ABCMdb

PMID: 22948143

Randak CO, Ver Heul AR, Welsh MJ
Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity.
J Biol Chem. 2012 Oct 19;287(43):36105-10. doi: 10.1074/jbc.M112.408450. Epub 2012 Sep 4., [PubMed]

Sentences

No. Mutations Sentence Comment

56 ABCC7 p.Ser1248Phe CFTR Adenylate Kinase Assay-Membranes containing either 30 ␮g of protein (from cells expressing wild-type CFTR) or 90 ␮g of protein (from cells expressing S1248F CFTR) were incubated gently shaking with nonradioactive 8- or 2-N3-AMP (at concentrations given in the figure legends), radioactive [␥-32 P]GTP (30 ␮Ci, 6000 Ci/mmol), 20 mM Hepes (pH 7.5), 50 mM NaCl, 3 mM MgCl2, and 1 mM Tricine (pH 7.6) for 5 min at 37 °C in a total volume of 30 ␮l followed by UV irradiation for 30 s (302 nm, 8-watt lamp) at a distance of 5 cm. Login to comment 57 ABCC7 p.Ser1248Phe CFTR Adenylate Kinase Assay-Membranes containing either 30 òe;g of protein (from cells expressing wild-type CFTR) or 90 òe;g of protein (from cells expressing S1248F CFTR) were incubated gently shaking with nonradioactive 8- or 2-N3-AMP (at concentrations given in the figure legends), radioactive [ॹ-32 P]GTP (30 òe;Ci, 6000 Ci/mmol), 20 mM Hepes (pH 7.5), 50 mM NaCl, 3 mM MgCl2, and 1 mM Tricine (pH 7.6) for 5 min at 37 &#b0;C in a total volume of 30 òe;l followed by UV irradiation for 30 s (302 nm, 8-watt lamp) at a distance of 5 cm. Login to comment 136 ABCC7 p.Ser1248Phe ABCC7 p.Ser1248Phe We chose a phenylalanine substitution for serine at position 1248 (S1248F) in the phosphate-binding loop of ATP-binding site 2. Login to comment 137 ABCC7 p.Ser1248Phe ABCC7 p.Ser1248Phe We chose a phenylalanine substitution for serine at position 1248 (S1248F) in the phosphate-binding loop of ATP-binding site 2. Login to comment 150 ABCC7 p.Ser1248Phe In lane 6, membranes containing 90 òe;g of protein from S1248F CFTR-expressing HeLa cells were employed. Login to comment 151 ABCC7 p.Ser1248Phe In lane 6, membranes containing 90 ␮g of protein from S1248F CFTR-expressing HeLa cells were employed. Login to comment 160 ABCC7 p.Ser1248Phe 30 òe;g (control membranes and membranes with wild-type CFTR, lanes 1-3) and 90 òe;g (membranes with S1248F CFTR, lane 4) of protein were used. Login to comment 161 ABCC7 p.Ser1248Phe 30 ␮g (control membranes and membranes with wild-type CFTR, lanes 1-3) and 90 ␮g (membranes with S1248F CFTR, lane 4) of protein were used. Login to comment 162 ABCC7 p.Ser1248Phe When we incubated membranes containing S1248F CFTR with [ॹ-32 P]GTP and nonradioactive N3-AMP, followed by UV irradiation, we found very little labeling (Fig. 4A, lane 6). Login to comment 163 ABCC7 p.Ser1248Phe When we incubated membranes containing S1248F CFTR with [␥-32 P]GTP and nonradioactive N3-AMP, followed by UV irradiation, we found very little labeling (Fig. 4A, lane 6). Login to comment 164 ABCC7 p.Ala462Phe We could not assess the effect of the homologous mutation in ATP-binding site 1 (A462F mutation) on adenylate kinase activity because that mutation affected intracellular CFTR processing to an extent that we were unable to detect the mutant CFTR protein in our membrane preparations by Western blot. Login to comment 165 ABCC7 p.Ala462Phe We could not assess the effect of the homologous mutation in ATP-binding site 1 (A462F mutation) on adenylate kinase activity because that mutation affected intracellular CFTR processing to an extent that we were unable to detect the mutant CFTR protein in our membrane preparations by Western blot. Login to comment 178 ABCC7 p.Ser1248Phe Substituting a phenylalanine into the phosphate-binding loop of NBD2 (the S1248F mutation) interfered with labeling. Login to comment 180 ABCC7 p.Ser1248Phe Previous observations support the interpretation that the S1248F mutation disrupted adenylate kinase activity. Login to comment 181 ABCC7 p.Ser1248Phe 1) A study characterizing the gating characteristics and the interaction of ATP with S1248F CFTR found that this mutation interfered with the interaction of nucleotides at ATP-binding site 2. Login to comment 183 ABCC7 p.Lys1250Ala ABCC7 p.Asp1370Asn 2) Patch clamp studies showed that mutations K1250A and D1370N, located within conserved motifs of ATP-binding site 2, abolished the effects of Ap5A and AMP on CFTR current. Login to comment