PURIFICATION AND CHARACTERIZATION OF A KINASE SPECIFIC FOR THE SERINE-RICH AND ARGININE-RICH PRE-MESSENGER-RNA SPLICING FACTORS
PURIFICATION AND CHARACTERIZATION OF A KINASE SPECIFIC FOR THE SERINE-RICH AND ARGININE-RICH PRE-MESSENGER-RNA SPLICING FACTORS
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DOI：
10.1073/pnas.91.23.10824
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发表时间：
1994-11-08
影响因子：
11.1
通讯作者：
FU, XD
中科院分区：
文献类型：
--
作者：
GUI, JF;TRONCHERE, H;FU, XD
前 mRNA 剪接因子 SR 家族的成员是磷蛋白，它们共享单克隆抗体 (mAb) 104 特异性识别的磷酸表位。最近的研究表明，磷酸化可以调节这些剪接因子的活性和细胞内定位。在这里，我们报告了 SR 蛋白激酶 1 (SRPK1) 的纯化和动力学特性，SR 蛋白激酶 1 是 SR 家族成员特异的激酶。我们证明该激酶特异性识别 SR 结构域，其中包含丝氨酸/精氨酸重复序列。先前的研究表明，去磷酸化的 SR 蛋白不与 mAb 104 发生反应，并且在 SDS 凝胶中比来自哺乳动物细胞的 SR 蛋白迁移得更快。我们发现 SRPK1 恢复了细菌中产生的 SR 蛋白 SF2/ASF（剪接因子 2/选择性剪接因子）的移动性和 mAB 104 反应性，这表明 SRPK1 负责体内 mAb 104 特异性磷酸表位的生成。最后，我们将 SF2/ASF SR 结构域中的突变对剪接的影响与 SRPK1 对该蛋白磷酸化的影响相关联，表明剪接需要 SR 蛋白的磷酸化。
Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.