Characterization of phosphorylation of NCC at serine 124

Abstract

LC‐MS/MS‐based phosphoproteomic profiling identified phosphorylation of NCC at S124 (Feric M et al. Am J Physiol Cell 300: C755–770, 2011). We previously showed pS124‐NCC to be predominantly associated with the apical plasma membrane of DCT cells, where its abundance was regulated by AVP and ANGII. Semi‐quantitative 36Cl uptake studies of various NCC mutants in X. Laevis oocytes showed decreased activity of NCC when replacing S124 with Ala. SPAK or OSR1 protein kinases were unable to phosphorylate synthetic peptides corresponding to the region surrounding S124. Novel tetracycline inducible MDCKII cell lines expressing WT‐NCC and various mutants were generated and used for characterization of the S124 phosphorylation site. WT‐cells displayed thiazide‐sensitive 36Cl uptake, whereas a NCC‐S124A mutant demonstrated decreased activity. WT cells showed robust phosphorylation of NCC at T53 and T58 and membrane targeting following hypotonic low chloride stimulation. Treatment of WT cells with PMA decreased total NCC in the plasma membrane, but the ratio of pNCC to total NCC at the membrane increased, suggesting that non‐phosphorylated NCC is preferentially internalized. Funded by the Lundbeck Foundation.