ABCMdb

ABCA1 p.Thr1243Ala

Predicted by SNAP2:	A: D (75%), C: D (80%), D: D (85%), E: D (91%), F: D (85%), G: D (80%), H: D (85%), I: D (85%), K: D (85%), L: D (85%), M: D (85%), N: D (59%), P: D (66%), Q: D (80%), R: D (85%), S: N (72%), V: D (85%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: N, P: N, Q: D, R: D, S: N, V: D, W: D, Y: D,

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[hide] Phosphorylation by protein kinase CK2 modulates th... J Biol Chem. 2004 Sep 3;279(36):37779-88. Epub 2004 Jun 24. Roosbeek S, Peelman F, Verhee A, Labeur C, Caster H, Lensink MF, Cirulli C, Grooten J, Cochet C, Vandekerckhove J, Amoresano A, Chimini G, Tavernier J, Rosseneu M
Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter.
J Biol Chem. 2004 Sep 3;279(36):37779-88. Epub 2004 Jun 24., [PMID:15218032]

Abstract [show]
In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters. These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2. To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1+R1 and NBD2+R2. We demonstrated that in vitro ABCA1 NBD1+R1, and not NBD2+R2, is phosphorylated by CK2, and we identified Thr-1242, Thr-1243, and Ser-1255 as the phosphorylated residues in the R1 domain by mass spectrometry. We further investigated the functional significance of the threonine and serine phosphorylation sites in NBD1 by site-directed mutagenesis of the entire ABCA1 followed by transfection into Hek-293 Tet-Off cells. The ABCA1 flippase activity, apolipoprotein AI and AII binding, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine and serine residues. This was confirmed by the effect of specific protein kinase CK2 inhibitors upon the activity of wild type and mutant ABCA1 in transfected Hek-293 Tet-Off cells. The activities of the mutants mimicking threonine phosphorylation were close to that of wild type ABCA1. Our data, therefore, suggest that besides protein kinase A and C, protein kinase CK2 might play an important role in vivo in regulating the function and transport activity of ABCA1 and possibly of other members of the ABCA subfamily.

