Phosphorylation and cell cycle-dependent regulation of Na+/H+ exchanger regulatory factor-1 by Cdc2 kinase

Na+/H+ exchanger regulatory factor (NHERF)-1 is a PDZ domain-containing ada ptor protein known to bind to various receptors, channels, cytoskeletal ele ments, and cytoplasmic signaling proteins. We report here that the phosphor ylation state of NHERF-1 is profoundly regulated by the cell cycle: NHERF-1 in HeLa cells is hyperphosphorylated in mitosis phase and much less phosph orylated at other points of the cell cycle. This mitosis phase-dependent ph osphorylation of NHERF-1 could be blocked by roscovitine, consistent with p hosphorylation by cyclin-dependent kinases. In vitro studies with purified NHERF-1 fusion proteins and purified kinases revealed that NHERF-1 was robu stly phosphorylated by the cyclin-dependent kinase Cdc2. In contrast, the N HERF-1 relative NHERF-2 was not phosphorylated at all by Cdc2. NHERF-1 poss esses two serines (Ser(279) and Ser(301)) that conform to the SPX(K/R) moti f preferred for phosphorylation by Cdc2. Mutation of either of these serine s reduced Cdc2-mediated phosphorylation of NHERF-1 in vitro, and mutation o f both residues together completely abolished Cdc2-mediated phosphorylation . When the S279A/S301A NHERF-1 mutant was expressed in cells, it failed to exhibit the mitosis phase-dependent phosphorylation observed with wild-type NHERF-1. Mutation of both Ser(279) and Ser(301) to aspartate, to mimic Cdc 2 phosphorylation of NHERF-1, resulted in a NHERF-1 mutant with a markedly impaired ability to oligomerize in vitro. Similarly, endogenous NHERF-1 fro m lysates of mitosis phase HeLa cells exhibited a markedly reduced ability to oligomerize relative to endogenous NHERF-1 from lysates of interphase He La cells. Mitosis phase NHERF-1 furthermore exhibited the ability to associ ate with Pint, a WW domain-containing peptidylprolyl isomerase that does no t detectably bind to NHERF-1 in interphase lysates. The association of NHER F-1 with Pint facilitated dephosphorylation of NHERF-1, as shown in experim ents in which cellular Pin1 activity was blocked by the selective inhibitor juglone. These data reveal that cellular NHERF-1 is phosphorylated during mitosis phase by Cdc2 at Ser(279) and Ser(301) and that this phosphorylatio n regulates NHERF-1 oligomerization and association with Pin1.