The Protein Kinase Clk/Sty Directly Modulates SR Protein Activity: Both Hyper- and Hypophosphorylation Inhibit Splicing
The Protein Kinase Clk/Sty Directly Modulates SR Protein Activity: Both Hyper- and Hypophosphorylation Inhibit Splicing
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DOI：
10.1128/mcb.19.10.6991
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发表时间：
1999-10
影响因子：
5.3
通讯作者：
J. Prasad;K. Colwill;T. Pawson;J. Manley
中科院分区：
文献类型：
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作者：
J. Prasad;K. Colwill;T. Pawson;J. Manley
摘要哺乳动物信使核糖核酸前体的剪接既需要蛋白质的磷酸化，也需要去磷酸化，可能涉及到剪接因子SR蛋白家族成员的修饰。几种在体外可以磷酸化SR蛋白的激酶已经被鉴定出来，并且转基因实验已经提供了证据，至少有一种，CLK/Sty，可以调节体内的剪接。但是，目前还没有证据表明特定的激酶可以直接影响SR蛋白的剪接活性。在这里，通过使用纯化的重组CLK/Sty，一个催化失活的突变体，和单个SR蛋白，我们证明了在重组剪接实验中，CLK/Sty直接影响SR蛋白的活性，而不是其他重要的剪接因子。我们还提供了证据表明，高磷酸化和低磷酸化都抑制了SR蛋白的剪接活性，抑制了结构性剪接和切换选择性剪接位点的选择。这些发现表明，CLK/Sty直接和特异性地影响SR蛋白剪接因子的活性，更重要的是，表明SR蛋白的低和过度磷酸化都可以调节剪接。
ABSTRACT The splicing of mammalian mRNA precursors requires both protein phosphorylation and dephosphorylation, likely involving modification of members of the SR protein family of splicing factors. Several kinases have been identified that can phosphorylate SR proteins in vitro, and transfection assays have provided evidence that at least one of these, Clk/Sty, can modulate splicing in vivo. But evidence that a specific kinase can directly affect the splicing activity of SR proteins has been lacking. Here, by using purified recombinant Clk/Sty, a catalytically inactive mutant, and individual SR proteins, we show that Clk/Sty directly affects the activity of SR proteins, but not other essential splicing factors, in reconstituted splicing assays. We also provide evidence that both hyper- and hypophosphorylation inhibit SR protein splicing activity, repressing constitutive splicing and switching alternative splice site selection. These findings indicate that Clk/Sty directly and specifically influences the activity of SR protein splicing factors and, importantly, show that both under- and overphosphorylation of SR proteins can modulate splicing.