Mechanism of the induction of zif268 gene expression by neuro-transmitter receptors and function of the zif268 gene product

由该基金资助的研究重点是zif268的调控和功能，zif268是一种快速诱导基因，编码一种含锌指的转录因子，该因子被认为在神经元可塑性中发挥作用。本研究的两个目的是：1)分析在刺激PC12D细胞毒蕈碱乙酰胆碱受体后介导zif268基因表达的细胞内信号通路；2)研究猕猴视觉记忆获得与下颞叶皮层zif268表达之间的关系。本研究的主要发现如下：1)PC12D细胞中m1毒毒碱乙酰胆碱受体的刺激诱导zif268 mRNA的快速增加，并通过半独立的细胞内信号通路激活NAP激酶，该信号通路需要蛋白激酶C （PKC）和细胞内钙的增加。2)毒蕈碱受体介导的MAPK激活，而不是zif268基因的表达，更多的是，被H89部分阻断，H89是一种对蛋白激酶a具有高选择性的蛋白激酶抑制剂。3)蛋白激酶抑制剂H7阻断NGF、毒蕈碱受体激动剂、phorbol酯和增加细胞内钙的化合物对zif268 RNA的诱导，但不阻断这些药物对MAP激酶的激活。4)膜去极化浓度的KCl可有效激活MAPK，但仅刺激zif268 mRNA的少量增加。这些发现（2-4）表明MAPK激活和zif268 mRNA的增加并不总是相关的，这意味着MAPK激活和zif268基因表达之间的关系比目前大多数模型所提出的更为复杂。5)我们已经检测到一种新的激酶活性，它可以与从细胞中分离出来的受磷酸乙酯碳醇刺激的Raf-1共沉淀，而不是从受NGF或高浓度KC1刺激的细胞中分离出来的Raf-1。这种激酶活性能迅速磷酸化一个100 kDa的蛋白，磷酸化的峰值在丙二醇刺激后的30 - 60秒内达到。6)在与东京大学的H.Okuno和Y.and Miyashita的合作研究中，Zif268被证明在被训练执行配对联想任务的猕猴的下颞回神经元斑块中有选择性地表达，但在被训练执行视觉识别任务的猴子中没有。少

The research funded by this grant focused on the regulation and function of zif268, a rapidly inducible gene encoding a zinc finger-containing transcription factor that has been postulated to play a role in neuronal plasticity. Two goals of the present study were to :1) analyze the intracellular signalling pathways that mediate induction of zif268 gene expression following stimulation of muscarinic acetylcholine receptors in PC12D cells, and2) examine the relationship between the acquisition of visual memory and Zif268 expression in the inferior temporal cortex in Macaque monkey. The primary findings of this research are as follows :1) Stimulation of m1 muscarinic acetylcholine receptors in PC12D cells induces rapid increases in zif268 mRNA and activates NAP kinase via semi-independent intracellular signalling pathways that require protein kinase C (PKC) and increases in intracellular calcium.2) Muscarinic receptor^-mediated activation of MAPK activation, but not zif268 gene expression … More , was partially blocked by H89, a protein kinase inhibitor with high selectivity for protein kinase A.3) The protein kinase inhibitor H7 blocks induction of zif268 RNA induction by NGF,muscarinic receptor agonists, phorbol esters, and compounds that increase intracellular calcium, but does not block activation of MAP kinase by these agents.4) Membrane depolarizing concentrations of KCl efficiently activate MAPK,but stimulate only minor increases in zif268 mRNA.These findings (2-4) reveal that MAPK activation and increases in zif268 mRNA are not always correlated, and imply that the relationship between MAPK activation and zif268 gene expression is more complicated than proposed in most current models.5) We have detected a novel kinase activity that coprecipitates with Raf-1 isolated from cells stimulated with carbachol of phorbol esters, but not from cells stimulated with NGF or high concentrations of KC1. This kinase activity rapidly phosphorylates a 100 kDa protein, with the peak of phosphorylation being reached within 30 to 60 seconds following stimulation with carbachol.6) In collaborative work with H.Okuno and Y.and Miyashita (Tokyo University), Zif268 was shown to be selectively expressed in patches of neurons within the inferior temporal gyrus of Macaque monkeys that were trained to perform a pair^-association task, but not in monkeys that were trained to perform a visual discrimination task. Less