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

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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