ABCMdb

ABCA1 p.Val1251Arg

Predicted by SNAP2:	A: D (59%), C: N (61%), D: D (75%), E: D (71%), F: D (59%), G: D (75%), H: D (63%), I: D (53%), K: D (75%), L: D (53%), M: N (53%), N: D (71%), P: D (75%), Q: D (66%), R: D (71%), S: D (63%), T: D (59%), W: D (75%), Y: D (63%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, W: D, Y: D,

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Publications
[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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Sentences [show]
No. Sentence Comment
144 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
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
142 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
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