- 學位論文
Tristetraprolin磷酸化與結合蛋白之研究
Functional regulation of tristetraprolin through protein phosphorylation and protein-protein interaction
摘要
Tristetraprolin (TTP)是一個核糖核酸(RNA)結合蛋白，可以調控含有多腺嘌呤尿嘧啶序列(AU-rich element)的核糖核酸的降解，且知TTP的功能受到結合蛋白的不同及本身蛋白磷酸化程度所調控。TTP會藉由CCR4-NOT脫腺苷化酶複合蛋白(CCR4-NOT deadenylase complex)進行核糖核酸的poly(A)降解。然而我們發現在CCR4-NOT腺苷化酶複合蛋白中的兩個腺苷化酶對於poly(A)降解是必要的。TTP的磷酸化除了已被廣泛研究的p38路徑外，ERK訊息傳遞路徑也可以去磷酸化TTP進而調控其穩定性、細胞內的位置和功能。先前利用質譜分析得知TTP的第316號絲氨酸會因ERK訊息傳遞路徑的活化而被磷酸化，且知第316號絲氨酸的磷酸化會降低TTP與CCR4-NOT腺苷化酶複合蛋白之間的結合。接著，我們進一步去研究TTP第316號絲氨酸磷酸化在生理條件下的功能及影響。在受到脂多醣(lipopolyssacharide,LPS)刺激的小鼠巨噬細胞RAW264.7中觀察到除了TTP的表現量增加外也伴隨著第316號絲氨酸被磷酸化，且此磷酸化降低了TTP和Cnot1蛋白的結合反而增加了和14-3-3的結合。另外，在脂肪前驅細胞3T3-L1的分化過程中利用MAPK去磷酸化酶(MKP-1)的抑制劑sanguinarine去促進ERK的活化和TTP第316號絲氨酸的磷酸化，進而抑制了3T3-L1脂肪前驅細胞的分化。此外同時也在NIH3T3胚胎纖維母细胞觀察到第316號絲氨酸的磷酸化使TTP較不容易進入細胞核。另外一方面，質譜分析發現和TTP結合的蛋白之一的組蛋白去乙酰酶(histone deacetylases3)是轉譯抑制分子，已知兩者皆參與負調控NF-κB訊息傳遞路徑的活化。我們發現TTP負調控NF-κB訊息傳遞路徑的可能因素，包含TTP會增加HDAC3的蛋白穩定性、TTP的磷酸化會使其較不易進細胞核還有TTP蛋白的不同區域對於轉譯調控有不同的影響。綜合以上研究結果，我們提供證據證明TTP的磷酸化和結合蛋白改變在基因調控上的分子機制。
並列摘要
Tristetraprolin (TTP) is an RNA-binding protein that mediates the degradation of AU-rich element containing mRNA. The function of TTP is tightly regulated by its protein phosphorylation status and protein-protein interaction. TTP-mediated mRNA decay is through the recruitment of CCR4-NOT deadenylase complex, which consists two deadenylases Cnot6 and Cnot7. Our result reveal that both enzymes of Cnot7 and Cnot6 are required for in vitro deadenylation. In addition to extensively studied p38 pathway, previous reports showed that ERK signaling also can regulate the protein stability, subcellular localization, and function of TTP. By mass spectrometry analysis Ser-316 of TTP was identified to be phosphorylated in response to ERK signaling. Ser-316 phosphorylation of TTP would decrease its interaction with the CCR4-NOT deadenylase complex. Next, we aim to investigate the functional effect of Ser-316 phosphorylation under the physiological condition. We demonstrates that ERK signals induce TTP expression and phosphorylation, which result in changing the binding protein of TTP in LPS-stimulated RAW264.7 and inhibiting 3T3-L1 adipogenesis. Additionally, we observed that Ser-316 phosphorylation can affect the subcellular localization of TTP. On the other hand, transcription co-repressor HDAC3 was identified as TTP-associated proteins in the mass spectrometry analysis. Both of TTP and HDAC3 have been known can negatively regulate NF-κB signaling-mediated transcriptional activation. We demonstrate possible molecular mechanism including that TTP deletion mutants has Transcriptional activity; TTP increases Hdac3 protein stability; and p38-mediated TTP phosphorylation affects its subcellular localization. Collectively, our results might provide evidence to demonstrate the molecular mechanism of TTP phosphorylation and protein-protein interaction on gene regulation.
參考文獻
延伸閱讀
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