Modulation of ion transport by direct targeting of protein phosphatase type 1 to the Na-K-Cl cotransporter

The specificity of major protein phosphatases is conferred via targeting su bunits, 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, th e catalytic subunit. Here we report that a substrate of PPI, the Na-K-Cl co transporter (NKCC1), bears this motif in its N terminus near sites of regul atory phosphorylation and that direct binding of PP1 to NKCC1 is functional ly important in determining the set point for intracellular chloride regula tion. NKCC1 mutants in which the motif is destroyed or improved exhibit dra matically shifted activation curves because of a change in the rate of cotr ansporter dephosphorylation. Furthermore, direct interaction of NKCC1 and P P1c observed by coprecipitation of the two proteins is not seen in a mutant lacking the site. This establishes a new paradigm of phosphatase specifici ty, one in which a substrate protein containing an RVXFXD motif binds direc tly to PP1c; we propose that this may be a quite general mechanism.