Identification of an effector protein and gain-of-function mutants that activate Pfmrk, a malarial cyclin-dependent protein kinase
Identification of an effector protein and gain-of-function mutants that activate Pfmrk, a malarial cyclin-dependent protein kinase
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DOI：
10.1016/j.molbiopara.2006.04.004
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发表时间：
2006-09-01
影响因子：
1.5
通讯作者：
Waters, Norman C.
中科院分区：
文献类型：
--
作者：
Chen, Yueqin;Jirage, Dayadevi;Waters, Norman C.
细胞周期蛋白依赖性蛋白激酶（CDKs）是细胞周期调控的关键调节因子。在人类中，CDK 7作为负责调节许多CDK的CDK活化激酶（CAK）和作为参与转录调节的RNA聚合酶II羧基末端结构域（CTD）激酶发挥双重作用。效应蛋白，人MAT 1的结合刺激CDK 7激酶活性并影响底物特异性。在恶性疟原虫中，CDK及其在调节生长和发育中的作用知之甚少。在这项研究中，我们的特点Pfmrk，一个假定的同源物的人CDK 7的调节机制。我们确定了一个效应，PfMAT 1，刺激Pfmrk激酶活性的细胞周期蛋白依赖性的方式。PfMAT 1的加入刺激RNA聚合酶II CTD磷酸化，并且对Pfmrk不能磷酸化PfPK 5（一种假定的CDK 1同源物）没有影响，这表明Pfmrk可能是CTD激酶而不是CAK。为了消除对PfMAT 1刺激的需求，我们突变了Pfmrk活性位点内的氨基酸。我们发现两个独立的突变体，S138 K和F143 L，产生了4-10倍的Pfmrk活性增加。在细胞周期蛋白或PfMAT 1不存在的情况下观察到这些突变体的显著激酶活性。最后，我们观察到Pfmrk的自磷酸化，不受细胞周期蛋白或PfMAT 1的影响。(c)2006 Elsevier B. V.保留所有权利。
Cyclin-dependent protein kinases (CDKs) are key regulators of cell cycle control. In humans, CDK7 performs dual roles as the CDK activating kinase (CAK) responsible for regulating numerous CDKs and as the RNA polymerase II carboxyl-terminal domain (CTD) kinase involved in the regulation of transcription. Binding of an effector protein, human MAT1, stimulates CDK7 kinase activity and influences substrate specificity. In Plasmodium falciparum, CDKs and their roles in regulating growth and development are poorly understood. In this study, we characterized the regulatory mechanisms of Pfmrk, a putative homolog of human CDK7. We identified an effector, PfMAT1, which stimulates Pfmrk kinase activity in a cyclin-dependent manner. The addition of PfMAT1 stimulated RNA polymerase II CTD phosphorylation and had no effect on the inability of Pfmrk to phosphorylate PfPK5, a putative CDK1 homolog, which suggests that Pfmrk may be a CTD kinase rather than a CAK. In an attempt to abrogate the requirement for PfMAT1 stimulation, we mutated amino acids within the active site of Pfmrk. We found that two independent mutants, S138K and F143L, yielded a 4-10-fold increase in Pfmrk activity. Significant kinase activity of these mutants was observed in the absence of either cyclin or PfMAT1. Finally, we observed autophosphorylation of Pfmrk that is unaffected by the addition of either cyclin or PfMAT1. (c) 2006 Elsevier B.V. All rights reserved.