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，是信号传递的重要介质，将Ca2+依赖性信号传导到各种细胞反应中。钙调磷酸酶是免疫抑制药物FK506在裂变酵母体内的靶点，通过基因破坏或在培养基中添加FK506抑制钙调磷酸酶活性并不影响裂变酵母的营养生长。我们进行了遗传筛选，以寻找对免疫抑制药物FK506敏感的突变，并确定了8个互补组。我们鉴定出PI4P5K Ypt3小GTPase，网格蛋白接头复合物的亚基，并证明这些分子和钙调磷酸酶在细胞分裂、细胞完整性和膜运输等细胞事件中具有重要功能。我们还发现了一个下游靶点calcinerin；Prz1，锌指型转录因子。Prz1可被钙调磷酸酶去磷酸化，其转录活性也受钙调磷酸酶介导的信号传导控制。另一种鉴定与钙调磷酸酶信号相互作用的分子的方法是利用我们的发现，钙调磷酸酶和Pmk1 MAPK信号在裂变酵母的Cl-稳态中起拮抗作用。使用这种方法，我们鉴定了编码MAPK磷酸酶的pmp1+，编码MAPK激酶的pek1+和编码rna结合蛋白的rnc1+。我们还证明Pmp1使Pmk1去磷酸化并失活，从而负向调节Pmk1信号。Rnc1是一种kh型rna结合蛋白，结合并稳定Pmp1 mRNA，从而作为MAPK信号传导的负反馈调节因子。

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.