Casein kinase 2 binds to the C terminus of Na⁺/H⁺ exchanger 3 (NHE3) and stimulates NHE3 basal activity by phosphorylating a separate site in NHE3

Na⁺/H⁺ exchanger 3 (NHE3) is the epithelial-brush border isoform responsible for most intestinal and renal Na⁺ absorption. Its activity is both up- and down-regulated under normal physiological conditions, and it is inhibited in most diarrheal diseases. NHE3 is phosphorylated under basal conditions and Ser/Thr phosphatase inhibitors stimulate basal exchange activity; however, the kinases involved are unknown. To identify kinases that regulate NHE3 under basal conditions, NHE3 was immunoprecipitated; LC-MS/MS of trypsinized NHE3 identified a novel phosphorylation site at S⁷¹⁹ of the C terminus, which was predicted to be a casein kinase 2 (CK2) phosphorylation site. This was confirmed by an in vitro kinase assay. The NHE3-S719A mutant but not NHE3-S719D had reduced NHE3 activity due to less plasma membrane NHE3. This was due to reduced exocytosis plus decreased plasma membrane delivery of newly synthesized NHE3. Also, NHE3 activity was inhibited by the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo- 1H-benzimidazole DMAT when wild-type NHE3 was expressed in fibroblasts and Caco-2 cells, but the NHE3-S⁷¹⁹ mutant was fully resistant to DMAT. CK2 bound to the NHE3 C-terminal domain, between amino acids 590 and 667, a site different from the site it phosphorylates. CK2 binds to the NHE3 C terminus and stimulates basal NHE3 activity by phosphorylating a separate single site on the NHE3 C terminus (S⁷¹⁹), which affects NHE3 trafficking.