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

钙调磷酸酶（蛋白磷酸酶2B）是唯一受Ca 2 +/钙调蛋白控制的丝氨酸/苏氨酸磷酸酶，是信号传递中的重要介质，将Ca 2+依赖性信号传导与多种细胞反应联系起来。钙调磷酸酶是免疫抑制剂FK 506在裂殖酵母中的体内靶点，通过基因破坏或向培养基中加入FK 506抑制钙调磷酸酶活性不会影响裂殖酵母的营养生长。我们进行了遗传筛查，以寻找对免疫抑制药物FK 506敏感的突变，并确定了8个互补组。我们鉴定了PI 4P 5 KYpt 3小GT3，网格蛋白-接头复合物的亚基，并证明这些分子和钙调神经磷酸酶在细胞事件中共享基本功能，如胞质分裂，细胞完整性和膜运输。我们还确定了calcinerin的下游靶点; Prz 1，一种锌指型转录因子。Prz 1被钙调神经磷酸酶去磷酸化，其转录活性也受到钙调神经磷酸酶介导的信号转导的控制。另一种鉴定与钙调神经磷酸酶信号传导功能相互作用的分子的方法利用了我们的发现，即钙调神经磷酸酶和Pmk 1 MAPK信号传导在裂殖酵母的Cl-稳态中具有拮抗作用。使用这种方法，我们鉴定了编码MAPK磷酸酶的pmp 1+、编码MAPK激酶的pek 1+和编码RNA结合蛋白的rnc 1+。我们还证明了Pmp 1去磷酸化和失活Pmk 1，从而负调控Pmk 1信号转导。Rnc 1是一种KH型RNA结合蛋白，可结合并稳定Pmp 1 mRNA，从而作为MAPK信号传导的负反馈调节剂。

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.