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International audience; 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 1 (PP1), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser(290). Mutating (VPF259)-V-257 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 PP1-mediated Ser(290) dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that -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 PP1 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.

The cytokine erythropoietin (EPO) is the master regulator of erythropoiesis. Intriguingly, many studies have shown that the cognitive performance of patients receiving EPO for its hematopoietic effects is enhanced, which prompted the growing interest in the use of EPO-based strategies to treat neuropsychiatric disorders. EPO plays key roles in brain development and maturation, but also modulates synaptic transmission. However, the mechanisms underlying the latter have remained elusive. Here, we show that acute (40-60 min) exposure to EPO presynaptically downregulates spontaneous and afferent-evoked excitatory transmission, without affecting basal firing of action potentials. Conversely, prolonged (3 h) exposure to EPO, if followed by a recovery period (1 h), is able to elicit a homeostatic increase in excitatory spontaneous, but not in evoked, synaptic transmission. These data lend support to the emerging view that segregated pathways underlie spontaneous and evoked neurotransmitter release. Furthermore, we show that prolonged exposure to EPO facilitates a form of hippocampal long-term potentiation that requires noncanonical recruitment of calcium-permeable AMPA receptors for its maintenance. These findings provide important new insight into the mechanisms by which EPO enhances neuronal function, learning, and memory.