人類Ninein蛋白調控機制及其功能性之研究:Ninein蛋白及其連結蛋白在中心體上所扮演的角色探討

Regulation and function of hNinein: A role for a hNinein and its partners in centrosome

鄭大山(Tai-Shan Cheng)

指導教授 ： 洪純隆

高雄醫學大學/醫學院/醫學研究所/博士(2007年)

https://doi.org/10.6832/KMU.2007.00142

摘要

人類中心體蛋白Ninein(hNinein)已被指出參與了中心體放射出之微管束生成及連結固定，並可能同時扮演著支架蛋白(scaffold protein)的角色，但是其在中心體上之基本架構及生理功能仍有待釐清，因此需篩選及了解更多的hNinein接合蛋白用以獲知hNinein在細胞中完整的功能。
第一章，我們發現SUMO-1、Ubc9 (SUMO-conjugating E2 enzyme)和PIAS1及PIASxα (SUMOylation enhancing enzymes)為hNinein之接合蛋白，更進一步我們證實了在試管反應下hNinein的C端片段可被SUMO修飾(SUMOylation)，並且在細胞中表達hNinein的重組蛋白GFP-hNinein亦觀察到相同的情形，此成果為首次揭露出中心體核心結構蛋白有被SUMO修飾之現象，而經SUMO修飾之hNinein蛋白將會由中心體移轉至核內，藉由此結果我們推測SUMO修飾hNinein或直接修飾其他中心體蛋白將可影響中心體之活性及功能。
第二章，我們詳細地描述hNinein如何與紡錘絲連結蛋白(spindle-associated protein)Astrin相結合，其於細胞周期中之S phase及 G2 phase同時結合座落在中心體上而分離於M phase，我們亦利用顯著抑制型(dominant-negative mutant)之hNinein片段重組蛋白進行研究，結果顯示此一顯著抑制型蛋白會抑制Astrin座落在中心體上，相同的結果亦顯示於hNinein siRNA的實驗中，然而壓抑Astrin之表現並不會影響hNinein於細胞中之位置，更進一步觀察缺少hNinein且處於M phase的細胞，Astrin不但無法與紡錘絲相連結且紡錘絲呈現不正常排列。綜合上述結果，我們推測在細胞周期之S phase及 G2 phase時期中hNinein為Astrin座落在中心體上所必須，藉由此推論我們假設出一個調控模式，即在細胞周期中hNinein不但與Astrin相結合並調控著Astrin之運行，且此一機制亦參與調控中心體及紡錘絲之完整性。
第三章，我們針對hNinein結合酵素GSK3β與Astrin之關係進行研究，結果指出Astrin不僅可與GSK3β相連結且為GSK3β之受質，抑制GSK3?狺宏簿嶼〝吤蝳P時干擾了Astrin與紡錘絲相連結，然而壓抑Astrin之表現並不會造成GSK3β紡錘絲位置的變化。另一方面有趣的是，Astrin會增強GSK3β之酵素活性，增加GSK3β磷酸化不同受質的能力。總而言之GSK3β會與Astrin相連結且磷酸化Astrin，且此一反應為Astrin座落在紡錘絲所必須，相對的， Astrin會增強GSK3β之酵素活性以利紡錘絲之組成及可能參與了阿茲海默症的生成。
而在未來我們將試圖勾畫出hNinein、GSK3β和Astrin三者在中心體之功能及紡錘絲之生成的互動藍圖。

