TI - G protein -coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via a PDZ domain -mediated interaction . AB - The Na(+)/H(+) exchanger regulatory factor ( NHERF ) is constitutively phosphorylated in cells , but the sites of this phosphorylation and the kinases responsible for it have not been identified . We show here that the primary site of constitutive NHERF phosphorylation in human embryonic kidney 293 ( HEK-293 ) cells is Ser(289) , and that the stoichiometry of phosphorylation is near 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 the possibility that kinases containing this motif might associate with and phosphorylate NHERF . Overlay experiments and co-immunoprecipitation studies revealed that NHERF binds with high affinity to a splice variant of the G protein -coupled receptor kinase 6 , GRK6A , which terminates in the motif T-R-L . NHERF does not associate with GRK6B or GRK6C , alternatively spliced variants that differ from GRK6A at their extreme carboxyl termini . GRK6A phosphorylates 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 -mediated interaction and that GRK6A is the kinase in HEK-293 cells responsible for the constitutive phosphorylation of NHERF .