Functional analysis of calcineurin-mediated signalling pathways using fission yeast as a model organism

使用裂殖酵母作为模型生物对钙调神经磷酸酶介导的信号通路进行功能分析

基本信息

批准号：
14580698
负责人：
SUGIURA Reiko
金额：
$ 1.86万
依托单位：
Kinki University (2004)Kobe University (2002-2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

Calcineurin (protein phosphatase 2B), the only serine/threonine phosphatase under the control of Ca2+/calmodulin, is an important mediator in signal transmission, connecting the Ca2+-dependent signalling to a wide variety of cellular responses. Calcineurin is an in vivo target of the immunosuppressant drug FK506 in fission yeast, and inhibition of calcineurin activity by gene disruption or by the addition of FK506 to the media does not affect the vegetative growth of fission yeast. We performed a genetic screen to search for the mutations that show sensitivity to the immunosuppressive drug FK506, and identified eight complementation groups. We identified PI4P5K Ypt3 small GTPase, subunits of clathrin-adaptor complex and demonstrated that these molecules and calcineurin share an essential function in cellular events such as cytokinesis, cell integrity and membrane trafficking. We also identified a downstream target of calcinerin ; Prz1, a zinc finger-type transcription factor. Prz1 was shown to be dephosphorylated by calcineurin and its transcriptional activity was also under the control of calcineurin-mediated signalling. Another approach to identify molecules that functionally interact with calcineurin signalling utilizes our discovery that calcineurin and the Pmk1 MAPK signalling function antagonistically in the Cl- homeostasis of fission yeast. Using this approach, we identified pmp1+ encoding MAPK phosphatase, pek1+ encoding MAPK kinase, and rnc1+, encoding an RNA-bindign protein. We also demonstrated that Pmp1 dephosphorylates and inactivates Pmk1, thereby negatively regulates Pmk1 siganlling. Rnc1 is a KH-type RNA-binding protein that binds and stabilizes the Pmp1 mRNA, thus acting as a negative feedback regulator of MAPK signalling.

钙调蛋白（蛋白质磷酸酶2B）是Ca2+/钙调蛋白控制下唯一的丝氨酸/苏氨酸磷酸酶，是信号传递中的重要介体，将CA2+依赖性信号连接到各种细胞反应。钙调蛋白是裂变酵母中免疫抑制剂FK506的体内靶标，并且通过基因破坏或通过向培养基中添加FK506对钙调蛋白活性抑制不会影响裂变酵母的营养生长。我们进行了一个遗传筛查，以寻找对免疫抑制药物FK506敏感的突变，并确定了八个互补组。我们确定了pi4p5k YPT3小GTPase，网状蛋白适应器复合物的亚基，并证明这些分子和钙调蛋白在细胞因子，细胞完整性和膜运输等细胞事件中具有重要功能。我们还确定了钙林林的下游靶标。 PRZ1，锌指转录因子。 PRZ1被证明是通过钙调神经蛋白去磷酸化的，其转录活性也受钙调神经素介导的信号传导的控制。鉴定在功能上与钙调神经蛋白信号传导相互作用的分子的另一种方法利用了我们的发现，即钙调蛋白和PMK1 MAPK信号传导在裂变酵母的Cl-稳态中拮抗作用。使用这种方法，我们确定了编码MAPK磷酸酶，PEK1+编码MAPK激酶和RNC1+的PMP1+编码RNA-BINGIGN蛋白。我们还证明了PMP1脱磷酸化并灭活PMK1，从而负调节PMK1 Siganlling。 RNC1是一种KH型RNA结合蛋白，可结合和稳定PMP1 mRNA，因此充当MAPK信号传导的负反馈调节剂。