G protein-coupled receptor kinase 6A phosphorylates the Na+/H+ exchanger regulatory factor via a PDZ domain-mediated interaction

The Na+/H+ exchanger regulatory factor (NHERF) is constitutively phosphoryl ated in cells, but the site(s) of this phosphorylation and the kinase(s) re sponsible for it have not been identified. We show here that the primary si te of constitutive NHERF phosphorylation in human embryonic kidney 293 (HEK -293) cells is Ser(289), and that the stoichiometry of phosphorylation is n ear 1 mol/mol, NHERF contains two PDZ domains that recognize the sequence S /T-X-L at the carboxyl terminus of target proteins, and thus we examined th e possibility that kinases containing this motif might associate with and p hosphorylate NHERF, Overlay experiments and co-immunoprecipitation studies revealed that NRERF binds with high affinity to a splice variant of the G p rotein-coupled receptor kinase 6, GRK6A, which terminates in the motif T-R- L, NHERF does not associate with GRK6B or GRK6C, alternatively spliced vari ants that differ from GRK6A at their extreme carboxyl termini, GRK6A phosph orylates NHERF efficiently on Ser(289) in vitro, whereas GRK6B, GRK6C, and GRK2 do not, Furthermore, the endogenous "NHERF kinase" activity in HEK-293 cell lysates is sensitive to treatments that alter the activity of GRK6A. These data suggest that GRK6A phosphorylates NHERF via a PDZ domain-mediate d interaction and that GRK6A is the kinase in HER-293 cells responsible for the constitutive phosphorylation of NHERF.