鑑定高分子量Phostensin與其結合蛋白並探討其在細胞胞吞運輸上之功能

Phostensin-α為KIAA1949 基因所轉錄的蛋白質含有165胺基酸，原先鑑定為一型蛋白質磷酸水解酶 (PP1) 結合蛋白。過去研究分析其蛋白質序列在C端具有一肌動蛋白的結合序列，為F-型肌動蛋白(-)端的帽蓋蛋白，具有調節肌動蛋白變化之功能。Phostensin 基因位在人類的第六號染色體上介於HLA-C和HLA-E基因叢之間，其mRNA主要分布在脾臟胸腺和週邊血液白血球。本篇研究中首先利用會辨識phostensin的單株抗體(PT2)研究其在不同類型白血球中及在細胞內的位置的分佈。結果發現phostensin主要分布於細胞邊緣的細胞骨架上並且可以在輔助型Ｔ淋巴球、毒殺型T淋巴球、單核球、巨噬細胞、B淋巴球、自然殺手細胞、顆粒球這些不同類型的白血球中表現；因此我們認為phostensin是一個在白血球中普遍分佈並可能具有調節白血球細胞功能的蛋白質。為了了解phostensin在細胞層級功能，我們利用共免疫沉澱合併蛋白質體學的方法尋找phostensin結合蛋白。我們鑑定出Eps15同源结構蛋白1 (EHD1)、EHD4、肌動蛋白、一型蛋白質磷酸水解酶 (PP1-α)與一個高分子量的phostensin (phostensin-β)可被PT2沉澱下來。Phostensin-β亦是由KIAA1949所轉錄之產物，由613個胺基酸組成，但其在十二烷基硫酸鈉聚丙烯酰胺凝膠電泳(SDS-PAGE)所顯示之分子量卻是遠大於理論值的110kD。Phostensin-α氨基酸序列是在phostensin-β的C-端區段。然而，Phostensin-α不是由phostensin-β降解而來。Phostensin-β亦會與PP1和actin結合，在多種細胞株中分佈。Phostensin與EHD蛋白的聚集亦被免疫螢光顯微鏡觀察到並可共聚在胞吞泡囊上，因EHD蛋白在胞吞泡囊運輸上扮演重要調控角色，我們的結果推測phostensin可能在細胞內參與胞吞運輸作用。

關鍵字

第一型蛋白質磷酸水解脢

並列摘要

Phostensin-α is a protein of 165 amino acids encoded by KIAA1949, and originally identified as a protein phosphatase-1(PP1) binding protein. It also contains one actin-binding motif at its C-terminal region and binds to the pointed ends of actin filaments, modulating actin dynamics. The genomic location of phostensin is between the HLA-C and HLA-E gene clusters on human chromosome 6, and its mRNA is predominantly distributed in the spleen, thymus, and peripheral leukocytes. In this study, an anti-phostensin monoclonal antibody (PT2) was used to examine the subcellular localization and distribution of phostensin in white blood cell populations. Our results indicated that phostensin is mainly concentrated at the cell periphery and co-localizes with actin filaments. In addition, phostensin was abundant in helper T-lymphocytes, cytotoxic T-lymphocytes, mature monocytes, macrophages, B-lymphocytes, natural killer cells, and granulocytes. Taken together, phostensin is a ubiquitous protein in leukocytes, and it may play a role in modulating the cellular functions of leukocytes. In order to explore the cellular functions of phostensin, co-immunoprecipitation in combination with proteomic approach was applied to search phostensin-binding proteins. Eps 15 homology domain-containing protein 1 (EHD1), EHD4, actin, PP1 and a high-molecular weight of phostensin (phostensin-β) were co-immunoprecipitated by PT2. Phostensin-β is also encoded by KIAA1949 and consists of 613 amino acids with an apparent molecular weight of 110 kDa on SDS-PAGE. The sequence of phostensin-α is the C-terminal region of phostensin-β. However, phostensin-α is not degraded from phostensin-β. Phostensin-β is capable of associating with PP1 and actin, and is present in many cell lines. Phostensin associated with EHD proteins was also observed by immunofluorescence microscopy. Our results also demonstrated that phostensin/EHD protein complex is co-localized at the endocytic vesicles. Since EHD proteins play a critical role in regulation of endocytic transport, our results suggested that phostensin may participate in endocytic trafficking.

並列關鍵字

Protein Phosphatase 1 ； KIAA1949 ； Phostensin

參考文獻

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Blume JJ, Halbach A, Behrendt D, Paulsson M, Plomann M (2007) EHD proteins are associated with tubular and vesicular compartments and interact with specific phospholipids. Exp Cell Res 313: 219-231

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鑑定高分子量Phostensin與其結合蛋白並探討其在細胞胞吞運輸上之功能

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