Interaction between the N-terminal domain of human DNA topoisomerase I and the arginin-serine domain of its substrate determines phosphorylation of SF2/ASF splicing factor
Interaction between the N-terminal domain of human DNA topoisomerase I and the arginin-serine domain of its substrate determines phosphorylation of SF2/ASF splicing factor
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DOI：
10.1093/nar/26.12.2955
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发表时间：
1998-06-15
影响因子：
14.9
通讯作者：
Tazi, J
中科院分区：
文献类型：
--
作者：
Labourier, E;Rossi, F;Tazi, J
人DNA拓扑异构酶I可能是体内SR蛋白剪接因子家族的磷酸化激酶之一，具有DNA松弛活性。我们以SR蛋白SF 2/ASF（SRp 30 a）为模型底物，证明了拓扑异构酶I/激酶磷酸化的丝氨酸残基仅位于SF 2/ASF RS结构域的最长的丝氨酸-丝氨酸重复序列中。与其他激酶如cdc 2和SRPK 1不同，它们也在RS结构域磷酸化丝氨酸，topo I/激酶需要几个SR二肽重复序列，这些重复序列可能有助于RS结构域中的通用结构，从而促进磷酸化。此外，使用SF 2/ASF突变体的远Western、荧光光谱和激酶测定表明，激酶活性和结合是紧密耦合的。由于Topo I的N-末端174个氨基酸的缺失破坏了SF 2/ASF结合和激酶活性，但不破坏ATP结合，因此我们得出结论，Topo I的至少两个不同的结构域是激酶活性所必需的：一个在C-末端区域中有助于ATP结合位点，另一个在N-末端区域中允许SF 2/ASF结合。
Human DNA topoisomerase I, known for its DNA-relaxing activity, is possibly one of the kinases phosphorylating members of the SR protein family of splicing factors, in vivo. Little is known about the mechanism of action of this novel kinase, Using the prototypical SR protein SF2/ASF (SRp30a) as model substrate, we demonstrate that serine residues phosphorylated by topo I/kinase exclusively located within the most extended arginine-serine repeats of the SF2/ASF RS domain. Unlike other kinases such as cdc2 and SRPK1, which also phosphorylated serines at the RS domain, topo I/kinase required several SR dipeptide repeats, These repeats possibly contribute to a versatile structure in the RS domain thereby facilitating phosphorylation, Furthermore, far-western, fluorescence spectroscopy and kinase assays using the SF2/ASF mutants, demonstrated that kinase activity and binding were tightly coupled. Since the deletion of N-terminal 174 amino acids of Topo I destroys SF2/ASF binding and kinase activity but not ATP binding, we conclude that at least two distinct domains of Topo I are necessary for kinase activity: one in the C-terminal region contributing to the ATP binding site and the other one in the N-terminal region that allows binding of SF2/ASF.