Regulation of Binding of Lamin B Receptor to Chromatin by SR Protein Kinase and cdc2 Kinase in Xenopus Egg Extracts*
Regulation of Binding of Lamin B Receptor to Chromatin by SR Protein Kinase and cdc2 Kinase in Xenopus Egg Extracts*
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DOI：
10.1074/jbc.m308854200
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发表时间：
2004-03
影响因子：
4.8
通讯作者：
M. Takano;Yuhei Koyama;Hiromi Ito;S. Hoshino;H. Onogi;M. Hagiwara;K. Furukawa;T. Horigome
中科院分区：
文献类型：
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作者：
M. Takano;Yuhei Koyama;Hiromi Ito;S. Hoshino;H. Onogi;M. Hagiwara;K. Furukawa;T. Horigome
先前提出了多种激酶参与调节核纤层蛋白B受体（LBR）与染色质的结合（Takano，M.，Takeuchi，M.，伊藤，H.，Furukawa，K.，杉本，K.，Omata，S.，Horigome，T.（2002）Eur. 269，943-953）。为了确定这些激酶，调节LBR的核质区（NK，氨基酸残基1-211）的精子染色质的结合进行了研究，使用细胞周期依赖性爪蟾卵提取物在体外。的结合刺激特定的磷酸化的NK片段的S期鸡蛋提取物。用带有结合SR蛋白激酶的SF 2/ASF的珠去除蛋白质，消除了这种刺激，表明SR蛋白激酶负责LBR的活化。这一点通过重组SR蛋白特异性激酶1的直接磷酸化和活化得到证实。NK片段与S期提取物预处理的染色质的结合通过与M期提取物孵育而受到抑制。酶抑制剂实验表明，多种激酶参与抑制。其中一种激酶被证明是cdc 2激酶，使用特异性抑制剂roscovitine，并使用带有p13的珠子进行蛋白质消耗，p13特异性结合cdc 2激酶。涉及突变NK片段的实验表明，由cdc 2激酶磷酸化丝氨酸71是负责的抑制。
Participation of multiple kinases in regulation of the binding of lamin B receptor (LBR) to chromatin was suggested previously (Takano, M., Takeuchi, M., Ito, H., Furukawa, K., Sugimoto, K., Omata, S., and Horigome, T. (2002) Eur. J. Biochem. 269, 943-953). To identify these kinases, regulation of the binding of the nucleoplasmic region (NK, amino acid residues 1-211) of LBR to sperm chromatin was studied using a cell cycle-dependent Xenopus egg extract in vitro. The binding was stimulated on specific phosphorylation of the NK fragment by an S-phase egg extract. Protein depletion with beads bearing SF2/ASF, which binds SR protein kinases, abolished this stimulation, suggesting that an SR protein kinase(s) is responsible for the activation of LBR. This was confirmed by direct phosphorylation and activation with recombinant SR protein-specific kinase 1. The binding of the NK fragment to chromatin pretreated with an S-phase extract was suppressed by incubation with an M-phase extract. Enzyme inhibitor experiments revealed that multiple kinases participate in the suppression. One of these kinases was shown to be cdc2 kinase using a specific inhibitor, roscovitine, and protein depletion with beads bearing p13, which specifically binds cdc2 kinase. Experiments involving a mutant NK fragment showed that the phosphorylation of serine 71 by cdc2 kinase is responsible for the suppression.