關鍵字

中心體 ； 紡錘體 ； 細胞周期 ； 有絲分裂 ； Ninein ； SUMO ； Astrin ； GSK3beta

並列摘要

Human Ninein (hNinein), a centrosomal-associated protein, is implicated in centrosomal microtubule nucleation and microtubule anchoring in interphase cells and may act as a scaffold protein, but the underlying structure and physiological functions remain unexplored in the centrosome. A full appreciation of how hNinein contribute to cellular functions requires the isolation and characterization of unknown hNinein-associated proteins.
In the first part, we identify the SUMO-1, SUMO-conjugating E2 enzyme, Ubc9, and SUMOylation enhancing enzymes, including PIAS1 and PIASxα, as binding partners of hNinein. In SUMOylation assay, the C-terminal region of hNinein can be SUMOylated in vitro and in vivo. Our findings firstly place SUMOylation target on the centrosome structure protein hNinein, resultting in the switch localization from centrosome to nucleus. Overall, our data suggest the importance of the SUMOylation of hNinein and probably other centrosomal proteins may also be involved in the centrosome activity.
In the second part, we show clearly that a spindle-associated protein, Astrin, interacts with hNinein at the centrosome during the S and G2 phases, and this complex may dissociate in the M phase. We also demonstrate that the truncated forms of hNinein, which could interfere with ??-tubulin and function as dominant-negative mutants, are able to affect Astrin localization to the centrosome. Moreover, siRNA-mediated knockdown of hNinein in HeLa cells causes Astrin to fail to target to the centrosome, whereas hNinein can localize at the centrosome in the absence of Astrin. In addition, reduction in hNinein protein levels causes mislocalization of Astrin with the spindle apparatus, resultting in the formation of an aberrant mitotic spindle. Collectively, these data suggest that hNinein is required for targeting Astrin to the centrosome during the S and G2 phases. We therefore propose a model wherein hNinein regulates the dynamic movement of Astrin throughout the cell cycle and this interaction, in turn, is required for maintenance of centrosome/spindle pole integrity.
In the third part, we report that GSK3β interacts with the spindle associated protein Astrin both in vitro and in vivo throughout the cell cycle. Inhibition of GSK3β impaired spindle accumulation of Astrin and spindle formation at mitosis, suggesting that GSK3β-mediated phosphorylation is required for the targeting of Astrin to the spindle microtubules. Conversely, depletion of Astrin by siRNA has no detectable influence on the localization of GSK3β. Interestingly, in vitro assays using recombinant proteins directly demonstrated that Astrin enhances GSK3β-mediated phosphorylation of various substrates. Moreover, we show that co-overexpression of Astrin and GSK3β differentially increase GSK3β-mediated Tau phosphorylation on unprimed site. Collectively, these data indicate that GSK3β interacts with and phosphorylates the spindle-associated protein Astrin, result in targeting Astrin to the spindle MTs. In turn, the GSK3β-Astrin complex may facilitate both physiological and pathological phosphorylation.
Future investigations should be aimed at understanding whether the interplay of these three proteins, hNinein, Astrin and GSK3β, involved in centrosome function and spindle microtubule assembly.

並列關鍵字

centrosome ； spindle ； cell cycle ； mitosis ； Ninein ； SUMO ； Astrin ； GSK3beta

參考文獻

Abal, M., Piel, M., Bouckson-Castaing, V., Mogensen, M., Sibarita, J. B., and Bornens, M., 2002. Microtubule release from the centrosome in migrating cells. J. Cell Biol. 159, 731-737

Ahn, J.H., Xu, Y., Jang, W.J., Matunis, M.J., Hayward, G.S., 2001. Evaluation of interactions of human cytomegalovirus immediate-early IE2 regulatory protein with small ubiquitin-like modifiers and their conjugation enzyme Ubc9. Journal of Virology 75, 3859– 3872.

Azimzadeh, J. and Bornens, M. 2007 Structure and duplication of the centrosome. J Cell Sci. 120, 2139-42.

Baron, A. T., Greenwood, T. M., Bazinet, C. W., and Salisbury, J. L., 1992. Centrin is a component of the pericentriolar lattice. Biology of the Cell 76, 383– 388.

Bouckson-Castaing, V., Moudjou, M., Ferguson, D. J., Mucklow, S., Belkaid, Y., Milon, G., and Crocker, P. R., 1996. Molecular characterisation of ninein, a new coiled-coil protein of the centrosome. J. Cell Sci. 109, 179–190.