TRIM28的研究：(1)TRIM28於肝癌臨床表現的關係 (2)TRIM28/S681和S757的磷酸化研究

Abstract

TRIM28已知為含有KRAB單元(Krüpple-associated box)的zinc finger抑制子的共通輔助抑制子。它藉由和異染色質蛋白1、組蛋白甲基轉移酶SETDB1以及兩種含有修飾染色質和乙醯化組蛋白活性的複合物N-CoR1和NuRD所組成之蛋白質複合體協調KRAB負責的轉錄抑制。TRIM28協調的抑制複合物和異染色質的形成緊密地調控基因表現。除此之外，TRIM28也參與細胞增殖和細胞分化，並對於老鼠胚胎的發育不可或缺。然而，目前對於TRIM28在癌細胞生長與侵犯能力(例如肝癌) 中扮演的角色仍有待研究。在這篇研究中，我們發現TRIM28在肝癌組織中之表現量顯著上升，並可能與肝癌細胞的異常生長密切相關。在TRIM28後轉譯修飾對其功能之影響方面，例如TRIM28第824個胺基酸絲胺酸的磷酸化已知會參與DNA受損反應，而第473個胺基酸絲胺酸的磷酸化則被證明會調節TRIM28和異染色質蛋白1的結合以調控與細胞週期相關的基因表現。TRIM28第681個和第757個胺基酸絲胺酸在HeLa細胞中被發現有磷酸化修飾，但其對TRIM28之功能影響尚待釐清。我們產生了針對這兩個位置磷酸化的多株抗體，發現這兩個磷酸化都只出現在HeLa細胞有絲分裂的初期。我們發現第681個絲胺酸被突變成丙胺酸的突變株(S681A)在細胞分裂期間展現和野生型相同的行為：在有絲分裂初期會和濃縮的染色體分離，但在有絲分裂末期則會和鬆散的染色質重新結合。至於第757個絲胺酸的磷酸化，則發現會被Cdk1的抑制物Roscovitine抑制，這意味著Cdk1極可能為該絲胺酸的上游激酶。

TRIM28 (tripartite motif protein 28) is a corepressor for large family of Krüpple-associated box domain containing zinc finger transcription factors and coordinates KRAB-mediated transcription repression by recruitment of HP1 (heterochromatin protein1), histone methyltransferase SETDB1, and two chromatin-remodeling and histone deacetylation complexes N-CoR1 and NuRD. TRIM28-mediated repression complexes and heterochromatin formation are tightly coordinated leading to silencing of genes. Moreover, it was also reported that TRIM28 is essential for cell proliferation, differentiation and early embryonic development in mice. To study the relationship between the expression of TRIM28 and the growth and invasiveness of hepatocellular carcinoma (HCC), I performed immunohistochemical staining of clinical specimens. TRIM28 is overexpressed in HCC and is likely to involve in its abnormal growth characteristics. Post-translational modifications have important functional impacts on TRIM28. For example, phosphorylation of TRIM28/S824 was shown to be involved in DNA-damaged response while phosphorylation at S473 modulates its interaction with HP1 to differentially regulate cell cycle-related genes. Recently, the phosphorylation of S681 and S757 were identified in HeLa cells, but their functions remain unknown. We generated polyclonal antibodies against phosphorylated TRIM28/Ser681 and Ser757. These phosphorylation events appeared at prometaphase of HeLa cells. Similar to wild type TRIM28, S681A mutant dissociates from condensed chromosomes in prophase and re-associates with chromatin in anaphase. The potential function of phospho-S681 is discussed. The phosphorylation of S757 was completely abolished after Roscovitine treatment. It is likely that Cdk1 is responsible for the phosphorylation of S757.