1. Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport.
- Author
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Zhang Q, Xiao K, Paredes JM, Mamonova T, Sneddon WB, Liu H, Wang D, Li S, McGarvey JC, Uehling D, Al-Awar R, Joseph B, Jean-Alphonse F, Orte A, and Friedman PA
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray, Furans pharmacology, HEK293 Cells, Humans, Ion Transport physiology, Lipids pharmacology, Phosphoproteins chemistry, Phosphorylation, Protein Conformation, Receptors, Neuropeptide Y antagonists & inhibitors, Receptors, Neuropeptide Y metabolism, Serine metabolism, Sodium-Hydrogen Exchangers chemistry, Parathyroid Hormone physiology, Phosphates metabolism, Phosphoproteins metabolism, Sodium-Hydrogen Exchangers metabolism, Sodium-Phosphate Cotransporter Proteins, Type IIa physiology
- 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 Mutating257 VPF259 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., (© 2019 Zhang et al.)- Published
- 2019
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