Alternative Splicing Controls Myotonic Dystrophy Protein Kinase Structure, Enzymatic Activity, and Subcellular Localization
Alternative Splicing Controls Myotonic Dystrophy Protein Kinase Structure, Enzymatic Activity, and Subcellular Localization
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DOI：
10.1128/mcb.23.16.5489-5501.2003
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发表时间：
2003-08
影响因子：
5.3
通讯作者：
D. G. Wansink;R. V. van Herpen;M. Coerwinkel-Driessen;P. Groenen;B. Hemmings;B. Wieringa
中科院分区：
文献类型：
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作者：
D. G. Wansink;R. V. van Herpen;M. Coerwinkel-Driessen;P. Groenen;B. Hemmings;B. Wieringa
强直性肌营养不良蛋白激酶（DMPK）基因是Rho激酶家族的一员，其转录本受到细胞类型特异性选择性剪接的影响。DMPK剪接异构体分布的不平衡可能在DM 1（一种严重的多系统疾病）的发病机制中发挥作用。在这里，我们报告如何结构子域决定DMPK亚型的生化特性和亚细胞分布。一种新开发的激酶分析表明，DMPK是一个赖氨酸/精氨酸定向激酶。单个DMPK亚型显示出相当的转磷酸化活性和肽底物的序列偏好。然而，DMPK自身磷酸化和MYPT 1的磷酸化（作为DMPK的推定体内靶点）依赖于可变剪接的VSGGG基序的存在和C末端的性质。VSGGG基序对全长激酶迁移行为的凝胶内效应为该基序介导DMPK亚型的3-D构象变化的模型提供了证据。最后，不同的C末端赋予DMPK与内质网或线粒体结合或采用胞质定位的能力。我们的研究结果表明，DMPK亚型具有细胞类型和位置依赖性底物特异性，在细胞器和细胞结构动力学中发挥作用。
ABSTRACT Transcripts of the myotonic dystrophy protein kinase (DMPK) gene, a member of the Rho kinase family, are subject to cell-type specific alternative splicing. An imbalance in the splice isoform profile of DMPK may play a role in the pathogenesis of DM1, a severe multisystemic disorder. Here, we report how structural subdomains determine biochemical properties and subcellular distribution of DMPK isoforms. A newly developed kinase assay revealed that DMPK is a Lys/Arg-directed kinase. Individual DMPK isoforms displayed comparable transphosphorylation activity and sequence preference for peptide substrates. However, DMPK autophosphorylation and phosphorylation of MYPT1 (as putative in vivo target of DMPK), were dependent on presence of an alternatively spliced VSGGG motif and the nature of the C terminus. In-gel effects of the VSGGG motif on the migration behavior of full-length kinase provide evidence for a model in which this motif mediates 3-D-conformational changes in DMPK isoforms. Finally, different C termini endow DMPK with the ability to bind to either endoplasmic reticulum or mitochondria or to adopt a cytosolic location. Our results suggest that DMPK isoforms have cell-type and location dependent substrate specificities with a role in organellar and cytoarchitectural dynamics.