Functional analysis of cell cycle-related protein kinase in terminally differentiated neurons

pcaire是cdc2相关激酶亚家族的成员，已被证明在有丝分裂后的细胞中优先表达。为了研究PCTAIRE的神经功能，分离了编码PCTAIRE 1、2和3 （PCTAIRE）的大鼠cDNA克隆，并分析了它们在脑中的表达模式。在这三种PCTAIRE中，只有PCTAIRE 2在大脑中特异表达，且其表达在大脑发育过程中短暂增加，在出生后7 ~ 15天达到峰值。在大脑内，PCTAIRE 2主要集中在海马和嗅球的神经元层，这些神经元层主要由有丝分裂后神经元组成。在免疫细胞化学实验中，PCTAIRE 2在细胞体和神经元的延伸神经突中检测到免疫反应性，但在星形胶质细胞中未检测到。PCTAIRE 2蛋白以颗粒形式被回收，并且抵抗非离子洗涤剂的增溶作用，这表明PCTAIRE 2可能是一个大的蛋白质复合物。免疫沉淀分析显示PCTAIRE 2蛋白与组蛋白H1的丝氨酸/苏氨酸磷酸化活性相关，其活性依赖于与一些在高盐条件下可以释放的调节伙伴的关联。这些发现表明PCTAIRE 2是一种丝氨酸/苏氨酸激酶，可能在终末分化的神经元中发挥独特的作用。为了阐明PCTAIRE 2的神经功能，通过酵母双杂交筛选研究了与PCTAIRE 2相关的蛋白。发现一个阳性克隆编码一种新的蛋白，可以在体外和体内与PCTAIRE 2相互作用，并被命名为Trap （Tudor Repeat Associator with PCTAIRE 2）。Trap的整体结构与任何蛋白均无明显的同源性，但含有果蝇都铎蛋白和a激酶锚定蛋白中保守的5个重复结构域（tudor-like repeat）。Trap通过含有两个都铎重复序列的c端结构域与PCTAIRE 2的n端结构域结合。PCTAIRE 1，但不是PCTAIRE 3，也可以与Trap相关联。Trap主要在大脑和睾丸中表达，并在发育过程中逐渐增加，与PCTAIRE 2的表达模式一致。PCTAIRE 2和Trap的免疫反应在COS7细胞的神经元细胞体和线粒体上共定位。免疫组化分析显示PCTAIRE 2和Trap分布于大脑皮层和小脑的同一细胞层。这些发现表明，Trap在终末分化神经元中作为PCTAIRE 2的生理伴侣。少

PCTAIREs are members of a subfamily of Cdc2-related kinases that have been shown to be preferentially expressed in post-mitotic cells. To examine the neural functions of PCTAIREs, rat cDNA clones encoding PCTAIRE 1, 2, and 3 (PCTAIREs) were isolated and their expression patterns in the brain were analyzed. Among the three PCTAIREs, only PCTAIRE 2 was found to be specifically expressed in the brain, furthermore its expression was transiently increased during brain development, peaking from 7 to 15 days after birth. Within the brain, PCTAIRE 2 was concentrated in the neuronal layers of the hippocampus and olfactory bulb, which mostly consist of post-mitotic neurons. In an immunocytochemical experiment, immunoreactivity for PCTAIRE 2 was detected in the cell bodies and extended neurites of neurons, but not in astrocytes. The PCTAIRE 2 protein was recovered in particulate fraction and resistant to solubilization with nonionic detergent, suggesting that PCTAIRE 2 might be present as a compo … More nent of a large protein complex. An immunoprecipitation assay revealed that the PCTAIRE 2 protein was associated with Ser/Thr phosphorylating activity for histone H1 and that its activity depended on association with some regulatory partner that can be released under high salt conditions. These findings suggest that PCTAIRE 2 is a Ser/Thr kinase that might play a unique role in terminally differentiated neurons. To elucidate the neural function of PCTAIRE 2, proteins that can associate with PCTAIRE 2 were surveyed by yeast two-hybrid screening. A positive clone was found to encode a novel protein that can interact with PCTAIRE 2 in vitro and in vivo, and was designated as Trap (Tudor Repeat Associator with PCTAIRE 2). Whole structure of Trap shows no significant homology to any proteins, but contains five repeated domains (tudor-like repeat) conserved in Drosophila tudor protein and A-kinase anchoring protein. Trap associates with N-terminal domain of PCTAIRE 2 through its C-terminal domain containing two tudor-like repeats. PCTAIRE 1, but not PCTAIRE 3, can also associate with Trap. Trap is predominantly expressed in brain and testis, and gradually increases during development in consistent with the expression patterns of PCTAIRE 2. Immunoreactivities for PCTAIRE 2 and Trap were colocalized on the cell body of neurons and on mitochondria in COS7 cells. Immunohistochemical analyses showed that PCTAIRE 2 and Trap were distributed in the same cell layer of the cerebral cortex and cerebellum. These findings suggest that Trap acts as a physiological partner of PCTAIRE 2 in the terminal differentiated neurons. Less