-5 16077079 Curated Info
28	Flemmer AW, et al. (2002) Activation of the Na-K-Cl otransporter NKCC1 detected with a phospho-specific antibody. J Biol Chem 277, 37551-8 12145305 Curated Info
29	Darman RB, Forbush B (2002) A regulatory locus of phosphorylation in the N terminus of the Na-K-Cl cotransporter, NKCC1. J Biol Chem 277, 37542-50 12145304 Curated Info