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94 Similar measurements were performed in the presence of 50 ␮M 4,5,6,7-tetrabromobenzotriazole and apigenin CK2 inhibitors on Mock-transfected and Hek-293 cells transfected with WT ABCA1 and with the T1242A, T1243A, T1242A/T1243A, and T1242D/T1243D ABCA1 mutants. Login to comment
133 In both systems we engineered several mutants designed at preventing phosphorylation by mutation either of the target threonines and serine, T1242A, T1243A, T1242A/ T1243A, S1255A, or of the downstream cluster of acidic residues, E1245Q, E1246Q, E1245Q/E1246Q. Login to comment
143 Analysis of the T1242A, T1243A, and T1242A/T1243A NBD1 mutants supports the loss of either one or two threonine phosphorylation sites in the mutated peptides, whereas the Ser-1255 site is preserved. Login to comment
159 Compared with WT ABCA1, set as 100%, the PE and PS content in the outer leaflet of the plasma membrane increased up to 130 Ϯ 14 and 123 Ϯ 8% for the T1242A mutant, 154 Ϯ 15 and 148 Ϯ 11% for the T1243A mutant, 170 Ϯ 15 and 207 Ϯ 16% for the T1242A/T1243A mutant, 141 Ϯ 11 and 136 Ϯ 10% for the S1255A mutant, and 141 Ϯ 13 and 120 Ϯ 9% for the E1245Q/E1246Q mutant, respectively (Fig. 4). Login to comment
162 The effect of the specific CK2 inhibitors 4,5,6,7-tetrabromo- benzotriazole and apigenin on the flippase activity in Hek-293 Tet-Off cells transfected with WT ABCA1 and with the T1242A, T1243A, T1242A/T1243A, S1255A, and T1242D/ T1243D mutants was consistent with the above data. Login to comment
176 TABLE I Mass spectrometric analysis of NBD1ϩR1 phosphorylation NBD1ϩR1 Tryptic peptide Molecular mass Molecular mass after beta-elimination Phosphorylated residues Da Da WT Leu-1229-Arg-1269 4542.5 4249.5 Thr-1242, Thr-1243, Ser-1255 T1242A Leu-1229-Lys-1250 2483.6 2385.9 Thr-1243 T1242A Val-1251-Arg-1272 2411.6 2313.7 Ser-1255 T1243A Leu-1229-Lys-1250 2483.1 2385.2 Thr-1242 T1243A Val-1251-Arg-1272 2411.2 2313.2 Ser-1255 T1242A/T1243A Leu-1229-Lys-1250 2356.1 2356.1 T1242A/T1243A Val-1251-Arg-1272 2411.6 2313.7 Ser-1255 S1255A Val-1251-Arg-1272 2297.7 2297.7 compared with WT ABCA1. Login to comment
177 This increase was more pronounced for the T1243A mutation either alone or in combination with the T1242A mutation, whereas the effect of the T1242A and S1255A mutants was similar. Login to comment
188 The T1242A and T1243A mutations either alone or in combination and the E1245Q/ E1246Q mutation, which all decrease CK2 activity, increased phospholipid and cholesterol efflux (Fig. 7, A and B). Login to comment
92 Similar measurements were performed in the presence of 50 òe;M 4,5,6,7-tetrabromobenzotriazole and apigenin CK2 inhibitors on Mock-transfected and Hek-293 cells transfected with WT ABCA1 and with the T1242A, T1243A, T1242A/T1243A, and T1242D/T1243D ABCA1 mutants. Login to comment
131 In both systems we engineered several mutants designed at preventing phosphorylation by mutation either of the target threonines and serine, T1242A, T1243A, T1242A/ T1243A, S1255A, or of the downstream cluster of acidic residues, E1245Q, E1246Q, E1245Q/E1246Q. Login to comment
141 Analysis of the T1242A, T1243A, and T1242A/T1243A NBD1 mutants supports the loss of either one or two threonine phosphorylation sites in the mutated peptides, whereas the Ser-1255 site is preserved. Login to comment
157 Compared with WT ABCA1, set as 100%, the PE and PS content in the outer leaflet of the plasma membrane increased up to 130 afe; 14 and 123 afe; 8% for the T1242A mutant, 154 afe; 15 and 148 afe; 11% for the T1243A mutant, 170 afe; 15 and 207 afe; 16% for the T1242A/T1243A mutant, 141 afe; 11 and 136 afe; 10% for the S1255A mutant, and 141 afe; 13 and 120 afe; 9% for the E1245Q/E1246Q mutant, respectively (Fig. 4). Login to comment
160 The effect of the specific CK2 inhibitors 4,5,6,7-tetrabromo- benzotriazole and apigenin on the flippase activity in Hek-293 Tet-Off cells transfected with WT ABCA1 and with the T1242A, T1243A, T1242A/T1243A, S1255A, and T1242D/ T1243D mutants was consistent with the above data. Login to comment
174 TABLE I Mass spectrometric analysis of NBD1af9;R1 phosphorylation NBD1af9;R1 Tryptic peptide Molecular mass Molecular mass after beta-elimination Phosphorylated residues Da Da WT Leu-1229-Arg-1269 4542.5 4249.5 Thr-1242, Thr-1243, Ser-1255 T1242A Leu-1229-Lys-1250 2483.6 2385.9 Thr-1243 T1242A Val-1251-Arg-1272 2411.6 2313.7 Ser-1255 T1243A Leu-1229-Lys-1250 2483.1 2385.2 Thr-1242 T1243A Val-1251-Arg-1272 2411.2 2313.2 Ser-1255 T1242A/T1243A Leu-1229-Lys-1250 2356.1 2356.1 T1242A/T1243A Val-1251-Arg-1272 2411.6 2313.7 Ser-1255 S1255A Val-1251-Arg-1272 2297.7 2297.7 Protein Kinase CK2 Phosphorylation Modulates ABCA1 Activity compared with WT ABCA1. Login to comment
175 This increase was more pronounced for the T1243A mutation either alone or in combination with the T1242A mutation, whereas the effect of the T1242A and S1255A mutants was similar. Login to comment
186 The T1242A and T1243A mutations either alone or in combination and the E1245Q/ E1246Q mutation, which all decrease CK2 activity, increased phospholipid and cholesterol efflux (Fig. 7, A and B). Login to comment