Demonstration of phosphoryl group transfer indicates that the ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) exhibits adenylate kinase activity

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5'-monophosphate (AMP), CFTR Cl(-) channel function is coupled to adenylate kinase activity (ATP+AMP <==> 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the (32)P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase.

FIGURE 1.

Model of CFTR labeling through phosphoryl group transfer between [γ-³²P]GTP and N₃-AMP followed by UV-mediated cross-linking of the resulting N₃-[β-³²P]ADP and solubilization and immunoprecipitation (IP) of CFTR. P* indicates a radioactive phosphoryl group containing ³²P. In each NBD, the open rectangle represents the Walker A motif, and the open triangle represents the signature motif. The binding site for AMP is not known.

FIGURE 2.

Membrane-inserted CFTR catalyzes phosphotransfer from [γ-³²P]GTP to N₃-AMP. A, Western blot (WB) probed with antibody 13-1. Letters label highly (C) and core glycosylated (B) CFTR. Each lane represents 30 μg of membrane protein. B, autoradiograph and Western blot (probed with antibody M3A7) of the same gel. Experiments were performed as illustrated in Fig. 1. Experimental conditions are indicated below the lanes. N₃-AMP concentration was 65 μm. Comparing the autoradiograph and Western blot corroborated that the labeled band was CFTR. C, CFTR photolabeling with 8-N₃-AMP and 2-N₃-AMP. N₃-AMP concentration was 65 μm. To compare the results from different autoradiographs, data were normalized to CFTR radioactivity under conditions indicated below bar 4. Asterisks indicate p ≤ 0.001 when compared with bar 4, and double daggers indicate p ≤ 0.001 when compared with bar 3 (one-way analysis of variance followed by the Holm-Sidak method for multiple comparisons, n = 3).

FIGURE 3.

Excess nonradioactive AMP, ATP, and Ap₅A reduce photolabeling. A, autoradiographs from two different experiments. Experiments were performed as in Fig. 1. Concentration of 2-N₃-AMP was 50 μm. B, summary data. Amount of radioactivity incorporated into CFTR was normalized to CFTR radioactivity under conditions indicated below bar 1. Asterisks indicate p ≤ 0.001 when compared with bar 1 (one-way repeated measures analysis of variance followed by the Holm-Sidak method for multiple comparisons, n = 4–6).

FIGURE 4.

CFTR has intrinsic adenylate kinase activity. A, autoradiograph of immunoprecipitated CFTR fractionated on a 6% SDS-polyacrylamide gel. Experiments were performed as illustrated in Fig. 1. Membranes containing 30 μg of protein from CFTR-expressing HeLa cells (lanes 3–5) or control membranes (contr. membr.) containing 30 μg of protein from HeLa cells not expressing recombinant CFTR (lane 1) were used. In lane 6, membranes containing 90 μg of protein from S1248F CFTR-expressing HeLa cells were employed. Membranes were incubated together with 50 μm 2-N₃-AMP and 30 μCi of [γ-³²P]GTP (6000 Ci/mmol) for 5 min at 37 °C followed by UV irradiation for 30 s (302 nm, 8-watt lamp) at a distance of 5 cm as described under “Experimental Procedures.” The sample of lane 4 was not UV-irradiated. In lane 2, 30 μg of membranes from HeLa cells not expressing recombinant CFTR (control membranes) were incubated with 50 μm 2-N₃-AMP and 30 μCi of [γ-³²P]GTP (6000 Ci/mmol) for 5 min at 37 °C. Then 30 μg of membranes containing CFTR were added on ice before UV irradiation (30 s, 302 nm, 8-watt lamp). In all cases, CFTR was then solubilized and immunoprecipitated as described under “Experimental Procedures.” B, summary data. Radioactivity incorporated into CFTR was normalized to radioactivity for conditions indicated below bar 5. Asterisks indicate p = 0.029 when compared with bar 5 (Mann-Whitney rank sum test, n = 4). No significant differences were detected between bars 1–4 and 6 (Kruskal-Wallis one-way analysis of variance on ranks, n = 4). C, Western blot probed with CFTR antibody 13-1. 30 μg (control membranes and membranes with wild-type CFTR, lanes 1–3) and 90 μg (membranes with S1248F CFTR, lane 4) of protein were used.