Loureiro, Cláudia AlmeidaBarros, PatríciaMatos, PauloJordan, Peter2019-07-182020-05-012019-05-28Arch Biochem Biophys. 2019 Jul 15;669:61-70. doi:10.1016/j.abb.2019.05.022. Epub 2019 May 280003-9861http://hdl.handle.net/10400.18/6443Cellular chloride transport has a fundamental role in cell volume regulation and renal salt handling. Cellular chloride entry or exit are mediated at the plasma membrane by cotransporter proteins of the solute carrier 12 family. For example, NKCC2 resorbs chloride with sodium and potassium ions at the apical membrane of epithelial cells in the kidney, whereas KCC3 releases chloride with potassium ions at the basolateral membrane. Their ion transport activity is regulated by protein phosphorylation in response to signaling pathways. An additional regulatory mechanism concerns the amount of cotransporter molecules inserted into the plasma membrane. Here we describe that tyrosine phosphorylation of NKCC2 and KCC3 regulates their plasma membrane expression levels. We identified that spleen tyrosine kinase (SYK) phosphorylates a specific N-terminal tyrosine residue in each cotransporter. Experimental depletion of endogenous SYK or pharmacological inhibition of its kinase activity increased the abundance of NKCC2 at the plasma membrane of human embryonic kidney cells. In contrast, overexpression of a constitutively active SYK mutant decreased NKCC2 membrane abundance. Intriguingly, the same experimental approaches revealed the opposite effect on KCC3 abundance at the plasma membrane, compatible with the known antagonistic roles of NKCC and KCC cotransporters in cell volume regulation. Thus, we identified a novel pathway modulating the cell surface expression of NKCC2 and KCC3 and show that this same pathway has opposite functional outcomes for these two cotransporters. The findings have several biomedical implications considering the role of these cotransporters in regulating blood pressure and cell volume.Highlights: Protein kinase SYK phosphorylates NKCC2 and KCC3 at an N-terminal tyrosine residue; Cellular SYK activity modulates the expression of NKCC2 and KCC3 at the cell surface; SYK activity modulates cell surface expression of NKCC2 and KCC3 in opposite ways; SYK-modulated cell surface expression of NKCC2 contributes to cell-volume regulation.engChloride cotransportProtein PhosphorylationSYKPhosphotyrosineSHC1KCC3Membrane TrafficNKCC2Vias de Transdução de Sinal e Patologias Associadasjournal article10.1016/j.abb.2019.05.022