Applying the Brakes to Multisite SR Protein Phosphorylation: Substrate-Induced Effects on the Splicing Kinase SRPK1
Applying the Brakes to Multisite SR Protein Phosphorylation: Substrate-Induced Effects on the Splicing Kinase SRPK1
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DOI：
10.1021/bi2007993
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发表时间：
2011-08-16
期刊：
影响因子：
2.9
通讯作者：
Adams, Joseph A.
中科院分区：
文献类型：
--
作者：
Aubol, Brandon E.;Adams, Joseph A.
为了研究蛋白激酶如何在延长的多位点磷酸化过程中与其蛋白底物相互作用，使用快速猝灭流技术研究了参与mRNA剪接控制的蛋白激酶的动力学机制。蛋白激酶SRPK 1与SR蛋白SRSF 1的富含精氨酸-丝氨酸结构域（RS结构域）中的10个丝氨酸类似地以C末端至N末端方向磷酸化，这种修饰将这种必需的剪接因子从细胞质引导到细胞核。瞬态动力学实验表明，第一个磷酸盐被快速添加到SRSF 1的RS结构域（t（1/2）= 0.1 s），然后在剩余的丝氨酸缓慢，多位点磷酸化（t（1/2）= 15 s）。通过SR蛋白的广泛氢键和静电结构域以及激酶的活性位点和对接沟来维持突变有效的磷酸化速率。催化捕获和粘度实验表明，而磷酰基转移步骤是快速的，ADP释放限制多位点磷酸化。通过研究磷酸盐掺入到选择性预磷酸化形式的酶-底物复合物中，评估了沿着反应坐标的位点特异性磷酸化的动力学机制。SR蛋白的结合亲和力，磷酰基转移率，ADP交换率被发现作为一个功能的RS域中的渐进磷酸化显着下降。这些发现表明，蛋白质底物积极调节与延长的多位点磷酸化相关的起始、延伸和终止事件。实验表明，RS之间的网络
To investigate how a protein kinase interacts with its protein substrate during extended, multisite phosphorylation, the kinetic mechanism of a protein kinase involved in mRNA splicing control was investigated using rapid quench flow techniques. The protein kinase SRPK1 phosphorylates similar to 10 serines in the arginine-serine-rich domain (RS domain) of the SR protein SRSF1 in a C- to N-terminal direction, a modification that directs this essential splicing factor from the cytoplasm to the nucleus. Transient-state kinetic experiments illustrate that the first phosphate is added rapidly onto the RS domain of SRSF1 (t(1/2) = 0.1 s) followed by slower, multisite phosphorylation at the remaining serines (t(1/2) = 15 s). Mutagenesis efficient phosphorylation rates are maintained by an extensive hydrogen bonding and electrostatic domain of the SR protein and the active site and docking groove of the kinase. Catalytic trapping and viscosometric experiments demonstrate that while the phosphoryl transfer step is fast, ADP release limits multisite phosphorylation. By studying phosphate incorporation into selectively pre-phosphorylated forms of the enzyme-substrate complex, the kinetic mechanism for site-specific phosphorylation along the reaction coordinate was assessed. The binding affinity of the SR protein, the phosphoryl transfer rate, and ADP exchange rate were found to decline significantly as a function of progressive phosphorylation in the RS domain. These findings indicate that the protein substrate actively modulates initiation, extension, and termination events associated with prolonged, multisite phosphorylation. experiments suggest that network between the RS