Abstract
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 Ser46, Ser162, Ser181, Ser269, Ser280, Ser291, Thr293, Ser299, and Ser302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser290 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 Ser290 Mutating 257VPF259 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 Ser290 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 Ser290 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 Ser290 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.
Highlights
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
The results validate the use of FLAG– PTHR–TAP–NHERF1 GnTIϪ cells to investigate parathyroid hormone (PTH)-induced NHERF1 phosphorylation
To ensure that NHERF1 phosphorylation observed in GnTIϪ– FLAG–PTHR–TAP–NHERF1 cells did not arise from overexpression, we examined the pattern of NHERF1 phosphorylation in human renal proximal tubule cells (RPTEC), which constitutively express endogenous NHERF1 and PTHR [10]
Summary
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. Our results show that PP1␣ and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein– coupled receptor kinase 6A controls the Ser290 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. Introducing a second compensatory mutation of the NHERF1 PDZ ligand (ϪSNA) prevented formation of the closed conformation and overcame the nominally deleterious action of the inherited mutation on signaling and function These observations regarding the Ser-rich cluster within the linker domain, combined with the finding that the closed NHERF1 conformation prevents engagement of PKA regulatory subunit binding and function, suggest that phosphoryla-.
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