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

钙调神经磷酸酶2B是钙/钙调素调控下的唯一一种丝氨酸/苏氨酸磷酸酶，是信号传递的重要介体，将钙依赖的信号与多种细胞反应联系起来。钙调神经磷酸酶是免疫抑制剂FK506在裂解酵母中的体内靶点，通过基因干扰或在培养基中添加FK506来抑制钙调神经磷酸酶的活性并不影响分裂酵母的营养生长。我们进行了基因筛查，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。我们鉴定了PI4P5K Ypt3小GTP酶，这是笼蛋白-适配器复合体的亚单位，并证明了这些分子和钙调神经磷酸酶在细胞质分裂、细胞完整性和膜运输等细胞事件中共享重要功能。我们还确定了钙化蛋白的下游靶点：Prz1，一种锌指型转录因子。研究表明Prz1被钙调神经磷酸酶去磷酸化，其转录活性也受钙调神经磷酸酶介导的信号转导的控制。另一种识别与钙调神经磷酸酶信号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Pmk1 MAPK信号在分裂酵母的氯离子稳态中起拮抗作用。利用这种方法，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。我们还证明了Pmp1去磷酸化和失活Pmk1，从而负性调节Pmk1信号转导。Rnc1是一种KH型RNA结合蛋白，它结合并稳定Pmp1mRNA，从而作为MAPK信号的负反馈调节因子。

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.