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R-Domain Phosphorylation by Protein Kinase A Stimulates Dissociation of Unhydrolyzed ATP from the First

Luba A Aleksandrov¹, Jonathan F Fay¹, John R Riordan¹

¹University of North Carolina , Chapel Hill , North Carolina 27599 , United States.

|August 16, 2018

Abstract:

The cystic fibrosis transmembrane conductance regulator (CFTR) is an asymmetric ATP-binding cassette transporter in which ATP hydrolysis occurs only at the second of the two composite nucleotide-binding sites whereas there are noncanonical substitutions of key catalytic residues in the first site. Therefore, in widely accepted models of CFTR function, ATP is depicted as remaining bound at the first site while it is hydrolyzed at the second site. However, the long lifetime of ATP at nucleotide-binding domain 1 (NBD1) had been measured under conditions where the channel had not been activated by phosphorylation. Here we show that phosphorylation by protein kinase A (PKA), obligatory for channel activation, strongly accelerates dissociation of the unhydrolyzed ATP from NBD1 of both full-length and NBD2-deleted CFTR. This stimulation of nucleotide release results from phosphorylation of the CFTR regulatory domain (residues 634-835) (R-domain). Mimicking phosphorylation by mutating the eight phosphorylation sites in the R-domain (8SE) has the same robust effect on accelerating the dissociation of ATP from NBD1. These findings provide new insight into relationships between R-domain phosphorylation and ATP binding and hydrolysis, the two main CFTR regulatory pathways.

Insights

Phosphorylation by protein kinase A (PKA) accelerates ATP release from the cystic fibrosis transmembrane conductance regulator (CFTR) NBD1. This finding reveals how R-domain phosphorylation regulates CFTR ATP binding and hydrolysis.

Area of Science:

Biochemistry
Molecular Biology
Cell Physiology

Background:

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette transporter crucial for ion channel function.
CFTR function involves two nucleotide-binding domains (NBDs) and a regulatory domain (R-domain).
ATP hydrolysis at NBD2 is essential for CFTR channel gating, while NBD1 binds ATP non-hydrolytically in accepted models.

Purpose of the Study:

To investigate the effect of protein kinase A (PKA) phosphorylation on ATP binding and dissociation at NBD1 of CFTR.
To elucidate the role of the CFTR R-domain in regulating nucleotide interactions at NBD1.

Main Methods:

Measured ATP dissociation rates from NBD1 of full-length and NBD2-deleted CFTR under phosphorylated and non-phosphorylated conditions.
Utilized protein kinase A (PKA) for phosphorylation and analyzed the impact of R-domain phosphorylation.
Employed site-directed mutagenesis to mimic R-domain phosphorylation (8SE mutant).

Main Results:

PKA-mediated phosphorylation significantly accelerates the dissociation of unhydrolyzed ATP from NBD1.
This effect is observed in both full-length CFTR and a construct lacking NBD2.
Mimicking R-domain phosphorylation via the 8SE mutation replicates the accelerated ATP dissociation from NBD1.

Conclusions:

R-domain phosphorylation is a key regulator that enhances the release of ATP from NBD1.
This finding provides novel insights into the interplay between CFTR R-domain phosphorylation and nucleotide binding/hydrolysis.
The results refine current models of CFTR functional regulation.

Read full analysis

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