| First Author | Zhang Q | Year | 2019 |
| Journal | J Biol Chem | Volume | 294 |
| Issue | 12 | Pages | 4546-4571 |
| PubMed ID | 30696771 | Mgi Jnum | J:275957 |
| Mgi Id | MGI:6307296 | Doi | 10.1074/jbc.RA119.007421 |
| Citation | Zhang Q, et al. (2019) Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport. J Biol Chem 294(12):4546-4571 |
| abstractText | 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(46), Ser(162), Ser(181), Ser(269), Ser(280), Ser(291), Thr(293), Ser(299), and Ser(302) did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser(290) was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1alpha (PP1alpha), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser(290) Mutating (257)VPF(259) 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 PP1alpha-mediated Ser(290) dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that beta-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(290) rephosphorylation. Our results show that PP1alpha and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser(290) 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. |
