ABCMdb

PMID: 15218032

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., [PubMed]

Sentences

No. Mutations Sentence Comment

92 ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1243Asp ABCA1 p.Thr1242Asp 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 94 ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1243Asp ABCA1 p.Thr1242Asp 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 131 ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Glu1245Gln ABCA1 p.Glu1245Gln ABCA1 p.Glu1246Gln ABCA1 p.Glu1246Gln ABCA1 p.Ser1255Ala 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 132 ABCA1 p.Thr1243Asp ABCA1 p.Thr1243Asp ABCA1 p.Thr1242Asp ABCA1 p.Thr1242Asp Mutants were also designed to mimic phosphorylated residues as T1242D, T1243D, T1242D/T1243D. Login to comment 133 ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Glu1245Gln ABCA1 p.Glu1245Gln ABCA1 p.Glu1246Gln ABCA1 p.Glu1246Gln ABCA1 p.Ser1255Ala 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 134 ABCA1 p.Thr1243Asp ABCA1 p.Thr1243Asp ABCA1 p.Thr1242Asp ABCA1 p.Thr1242Asp Mutants were also designed to mimic phosphorylated residues as T1242D, T1243D, T1242D/T1243D. Login to comment 141 ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala 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 142 ABCA1 p.Ser1255Ala ABCA1 p.Val1251Arg Analysis of the S1255A mutant demonstrates the loss of one phosphorylation site in the Val-1251-Arg-1272 tryptic peptide (Table I). Login to comment 143 ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala 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 144 ABCA1 p.Ser1255Ala ABCA1 p.Val1251Arg Analysis of the S1255A mutant demonstrates the loss of one phosphorylation site in the Val-1251-Arg-1272 tryptic peptide (Table I). Login to comment 157 ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Glu1245Gln ABCA1 p.Glu1246Gln ABCA1 p.Ser1255Ala 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 159 ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Glu1245Gln ABCA1 p.Glu1246Gln ABCA1 p.Ser1255Ala 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 160 ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1243Asp ABCA1 p.Thr1242Asp ABCA1 p.Ser1255Ala 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 162 ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1243Asp ABCA1 p.Thr1242Asp ABCA1 p.Ser1255Ala 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 ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Ser1255Ala ABCA1 p.Val1251Arg ABCA1 p.Val1251Arg ABCA1 p.Val1251Arg ABCA1 p.Val1251Arg ABCA1 p.Leu1229Lys ABCA1 p.Leu1229Lys ABCA1 p.Leu1229Lys ABCA1 p.Leu1229Arg 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 ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Ser1255Ala 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 176 ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Glu1245Gln ABCA1 p.Glu1246Gln ABCA1 p.Ser1255Ala ABCA1 p.Val1251Arg ABCA1 p.Val1251Arg ABCA1 p.Val1251Arg ABCA1 p.Val1251Arg ABCA1 p.Leu1229Lys ABCA1 p.Leu1229Lys ABCA1 p.Leu1229Lys ABCA1 p.Leu1229Arg 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 ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1242Ala ABCA1 p.Thr1243Asp ABCA1 p.Thr1243Asp ABCA1 p.Thr1242Asp ABCA1 p.Thr1242Asp ABCA1 p.Ser1255Ala 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 178 ABCA1 p.Glu1245Gln ABCA1 p.Glu1246Gln The E1245Q/ E1246Q mutation of the downstream acidic residues, which also impairs CK2 activity, similarly increased cellular apoAI and apoAII binding. Login to comment 179 ABCA1 p.Thr1243Asp ABCA1 p.Thr1243Asp ABCA1 p.Thr1242Asp ABCA1 p.Thr1242Asp The T1242D, T1243D, T1242D/T1243D mutations, which mimic CK2 phosphorylation, had an opposite effect to the Thr-Ala mutations, as apoprotein binding to cells transfected with these mutants amounted to around 110% of WT ABCA1. Login to comment 186 ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Glu1245Gln ABCA1 p.Glu1246Gln 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 188 ABCA1 p.Thr1243Ala ABCA1 p.Thr1242Ala ABCA1 p.Glu1245Gln ABCA1 p.Glu1246Gln 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 194 ABCA1 p.Ser1255Ala of the S1255A mutant on lipid efflux was comparable with the single threonine mutants. Login to comment 195 ABCA1 p.Thr1243Asp ABCA1 p.Thr1243Asp ABCA1 p.Thr1242Asp The T1242D, T1243D, and T1242/ T1243D mutations had an opposite effect as the efflux induced by that mutant was close to WT ABCA1. Login to comment 196 ABCA1 p.Ser1255Ala of the S1255A mutant on lipid efflux was comparable with the single threonine mutants. Login to comment 197 ABCA1 p.Thr1243Asp ABCA1 p.Thr1243Asp ABCA1 p.Thr1242Asp The T1242D, T1243D, and T1242/ T1243D mutations had an opposite effect as the efflux induced by that mutant was close to WT ABCA1. Login to comment 211 ABCA1 p.Thr1242Ala ABCA1 p.Ser1255Ala This was then confirmed by the engineering of the S1255A mutant, whose activities are comparable with those of the T1242A mutant. Login to comment 213 ABCA1 p.Thr1242Ala ABCA1 p.Ser1255Ala This was then confirmed by the engineering of the S1255A mutant, whose activities are comparable with those of the T1242A mutant. Login to comment