Phosphorylation of serine 1106 in the catalytic domain of topoisomerase IIα regulates enzymatic activity and drug sensitivity
Phosphorylation of serine 1106 in the catalytic domain of topoisomerase IIα regulates enzymatic activity and drug sensitivity
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DOI：
10.1074/jbc.m300837200
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发表时间：
2003-04-11
影响因子：
4.8
通讯作者：
Ganapathi, MK
中科院分区：
文献类型：
--
作者：
Chikamori, K;Grabowski, DR;Ganapathi, MK
拓扑异构酶改变DNA拓扑结构，对维持基因组完整性至关重要。拓扑异构酶I和II也是广泛使用的抗肿瘤剂的靶标。我们以前证明，在人类白血病细胞系，HL-60，拓扑异构酶（拓扑）II靶向药物，如依托泊苷的耐药性与拓扑异构酶II α的位点特异性低磷酸化。这种效应可以在用细胞内Ca 2+螯合剂1，2-双（2-氨基苯氧基）乙烷-N，N，N '，N'-四乙酸（BAPTA-AM）处理的敏感细胞中模拟。在这里，我们确定Ser-1106作为一个主要的磷酸化位点的拓扑异构酶II α的催化结构域。该位点位于酸营养激酶酪蛋白激酶I和酪蛋白激酶II的共有序列内。丝氨酸1106突变为丙氨酸（S1106 A）消除了在用BAPTA-AM处理的耐药HL-60细胞或敏感细胞中被发现低磷酸化的磷酸肽的磷酸化。含有S1106 A取代的纯化的topo II α在脱连环动质体DNA中的活性比野生型topo II α低4倍，并且在依托泊苷稳定的DNA可切割复合物形成的水平上也表现出2-4倍的降低。表达S1106 A突变体拓扑异构酶II α蛋白的酿酒酵母（JN 394 t2 -4）细胞对依托泊苷或安吖啶的细胞毒性作用更有抗性。这些结果表明，Ca 2+调节磷酸化的Ser-1106的催化结构域的拓扑II α调节这种蛋白质的酶活性和敏感性的拓扑II靶向药物。
Topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity. Topoisomerases I and II are also targets for widely used antitumor agents. We demonstrated previously that in the human leukemia cell line, HL-60, resistance to topoisomerase (topo) II-targeting drugs such as etoposide is associated with site-specific hypophosphorylation of topo IIalpha. This effect can be mimicked in sensitive cells treated with the intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Here we identify Ser-1106 as a major phosphorylation site in the catalytic domain of topo IIalpha. This site lies within the consensus sequence for the acidotrophic kinases, casein kinase I and casein kinase II. Mutation of serine 1106 to alanine (S1106A) abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in resistant HL-60 cells or sensitive cells treated with BAPTA-AM. Purified topo IIalpha containing a S1106A substitution is 4-fold less active than wild type topo IIalpha in decatenating kinetoplast DNA and also exhibits a 2-4-fold decrease in the level of etoposide-stabilized DNA cleavable complex formation. Saccharomyces cerevisiae (JN394t2-4) cells expressing S1106A mutant topo IIalpha protein are more resistant to the cytotoxic effects of etoposide or amsacrine. These results demonstrate that Ca2+-regulated phosphorylation of Ser-1106 in the catalytic domain of topo IIalpha modulates the enzymatic activity of this protein and sensitivity to topo II-targeting drugs.