Na-H exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins， including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here， using tandem MS analysis， we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser， Ser， Ser， Ser， Ser， Ser， Thr， Ser， and Ser did not affect phosphate uptake， but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly， Ser was rapidly dephosphorylated and rephosphorylated after PTH stimulation， and we found that prot... More
Na-H exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins， including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here， using tandem MS analysis， we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser， Ser， Ser， Ser， Ser， Ser， Thr， Ser， and Ser did not affect phosphate uptake， but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly， Ser was rapidly dephosphorylated and rephosphorylated after PTH stimulation， and we found that protein phosphatase 1α (PP1α)， which binds NHERF1 through a conserved VxF/W PP1 motif， dephosphorylates Ser Mutating VPF eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM)， we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that β-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1， implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser phosphorylation status of NHERF1 and regulates PTH-sensitive， NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.
