| Summary: | 博士 === 國立陽明大學 === 微生物及免疫學研究所 === 96 === Abstract
The promyelocytic leukemia protein (PML) is a tumor suppressor. It is involved in many different physiological functions but the complicated action mechanisms are not yet fully understood. PML localizes in the nucleus to form speckles called PML nuclear bodies, containing regulatory proteins, such as p53, Daxx, CREB binding protein (CBP) and small ubiquitin-related modifier(SUMO-1). Nuclear factor of activated T cell (NFAT) is an important transcription factor, regulates many key T cell functions. In this study, we found that NFAT is an unexpected partner of PML: PML specifically enhanced the transcription activation of NFAT in T cells and 293 cells. In PML-null mouse embryonic fibroblasts, no transcription activity of NFAT could be detected, while PML expression restored NFAT activation. In constrast, PML was not regulated for NF-�羠 transcription. Sumoylation of PML was essential for PML to promote the transcription activity of NFAT. The additive effect of PML was not due to sequestration of Daxx by PML. There was a selective requirement of PML isoform in NFAT activation. PML-I and PML-VI, but not PML-IV, enhanced NFAT transactivation. The potential interaction between NFAT and PML was demonstrated by co-immunoprecipitation of NFATc with PML-I, PML-IV, or PML-VI. GST pull down assay further illustrated a direct binding between the NFAT-homology region (NHR) of NFATc and the RING finger, B-boxes of PML. Inside nucleus of activated T cells, NFATc was found co-localized with PML speckles. Even though PML overexpression did not increase nuclear localization of NFATc, knockdown of PML was moderately reduced nuclear presence of NFAT. Some, but not all, of NFAT activity-mediated genes expression were regulated by PML. The interaction of PML with NFATc in vivo was further confirmed by chromatin immunoprecipitation (ChIP) and DNA affinity precipitation assay (DAPA) analysis. Knockdown of PML was also associated with reduced promoter binding of NFAT and CBP. It is suggested that part of mechanisms of underlying enhancement of NFATc transactivation mediated by PML is due to stabilization of NFAT-CBP transcriptional complex. The unexpected coupling of PML with NFAT reveals a novel mechanism underlying the diverse physiological functions of PML.
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