Mechanism of dephosphorylation of the SR protein ASF/SF2 by protein phosphatase 1.
Mechanism of dephosphorylation of the SR protein ASF/SF2 by protein phosphatase 1.
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DOI：
10.1016/j.jmb.2010.08.024
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发表时间：
2010-10-29
影响因子：
5.6
通讯作者：
Adams JA
中科院分区：
文献类型：
--
作者：
Ma CT;Ghosh G;Fu XD;Adams JA
SR蛋白是一种重要的剪接因子，其功能由富含丝氨酸-丝氨酸重复序列的C-末端结构域（RS结构域）的多位点磷酸化控制。蛋白激酶SRPK 1已被证明多磷酸化SR蛋白ASF/SF 2的RS结构域（RS 1）的N-末端部分，这是一种促进该剪接因子进入核并参与剪接功能的修饰。随后，剪接体的成熟和其他RNA加工步骤需要去磷酸化。虽然磷酸盐通过SRPK 1以顺序方式连接到RS 1，但对它们如何被去除知之甚少。为了研究控制去磷酸化的因素，监测ASF/SF 2中磷酸化位点的区域特异性映射作为蛋白磷酸酶PP 1的函数。我们发现，通过SRPK 1添加到RS 1片段的10个磷酸盐以优选的N-to-C方式被去除，直接对抗SRPK 1的C-to-N磷酸化。ASF/SF 2中的两个N端RNA识别基序（RRM）控制对RS结构域的访问并指导定向机制。RNA与RRM的结合防止去磷酸化，这表明SR蛋白与外显子剪接增强子的接合可以调节RS结构域中的磷酰基含量。除了N-末端结构域的调节外，核蛋白激酶Clk/Sty对RS结构域（RS 2）的C-末端部分的磷酸化抑制RS 1去磷酸化并破坏定向机制。这些数据表明，RNA-蛋白质相互作用和侧翼序列中的磷酸化诱导ASF/SF 2的构象，从而增加RS结构域中磷酸盐的寿命。
SR proteins are essential splicing factors whose function is controlled by multi-site phosphorylation of a C-terminal domain rich in arginine-serine repeats (RS domain). The protein kinase SRPK1 has been shown to polyphosphorylate the N-terminal portion of the RS domain (RS1) of the SR protein ASF/SF2, a modification that promotes nuclear entry of this splicing factor and engagement in splicing function. Later, dephosphorylation is required for maturation of the spliceosome and other RNA processing steps. While phosphates are attached to RS1 in a sequential manner by SRPK1, little is known about how they are removed. To investigate factors that control dephosphorylation, region-specific mapping of phosphorylation sites in ASF/SF2 was monitored as a function of the protein phosphatase PP1. We showed that ten phosphates added to the RS1 segment by SRPK1 are removed in a preferred N-to-C manner, directly opposing the C-to-N phosphorylation by SRPK1. Two N-terminal RNA recognition motifs (RRMs) in ASF/SF2 control access to the RS domain and guide the directional mechanism. Binding of RNA to the RRMs protects against dephosphorylation suggesting that engagement of the SR protein with exonic splicing enhancers can regulate phosphoryl content in the RS domain. In addition to regulation by N-terminal domains, phosphorylation of the C-terminal portion of the RS domain (RS2) by the nuclear protein kinase Clk/Sty inhibits RS1 dephosphorylation and disrupts the directional mechanism. The data indicate that both RNA-protein interactions and phosphorylation in flanking sequences induce conformations of ASF/SF2 that increase the lifetime of phosphates in the RS domain.
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