Transgenic Analysis of the Leishmania MAP Kinase MPK10 Reveals an Auto-inhibitory Mechanism Crucial for Stage-Regulated Activity and Parasite Viability

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.

利什曼属（Leishmania）的原生动物病原体演化出独特的信号传导机制，能够感知宿主环境变化并触发适应性阶段分化——这一过程对于宿主细胞感染至关重要。尽管此前已有研究将利什曼丝裂原活化蛋白激酶（mitogen-activated protein kinases, MAPKs）与环境诱导的分化及毒力相关联，但该寄生虫发育背后的信号传导机制仍尚未得到充分阐明。本研究解析了利什曼丝裂原活化蛋白激酶10（MPK10）极为特殊的调控机制。通过转基因实验方法，我们证实MPK10呈阶段特异性调控：其激酶活性在前鞭毛体（promastigote）向无鞭毛体（amastigote）转化过程中逐步升高。然而，与经典丝裂原活化蛋白激酶通过激活环内调控性TxY基序的双磷酸化激活不同，MPK10的激活并不依赖于酪氨酸残基的磷酸化——该位点的磷酸化整体上呈组成型状态。截去MPK10的最后46个氨基酸残基后，重组蛋白与转基因蛋白的激酶活性均显著增强，这表明MPK10受到自抑制机制的调控。在转基因寄生虫中过表达该高活性突变体，会引发显性负效应，导致无鞭毛体分化阶段出现大量细胞死亡，这证明MPK10的自抑制作用对于寄生虫的存活至关重要。此外，磷酸化蛋白质组学（phosphoproteomics）分析在C端自抑制结构域中发现了一个位于395位点的新型调控性磷酸化丝氨酸残基，其可能参与激酶活性的调控。我们还发现了一条反馈环路，该环路通过对TxY基序的酪氨酸残基进行去磷酸化来限制MPK10的活性。综上，本研究揭示了利什曼原虫中蛋白激酶调控的全新机制，并提出MPK10可作为哺乳动物宿主环境的潜在信号感受器，其固有的预激活构象受到自抑制作用的调控。