FAP-1去磷酸酶為Mallory- Denk Body形成過程之ㄧ新穎調控因子

Fas-Associated Phosphatase-1 as a Novel Regulator for Mallory-Denk Body Formation

陳姿蓉(Tzu- Jung Chen)

指導教授 ： 葉秀慧

國立臺灣大學/醫學院/微生物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.03368

摘要

Fas-associated phosphatase-1 (FAP-1) 為一個大型的非穿膜型酪胺酸去磷酸酶。本實驗室先前之研究發現FAP-1位於一段常發生於肝癌中缺失之染色體片段─4q22上。我們並且發現在約50%之肝癌組織中FAP-1 RNA的表現量有明顯下降之趨勢，因此指出FAP-1在肝臟中可能扮演抑癌基因的角色，然而目前仍不清楚FAP-1在肝臟之病理機轉中所扮演的功能為何。為了瞭解其功能，本實驗室先前利用yeast-two hybrid找尋FAP-1在肝細胞中與之有交互作用之蛋白質，並且發現一個與FAP-1有交互作用的新蛋白質－角蛋白18 (keratin 18, K18)。角蛋白18與角蛋白8 (keratin 8, K8)為構成肝細胞內中間絲的組成成分，其對於肝細胞之結構極為重要。K8及K18並且是許多肝炎中會出現的不正常蛋白堆疊物─ Mallory- Denk body (MDB)的主要組成成分。
因此，在本研究中，我們著重於探討FAP-對K8與K18之調控，並且探討FAP-1參與在MDB形成過程之可能性及其分子機轉。我們首先利用loss of function之方式，在一株高度表現FAP-1之肝癌細胞株PLC5中探討FAP-1對於肝細胞中之K8及K18有何影響。我們發現當細胞中的FAP-1表現量降低時，K8的蛋白質穩定度上升並造成K8蛋白質表現量之累積。於此同時，我們並且發現當FAP-1表現量下降時，K8在絲胺酸73 (Ser73)位點上的磷酸化會明顯上升。此外，我們亦發現當FAP-1表現量下降後細胞中之transglutaminase 活性上升。而值得注意的是，K8之蛋白質表現量上升、K8絲胺酸73位點磷酸化上升及transglutaminase 活性上升皆為參與在MDB形成過程之要因。因此我們進一步探討FAP-1參與在MDB形成的可能性，在利用西方點墨法及免疫螢光染色的實驗中，皆指出FAP-1參與在MDB之形成過程。因此本研究提出FAP-1為一個新的MDB形成過程之調控因子。最後，在本研究中，我們並嘗試找出FAP-1調控絲胺酸73磷酸化及transglutaminase活性之訊息傳導路徑來了解FAP-1可能在肝臟中扮演的功能為何，而初步之結果顯示FAP-1可能透過p38之訊息傳導路徑調控K8於絲胺酸73位點之磷酸化。總結上述之結果，本研究提出FAP-1是一個新的MDB之調控因子，其藉由影響 K8 S73 的磷酸化與transglutaminse的活性來調控MDB在肝臟細胞中的形成，未來我們將在臨床檢體中對FAP-1此角色作進一步之確認。

關鍵字

FAP-1 ； MDB ； 角蛋白8/18 ； transglutaminse ； p38

並列摘要

Fas-associated phosphatase-1 (FAP-1) is a large non-transmembrane protein tyrosine phosphatase. In our previous study, FAP-1 was identified as a putative tumor suppressor gene at chromosome 4q22, a region with common allelic loss in most HCCs. The RNA expression of FAP-1 is significantly decreased in more than 50% of HCC, but its function in liver pathogenesis is remained unclear. To address this issue, we took the approach by searching for its associated protein(s) with specific functions in hepatocytes. Aided by yeast two- hybrid analysis, we have previously identified keratin 18 (K18) as a novel interacting protein for FAP-1, which together with keratin 8 (K8) constitutes the major components of the intermediate filaments of hepatocytes. The K8/K18 intermediate filaments not only contribute to the mechanical integrity of cells, they are also the major components of Mallory- Denk body (MDB), the abnormal protein aggregates identified in hepatocytes of many liver diseases.
In the current study, we focused to explore the role of FAP-1 in regulating the function of K18 and K8, and for its involvement in MDB formation. We first took the loss of function approach to examine the effect of FAP-1 on K8/K18 in PLC5 hepatoma cell line, which expresses high level of endogenous FAP-1. Knocking down of FAP-1 caused the accumulation of K8 protein through increasing the protein stability; and the phosphorylation of K8 at Ser73 was increased at the same time. Intriguingly, we found that si-FAP-1 can also enhance the transglutaminse activity. It is noteworthy that the upregulation of K8, increase of phosphorylation K8 at Ser73, and also the higher transglutaminse activity, all contribute to the MDB formation. Our results thus raised a possibility for FAP-1 as a novel regulator for MDB in hepatocytes, which has been supported by our results showing that si-FAP-1 can induce the MDB in PLC5 cells. Finally, we have tried to identify the signaling pathway(s) involved in regulating the function of FAP-1, for the phosphorylation of K8 at Ser73 and the transglutaminse activity. The preliminary results suggested p38 as one candidate kinase involved in regulating FAP-1 for the phosphorylation of K8 at Ser73. In conclusion, the current study has identified FAP-1 as a novel regulator in regulating MDB formation, through its influence on the phosphorylation of K8 at Ser73 and transglutaminse activity in hepatocytes, and the implication in clinical specimens warrants further investigation.

並列關鍵字

FAP-1 ； MDB ； keratin 8/18 ； transglutaminse ； p38

參考文獻

[1] T. Sato, S. Irie, S. Kitada, and J. C. Reed, "FAP-1: a protein tyrosine phosphatase that associates with Fas," Science, vol. 268, pp. 411-5, Apr 21 1995.

[2] F. Villa, M. Deak, G. B. Bloomberg, D. R. Alessi, and D. M. van Aalten, "Crystal structure of the PTPL1/FAP-1 human tyrosine phosphatase mutated in colorectal cancer: evidence for a second phosphotyrosine substrate recognition pocket," J Biol Chem, vol. 280, pp. 8180-7, Mar 4 2005.

[3] O. D. Abaan and J. A. Toretsky, "PTPL1: a large phosphatase with a split personality," Cancer Metastasis Rev, vol. 27, pp. 205-14, Jun 2008.

[4] G. Bompard, M. Martin, C. Roy, F. Vignon, and G. Freiss, "Membrane targeting of protein tyrosine phosphatase PTPL1 through its FERM domain via binding to phosphatidylinositol 4,5-biphosphate," J Cell Sci, vol. 116, pp. 2519-30, Jun 15 2003.

[5] W. A. Kimber, M. Deak, A. R. Prescott, and D. R. Alessi, "Interaction of the protein tyrosine phosphatase PTPL1 with the PtdIns(3,4)P2-binding adaptor protein TAPP1," Biochem J, vol. 376, pp. 525-35, Dec 1 2003.