Functional dissection of adenylate cyclase R, an inducer of spore encapsulation.
Functional dissection of adenylate cyclase R, an inducer of spore encapsulation.
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腺苷酸环化酶R的功能解剖，这是孢子封装的诱导剂。
DOI：
10.1074/jbc.m110.156380
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发表时间：
2010-12-31
期刊：
影响因子：
--
通讯作者：
Schaap P
中科院分区：
文献类型：
--
作者：
Chen ZH;Schilde C;Schaap P
环AMP作用于蛋白激酶A，控制群居和独居阿米巴原虫的产孢和成囊。在盘基网柄菌discoideum,腺苷酸环化酶R (ACR),对孢子封装至关重要。除了环化酶（AC）结构域外，ACR还含有7个跨膜螺旋结构域、一个组氨酸激酶结构域和两个受体结构域。我们研究了这些结构域在调节AC活性中的作用。acr - yfp融合蛋白在acr细胞中的表达挽救了它们的孢子发育缺陷表型，并揭示了acr与核膜和内质网有关。跨膜螺旋的丢失（ΔTM）导致AC活性降低60%，但ΔTM-ACR仍然挽救了acr -表型。分离的AC结构域表达正常，但无活性。组氨酸激酶结构域中三个必需atp结合残基的突变不影响AC活性或表型挽救。受体1、2或两者中必需的接受磷酸化的天冬氨酸的突变对AC活性只有适度的影响，不影响表型拯救，这表明AC活性不受磷接力的关键调节。值得注意的是，二聚化组氨酸磷酸化受体亚结构域在ACR中缺乏自磷酸化的典型组氨酸，对AC活性至关重要。带有ACR等位基因（ΔCRA）的野生型细胞在该结构域后被截断，其转化抑制了内源性ACR的AC活性，并复制了ACR -表型。结合对分离的AC结构域失活的观察，ΔCRA的显性负作用强烈表明，失活的磷受体结构域在加强AC结构域二聚化方面获得了新的作用。
Cyclic AMP acting on protein kinase A controls sporulation and encystation in social and solitary amoebas. In Dictyostelium discoideum, adenylate cyclase R (ACR), is essential for spore encapsulation. In addition to its cyclase (AC) domain, ACR harbors seven transmembrane helices, a histidine kinase domain, and two receiver domains. We investigated the role of these domains in the regulation of AC activity. Expression of an ACR-YFP fusion protein in acr− cells rescued their sporulation defective phenotype and revealed that ACR is associated with the nuclear envelope and endoplasmic reticulum. Loss of the transmembrane helices (ΔTM) caused a 60% reduction of AC activity, but ΔTM-ACR still rescued the acr− phenotype. The isolated AC domain was properly expressed but inactive. Mutation of three essential ATP-binding residues in the histidine kinase domain did not affect the AC activity or phenotypic rescue. Mutation of the essential phosphoryl-accepting aspartate in receivers 1, 2, or both had only modest effects on AC activity and did not affect phenotypic rescue, indicating that AC activity is not critically regulated by phosphorelay. Remarkably, the dimerizing histidine phosphoacceptor subdomain, which in ACR lacks the canonical histidine for autophosphorylation, was essential for AC activity. Transformation of wild-type cells with an ACR allele (ΔCRA) that is truncated after this domain inhibited AC activity of endogenous ACR and replicated the acr− phenotype. Combined with the observation that the isolated AC domain was inactive, the dominant-negative effect of ΔCRA strongly suggests that the defunct phosphoacceptor domain acquired a novel role in enforcing dimerization of the AC domain.