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Cooperativity between the Phosphorylation of Thr(95) and Ser(77) of NHERF-1 in the Hormonal Regulation of Renal Phosphate Transport

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Date
2013-01-06
Authors
Weinman, Edward J
Steplock, Deborah
Zhang, Yinghua
Biswas, Rajatsubhra
Bloch, Robert J
Shenolikar, Shirish
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Abstract
The phosphorylation of the sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) plays a key role in the regulation of renal phosphate transport by parathyroid hormone (PTH) and dopamine. Ser(77) in the first PDZ domain of NHERF-1 is a downstream target of both hormones. The current experiments explore the role of Thr(95), another phosphate acceptor site in the PDZ I domain, on hormone-mediated regulation of phosphate transport in the proximal tubule of the kidney. The substitution of alanine for threonine at position 95 (T95A) significantly decreased the rate and extent of in vitro phosphorylation of Ser(77) by PKC. In NHERF-1-null proximal tubule cells, neither PTH nor dopamine inhibited sodium-dependent phosphate transport. Infection of the cells with adenovirus expressing full-length WT GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of both PTH and dopamine. Infection with full-length NHERF-1 containing a T95A mutation, however, increased basal phosphate transport but not the responsiveness to either hormone. As determined by surface plasmon resonance, the substitution of serine for aspartic acid (S77D) in the PDZ I domain decreased the binding affinity to the sodium-dependent phosphate transporter 2a (Npt2a) as compared with WT PDZ I, but a T95D mutation had no effect on binding. Finally, cellular studies indicated that both PTH and dopamine treatment increased the phosphorylation of Thr(95). These studies indicate a remarkable cooperativity between the phosphorylation of Thr(95) and Ser(77) of NHERF-1 in the hormonal regulation of renal phosphate transport. The phosphorylation of Thr(95) facilitates the phosphorylation of Ser(77). This, in turn, results in the dissociation of NHERF-1 from Npt2a and a decrease in phosphate transport in renal proximal tubule cells.
Type
Journal article
Subject
Cells, Cultured
Cell Line
Animals
Humans
Mice
Phosphates
Dopamine
Parathyroid Hormone
Threonine
Serine
Sodium-Hydrogen Antiporter
Phosphoproteins
Blotting, Western
Electrophoresis, Polyacrylamide Gel
Protein Binding
Biological Transport
Phosphorylation
Sodium-Phosphate Cotransporter Proteins, Type IIa
Permalink
https://hdl.handle.net/10161/18121
Published Version (Please cite this version)
10.1074/jbc.M110.132423
Publication Info
Weinman, Edward J; Steplock, Deborah; Zhang, Yinghua; Biswas, Rajatsubhra; Bloch, Robert J; & Shenolikar, Shirish (2013). Cooperativity between the Phosphorylation of Thr(95) and Ser(77) of NHERF-1 in the Hormonal Regulation of Renal Phosphate Transport. JOURNAL OF THE ROYAL SOCIETY INTERFACE, 10(78). 10.1074/jbc.M110.132423. Retrieved from https://hdl.handle.net/10161/18121.
This is constructed from limited available data and may be imprecise. To cite this article, please review & use the official citation provided by the journal.
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Scholars@Duke

Shenolikar

Shirish Shenolikar

Professor Emeritus of Psychiatry and Behavioral Sciences
Protein phosphorylation controls a wide range of physiological processes in mammalian tissues. Phosphorylation state of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases that are regulated by hormones, neurotransmitters, growth factors and other environmental cues. Our research attempts to understand the communication between protein kinases and phosphatases that dictates cellular protein phosphorylation and the cell's response to hormones. Over the
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