Role of protein kinase A in Trypanosoma cruzi
Role of protein kinase A in Trypanosoma cruzi
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DOI：
10.1128/iai.00527-08
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发表时间：
2008-10-01
影响因子：
3.1
通讯作者：
Huang, Huan
中科院分区：
文献类型：
--
作者：
Bao, Yi;Weiss, Louis M.;Huang, Huan
蛋白激酶A（PKA）是真核细胞中许多信号转导通路的重要介质，并且它被认为是克氏锥虫阶段分化的调节因子。为了评估克氏锥虫PKA催化亚基（TcPKAc）的重要性，将一个编码含有特异性PKA假底物R - R - N - A的PKA抑制剂（PKI）的基因亚克隆到pTREX载体中，并通过电穿孔导入前鞭毛体。PKI的表达对这种寄生虫具有致死作用。同样，一种药物抑制剂H89在10μM的浓度下可杀死前鞭毛体。要了解PKA的生物学特性，鉴定这种酶的特定底物是至关重要的。利用酵母双杂交系统，鉴定出38个与TcPKAc相互作用的候选物。其中18个是功能未知的假设蛋白，而其他的则具有假定的或已知的功能。在最高严格筛选条件下，在酿酒酵母中证实了8个可能对调节克氏锥虫生长、适应和分化很重要的基因的整个开放阅读框与TcPKAc相互作用，这些基因包括III型PI3激酶（Vps34）、一种假定的PI3激酶、一种假定的丝裂原活化的细胞外信号调节激酶、一种环腺苷酸（cAMP）特异性磷酸二酯酶（PDEC2）、一种己糖激酶、一种假定的ATP酶、一种DNA切除修复蛋白和一种水通道蛋白，并且PKA使这些基因的重组蛋白磷酸化。综上所述，这些发现证明了cAMP - PKA信号在这种生物中的重要性。
Protein kinase A (PKA) is an important mediator of many signal transduction pathways that occur in eukaryotic cells, and it has been implicated as a regulator of stage differentiation in Trypanosoma cruzi. To evaluate the importance of the PKA catalytic subunit of T. cruzi (TcPKAc), a gene encoding a PKA inhibitor (PKI) containing a specific PKA pseudosubstrate, R-R-N-A, was subcloned into a pTREX vector and introduced into epimastigotes by electroporation. Expression of PKI has a lethal effect in this parasite. Similarly, a pharmacological inhibitor, H89, killed epimastigotes at a concentration of 10 mu M. To understand the biology of PKA, identification of the particular substrates of this enzyme is essential. Using a yeast two-hybrid system, 38 candidates interacting with TcPKAc were identified. Eighteen of these were hypothetical proteins with unknown functions, while the others had putative or known functions. The entire open reading frames of eight genes presumably important in regulating T. cruzi growth, adaptation, and differentiation, including a type III PI3 kinase (Vps34), a putative PI3 kinase, a putative mitogenactivated extracellular signal-regulated kinase, a cyclic AMP (cAMP)-specific phosphodiesterase (PDEC2), a hexokinase, a putative ATPase, a DNA excision repair protein, and an aquaporin were confirmed to interact with TcPKAc in the yeast Saccharomyces cerevisiae under the highest stringency selection conditions, and PKA phosphorylated the recombinant proteins of these genes. Taken together, these findings demonstrate the importance of cAMP-PKA signaling in this organism.