Modulation of Ion Transport by Direct Targeting of Protein Phosphatase Type 1 to the Na-K-Cl Cotransporter

Abstract

The specificity of major protein phosphatases is conferred via targeting subunits, each of which binds specifically to the phosphatase and targets it to the vicinity of substrate proteins. In the case of protein phosphatase 1 (PP1), an RVXFXD motif on a targeting subunit binds to a cleft in PP1c, the catalytic subunit. Here we report that a substrate of PP1, the Na-K-Cl cotransporter (NKCC1), bears this motif in its N terminus near sites of regulatory phosphorylation and that direct binding of PP1 to NKCC1 is functionally important in determining the set point for intracellular chloride regulation. NKCC1 mutants in which the motif is destroyed or improved exhibit dramatically shifted activation curves because of a change in the rate of cotransporter dephosphorylation. Furthermore, direct interaction of NKCC1 and PP1c observed by coprecipitation of the two proteins is not seen in a mutant lacking the site. This establishes a new paradigm of phosphatase specificity, one in which a substrate protein containing an RVXFXD motif binds directly to PP1c; we propose that this may be a quite general mechanism.A small number of protein phosphatases is responsible for dephosphorylation of the majority of cellular phosphoproteins (1, 2). The specificity of these otherwise promiscuous enzymes is conferred by targeting subunits, each of which binds specifically to the phosphatase and targets it to the vicinity of substrate proteins. For protein phosphatase 1 (PP1), 1 a cleft in the catalytic subunit (PP1c) binds proteins that contain the consensus motif, RVXFXD, illustrated both in a direct examination of the molecular structure (3) and by the results of peptide panning experiments (4). The present study began with the observation that the N terminus of the Na-K-Cl cotransporter (NKCC1) contains a highly conserved region with the sequence RVNFVD (residues 140 -145 in human NKCC1, 107-112 in shark NKCC1), posing the possibility that the cotransporter is directly targeted as a phosphatase substrate. The Na-K-Cl cotransporter is an integral membrane protein constituting a major regulated pathway for coupled inward movement of Na ϩ , K ϩ , and Cl Ϫ in many cell types. The transporter is a central element in electrolyte movement by secretory epithelia, where it functions in concert with cystic fibrosis transmembrane conductance regulator, potassium channels, and the sodium pump to bring about transcellular chloride transport (5, 6). Cotransport activity is fully controlled by direct phosphorylation of the NKCC1 protein (7-9) in response to decreases in cell volume or intracellular [Cl]. Phosphorylation of NKCC1 is mediated by a kinase whose identity is still unknown, but dephosphorylation appears to occur through the action of protein phosphatase 1 (10). The phosphoregulatory region, including three identified threonine phosphoacceptors, is in the cytoplasmic N terminus of the NKCC1 (7, 11-13). Functional characteristics of cotransporter regulation are highly conserved among vertebrates from shark to human (14), consistent with con...

Regulation of Na,K-ATPase by PLMS, the Phospholemman-like Protein from Shark

“…In accordance with this model, the present results have indicated co-localization of Na,K-ATPase and PLMS on basolateral membranes in the rectal gland cells. By the recognition of the new functional role of FXYD proteins in regulation of the Na,K-ATPase the transport pathways responsible for Cl Ϫ secretion seem all to be controlled by protein kinase/phosphatase regulation (62)(63)(64).…”

A Dominant Negative Mutant of the KCC1 K-Cl Cotransporter

“…The N-terminal cytoplasmic tail of dogfish NKCC1 harbors a functional consensus binding site for protein phosphatase I, which dephosphorylates NKCC1 aa 184 to inactivate ion transport (28). This consensus sequence is absent from both cytoplasmic termini of mKCC1.…”

WNK1 and OSR1 regulate the Na + , K + , 2Cl − cotransporter in HeLa cells

“…The WNK1 fragments containing at least one of the GRFXV motifs (streaks 2, 10, and 12) interacted with the C terminus of OSR1 but not with the N terminus of OSR1 (streaks 1, 9, and 11). OSR1 and its homolog SPAK bind to NKCC1 and, presumably, NKCC2 through a similar motif also present in these proteins (9,17).…”