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

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.

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10.1074/jbc.M110.132423

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Weinman, Edward J, Deborah Steplock, Yinghua Zhang, Rajatsubhra Biswas, Robert J Bloch and Shirish Shenolikar (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.

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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 last decade, our work has provided critical information about the role of protein phosphatase-1 (PP1) in controlling synaptic function, cell stress, gene expression and growth. We have generated a large repertoire of reagents to decipher PP1's role in signaling pathways in mammalian cells and tissues. Emerging evidence suggests that in many cells, PP1 activity is fine tuned by the protein, inhibitor-1 (I-1). A major focus of our research is to elucidate the role of I-1 in kinase-phosphatase cross-talk and impact of the altered I-1 gene expression seen in several human diseases. Our studies showed that recognition of cellular substrates by PP1 is also directed by its association with a variety of targeting subunits that are themselves also subject to physiological control. Thus, the overall focus of our research is to define the physiological mechanisms that regulate PP1 functions relevant to human health and disease.


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