Transgenic analysis of the Leishmania MAP kinase MPK10 reveals an auto-inhibitory mechanism crucial for stage-regulated activity and parasite viability.
Transgenic analysis of the Leishmania MAP kinase MPK10 reveals an auto-inhibitory mechanism crucial for stage-regulated activity and parasite viability.
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DOI：
10.1371/journal.ppat.1004347
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发表时间：
2014-09
期刊：
影响因子：
6.7
通讯作者：
Späth GF
中科院分区：
文献类型：
--
作者：
Cayla M;Rachidi N;Leclercq O;Schmidt-Arras D;Rosenqvist H;Wiese M;Späth GF
利什曼原虫属的原生动物病原体已经进化出独特的信号传导机制，该机制可以感知宿主环境的变化并触发宿主细胞感染所必需的适应性阶段分化。尽管利什曼原虫丝裂原活化蛋白激酶（MAPKs）以前已与环境诱导的分化和毒力有关，但寄生虫发育的信号传导机制在很大程度上仍然难以捉摸。在这里，我们解开利什曼原虫MAP激酶10（MPK 10）的高度不寻常的调节机制。使用转基因的方法，我们证明，MPK 10是阶段特异性调节，因为它的激酶活性增加，在前鞭毛体无鞭毛体转换。然而，与通过活化环中的调节TxY基序的双重磷酸化活化的典型MAPK不同，MPK 10活化独立于酪氨酸残基的磷酸化，其在很大程度上是组成性的。去除最后46个氨基酸导致重组蛋白和转基因蛋白的MPK 10活性显著增强，表明MPK 10受自抑制机制调节。在转基因寄生虫中过度表达这种过度活跃的突变体导致显性负效应，在无鞭毛体分化期间引起大量细胞死亡，证明MPK 10自身抑制寄生虫活力的本质。此外，磷酸蛋白质组学分析确定了一种新的监管磷酸丝氨酸残基的C-末端的自抑制结构域的位置395，可能涉及激酶的调节。最后，我们发现了一个反馈回路，通过TxY基序的酪氨酸残基的去磷酸化限制MPK 10的活性。我们的数据揭示了利什曼原虫蛋白激酶调控的新方面，并提出MPK 10作为哺乳动物宿主环境的潜在信号传感器，其内在的预激活构象受自抑制调节。利什曼病是由利什曼原虫引起的人类重要疾病。利什曼原虫感染性的一个重要方面是它能够感知不同的环境，并通过阶段分化适应昆虫载体和脊椎动物宿主内的生存。这一过程是由这些生物体中遇到的环境变化触发的，包括温度和pH值的变化，这些变化通常由包括蛋白激酶及其底物在内的信号级联反应感知和转导。在这项研究中，我们分析了利什曼原虫丝裂原活化蛋白激酶MPK 10的调节使用从转基因寄生虫纯化的蛋白质，并结合定点突变和活性测试。我们证明，这种激酶在寄生虫分化过程中被激活，并受到一种非典型机制的调控，这种机制涉及自抑制，这对寄生虫的生存能力至关重要。
Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition. Leishmaniasis is an important human disease caused by Leishmania parasites. A crucial aspect of Leishmania infectivity is its capacity to sense different environments and adapt for survival inside insect vector and vertebrate host by stage differentiation. This process is triggered by environmental changes encountered in these organisms, including temperature and pH shifts, which usually are sensed and transduced by signaling cascades including protein kinases and their substrates. In this study, we analyzed the regulation of the Leishmania mitogen-activated protein kinase MPK10 using protein purified from transgenic parasites and combining site-directed mutagenesis and activity tests. We demonstrate that this kinase is activated during parasite differentiation and regulated by an atypical mechanism involving auto-inhibition, which is essential for parasite viability.
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影响因子：
5.7
作者：
Horjales, Sofia;Schmidt-Arras, Dirk;Buschiazzo, Alejandro
通讯作者：
Buschiazzo, Alejandro
影响因子：
2.5
作者：
DAVIS, RJ
通讯作者：
DAVIS, RJ
影响因子：
10.5
作者：
Breitwieser, Wolfgang;Lyons, Steve;Jones, Nic
通讯作者：
Jones, Nic
DOI：
10.1073/pnas.95.3.1091
发表时间：
1998-02-03
影响因子：
11.1
作者：
Cheng, MG;Sexl, V;Roussel, MF
通讯作者：
Roussel, MF
影响因子：
4.8
作者：
Ferrell, JE;Bhatt, RR
通讯作者：
Bhatt, RR