Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter
Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter
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DOI：
10.1074/jbc.m401821200
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发表时间：
2004-09-03
影响因子：
4.8
通讯作者：
Rosseneu, M
中科院分区：
文献类型：
--
作者：
Roosbeek, S;Peelman, F;Rosseneu, M
在先前对ATP结合盒（ABC）转运蛋白的ABCA亚家族的表征中，我们鉴定了潜在的蛋白激酶2（CK 2）磷酸化位点，其在ABCA转运蛋白的真核和原核成员中是保守的（Peelman，F.，Labeur，C.，Vanloo，B.，Roosbeek，S.，德沃角，Duverger，N.，Denefle，P.，Rosier，M.，Vandekerckhove，J.，Rosseneu，M.等人（2003）J. Mol. 325，259 - 274）。这些磷酸化残基位于保守的胞质R1和R2结构域中，核苷酸结合结构域NBD 1和NBD 2的下游。为了研究CK 2对ABCA 1转运蛋白的可能调节，我们表达了ABCA 1、NBD 1 + R1和NBD 2 + R2的重组胞质结构域。我们证明，在体外ABCA 1 NBD 1 + R1，而不是NBD 2 + R2，是磷酸化的CK 2，我们确定的Thr-1242，Thr-1243，和Ser-1255作为磷酸化的残基在R1域的质谱。我们进一步研究了NBD 1中苏氨酸和丝氨酸磷酸化位点的功能意义，通过定点突变整个ABCA 1，然后转染到Hek-293 Tet-Off细胞中。ABCA 1翻转酶活性，载脂蛋白AI和AII结合，和细胞磷脂和胆固醇流出增强突变阻止CK 2磷酸化的苏氨酸和丝氨酸残基。这通过特异性蛋白激酶CK 2抑制剂对转染的Hek-293 Tet-Off细胞中野生型和突变体ABCA 1活性的影响得到证实。模拟苏氨酸磷酸化的突变体的活性接近野生型ABCA 1的活性。因此，我们的数据表明，除了蛋白激酶A和C，蛋白激酶CK 2可能发挥重要作用，在体内调节ABCA 1的功能和运输活动，并可能ABCA亚家族的其他成员。
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 (Peelman, F., Labeur, C., Vanloo, B., Roosbeek, S., Devaud, C., Duverger, N., Denefle, P., Rosier, M., Vandekerckhove, J., and Rosseneu, M. ( 2003) J. Mol. Biol. 325, 259 - 274). 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.