探討FAP-1去磷酸酶基因對於中間絲角質蛋白Keratin-8與Keratin-18的功能性影響

The Functional Effect of Fas-Associated Phosphatase-1 (FAP-1) on Keratin 8/18 Intermediate Filaments

盧珉如(Min-Ju Lu)

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(2008年)

https://doi.org/10.6342/NTU.2008.02581

摘要

Fas-associated phosphatase-1 (FAP-1)基因屬於protein tyrosine phosphatase的一員。根據本實驗室先前之位置選殖研究指出，FAP-1有可能是位於染色體4q21-23上之一可能參與肝癌之腫瘤抑制基因，但其參與肝癌過程真正的功能角色仍未釐清。
因為FAP-1結構中有很多protein-protein interacting domain，包括KIND domain、FERM domain、5個PDZ domain，於細胞中為一會與許多蛋白質交互作用之scaffold 蛋白。要了解FAP-1在肝臟疾病扮演的功能性角色，我們的策略是在肝細胞中尋找會與FAP-1有交互作用的蛋白質。本實驗室先前利用yeast two hybrid system在liver cDNA library以FAP-1的N’端為餌，找到一個新的FAP-1 associated protein，Keratin 18 (K18)，目前已知K18會與Keratin 8 (K8)形成非共價結合的heterodimer，共同組成肝細胞中主要的intermediate filaments (IFs)。

關鍵字

去磷酸 ； 中間絲 ； 角質蛋白

並列摘要

Fas-associated phosphates-1 (FAP-1) is a member of protein tyrosine phosphatases. According to our previous positional cloning studies, FAP-1 might serve as a potential candidate tumor suppressor gene at chromosome 4q21-23. But its function role in hepatocarcinogenesis still remains unknown.
FAP-1 contains several protein-protein interacting domains, including KIND domain, FERM domain, five PDZ domains, and thus might function as a scaffold protein. Therefore, aiming to study the functional role of FAP-1 in liver diseases, we took the approach by searching for its novel interacting protein in hepatocytes. Aided by yeast two hybrid screening of liver cDNA library, we have previously identified that Keratin 18 (K18) can interact with the N’ terminal domain of FAP-1. K18 forms the obligate non-covalent heterodimer together with its partner Keratin 8 (K8), which constitutes as the major component of the intermediate filaments (IFs) of hepatocytes.
To investigate the functional effect of FAP-1 on K8/K18 IFs, the specific objectives of this study contain (1) the effect of FAP-1 on K8/K18 protein expression and specific post translational modifications (PTM), mainly focused on phosphorylation and ubiquitination; (2) the molecular mechanism underlying how FAP-1 regulates K8/K18 IFs; (3) the functional effect of FAP-1 on the structure of K8/K18 IFs.
We took approach to knockdown the FAP-1 in PLC5 cells by lenti-si-RNA and then evaluate the resulting effect on the expression and PTMs of K8/K18 (loss of function approach). We found that FAP-1 knockdown can increase the protein level of K8/K18, but no significant effect on K8/K18 phosphorylation was identified. The results from quantitative PCR did not find obvious RNA changes in accordance with the changes of protein level, suggesting that FAP-1 regulates the protein level of K8/K18 might not only occur at the RNA level. Another line of evidence comes from the system we established in 293T cells, which express high level of endogenous FAP-1. By co-transfecting of K8 and K18 expression constructs into 293T cells, the K8/K18 IFs can be artificially estalished. Knockdown of FAP-1 in 293T cells caused dramatic enhance of K8/K18 protein expression level. Again, the results of quantitative PCR also supports that the effect on K8/K18 proteins might not only occur at RNA level.
To further address the molecular mechanisms underlying FAP-1 affects the protein level of K8/K18, we focused on its influence on the stability of K8/K18 proteins. The results both from cyclohexamide and MG132 treatments suggested that FAP-1 might affect the protein level through regulating their protein stability. Finally, to investigate the functional effect of FAP-1 on K8/K18 IFs, FAP-1 was transfected into Huh-7 cells, which did not express FAP-1, for its effect on the K8/K18 IFs. The preliminary results showed that overexpression of FAP-1 can change the structure of K8/K18 IFs, leading to peri-nuclear aggregations, which seems to be dependent on its phosphatase activity.
The results of current study supported the functional effect of FAP-1 on the protein level of K8/K18 and further suggested its influence on their protein stability as one possible mechanism. The functional role of FAP-1, through regulating the stability of K8/K18, in liver diseases is worthy to be further investigated.

並列關鍵字

FAP-1 ； Keratin-8 ； Keratin-18 ； intermediate filaments

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