BTB-kelch蛋白DIP-2的表現及功能特性分析

DIP-2為BTB-kelch蛋白家族的一員。許多BTB-kelch蛋白已被發現高度表現於神經系統中，並參與包括神經型態生成以及突觸訊息傳遞在內的神經發育機轉。但就DIP-2而言，儘管我們已發現它可作為Cul3泛素接合酶與受質間的轉接者，進而導致死亡相關蛋白激酶DAPK以及抑癌蛋白PML的泛素化和降解，但對於DIP-2的表現分佈以及其詳細的功能機制所知甚少。本篇論文中，我們觀察DIP-2於成鼠腦部的表現情形，發現DIP-2主要分佈於大腦皮層、海馬迴、嗅球及小腦皮層等區域。值得注意的是，DIP-2於絕大多數區域呈現以細胞體和樹突為主的表現型態，但於海馬迴CA3處則特別表現於軸突中。另一方面，為求更進一步瞭解DIP-2的功能，本篇論文亦深入探討DIP-2對PML進行蛋白降解的調控機制，從而發現細胞週期素依賴性激酶(CDKs)參與DIP-2對PML的調控。我們證實PML確實為CDK1/2/4/6的受質，而Ser 518則為主要發生磷酸化的殘基。實驗結果顯示，這些CDK都參與由DIP-2所誘發的PML蛋白降解。總的來說，這項研究不僅呈現DIP-2蛋白於鼠腦中的表現型態特徵，亦將DIP-2所媒介的PML蛋白降解與細胞週期對PML的調控機制作緊密結合，為抑癌蛋白PML穩定性的研究提供新的一層認知。

關鍵字

降解；泛素化；腦；細胞週期素依賴性激酶DIP-2 ； Pin1

並列摘要

DIP-2 is a member of the BTB-kelch protein family, of which several members are highly expressed in the nervous system and have been demonstrated to elicit neuronal functions, including neural morphogenesis and synapse transmission. However, little is known about the expression patterns of DIP-2 and its biological functions, although previous studies in our laboratory revealed that DIP-2 functions as a substrate adaptor of Cul3-based ubiquitin ligase to promote the ubiquitination of DAPK and PML. Here, we characterized the expression pattern of DIP-2 protein in the adult mouse brain and found its high expression levels in the cerebral cortex, the hippocampus, the olfactory bulb and the cerebellar cortex. Notably, DIP-2 displays both somatic and dendritic patterns in most regions observed, whereas it exhibits an axonal pattern in CA3 of hippocampus. In an attempt to study the function of DIP-2, we investigated the mechanism through which DIP-2 mediates PML protein degradation. First, we identified a role of CDK in DIP-2-mediated PML degradation. Furthermore, we demonstrated PML as the substrate of CDK1/2/4/6 in vitro and the Ser 518 residue as the major phosphorylation site. Finally, we showed that the each of these CDKs contributes to PML degradation induced by DIP-2. Together, this study not only presents the expression features of DIP-2 in mouse brain, but also provides a linkage between DIP-2-mediated PML degradation and cell cycle-dependent regulation of PML, thus adding another aspect of proper control for the stability of tumor suppressor PML protein.

並列關鍵字

brain ； BTB-kelch ； CDK ； DIP-2 ； Pin1 ； PML

參考文獻

Adams J, Kelso R, Cooley L (2000) The kelch repeat superfamily of proteins: propellers of cell function. Trends Cell Biol 10(1): 17-24

Alcalay M, Tomassoni L, Colombo E, Stoldt S, Grignani F, Fagioli M, Szekely L, Helin K, Pelicci PG (1998) The promyelocytic leukemia gene product (PML) forms stable complexes with the retinoblastoma protein. Mol Cell Biol 18(2): 1084-1093

Allen E, Ding J, Wang W, Pramanik S, Chou J, Yau V, Yang Y (2005) Gigaxonin-controlled degradation of MAP1B light chain is critical to neuronal survival. Nature 438(7065): 224-228

Alsheich-Bartok O, Haupt S, Alkalay-Snir I, Saito S, Appella E, Haupt Y (2008) PML enhances the regulation of p53 by CK1 in response to DNA damage. Oncogene 27(26): 3653-3661

Bernardi R, Scaglioni PP, Bergmann S, Horn HF, Vousden KH, Pandolfi PP (2004) PML regulates p53 stability by sequestering Mdm2 to the nucleolus. Nat Cell Biol 6(7): 665-672

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BTB-kelch蛋白DIP-2的表現及功能特性分析

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