- 學位論文
ERK2嵌合及磷酸化調控MKP-1穩定性之探討
Regulation of MKP-1 stability through ERK2 docking and phosphorylation
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摘要
ERK1/2為MAPK激酶家族成員，是調節細胞受到外在環境刺激下做出增生、分化、死亡等反應的重要訊號傳遞分子。MKP-1為 dual specificity MAPK phosphatase位於細胞核中，是一新陳代謝快速之蛋白質，主要功能是除去活化態MAPK的TXY磷酸化，以終止激酶訊號。活化態ERK2能直接與MKP-1羧基端之DEF區位嵌合 (docking) 並磷酸化其Ser296及Ser323二個SP序列，促使MKP-1泛素化 (ubiquitination) 而加快其降解。未活化的ERK也可與MKP-1胺基端之KIM區域嵌合，藉此加強去磷酸酶的活性。本篇論文探討活化態ERK2嵌合KIM區域是否造成MKP-1磷酸化、是否與特定SP序列有關、是否影響MKP-1的穩定性。我們利用一系列DEF、KIM及SP序列缺陷之MKP-1點突變蛋白質，發現於活體外，活化態ERK2也能嵌合MKP-1之KIM區域，但其親和力小於未活化態ERK2與KIM之嵌合。活化態ERK2嵌合KIM區域之親和力也比嵌合DEF區位弱。透過KIM區域MKP-1與活化態ERK2嵌合而被磷酸化，且其Ser359和Ser364二個SP序列是不可或缺。再者，強制表現Ser359/Ser364 之模擬磷酸化 (S359D/S364D) MKP-1突變蛋白質於人類胚胎腎細胞293中，在有無血清刺激下，其蛋白質穩定性皆高於正常MKP-1。有趣的是，S359D/S364D降低活化態ERK2嵌合於DEF區位之能力以及MKP-1之Ser296的磷酸化。由本篇論文的發現，我們推測活化態ERK2能透過嵌合KIM功能區而磷酸化MKP-1之Ser359和Ser364，並降低其Ser296的磷酸化，間接增加MKP-1的穩定性。綜合而言，活化態ERK2透過KIM或DEF嵌合以磷酸化MKP-1特定之SP序列，進而正向或負向調控去磷酸酶的穩定性。活化態ERK2因此可精確地微調MKP-1蛋白質的含量，以自我調節訊號活化的強度以及持久性。
並列摘要
Extracellular signal-regulated kinases (ERK1/2), belong to the family of mitogen-activated protein kinases (MAPK), are important intracellular signaling molecules to regulate cell proliferation, differentiation, and death in response to extracellular stimuli. The dual specificity MAPK phosphatase MKP-1 is an inducible and liable nuclear protein functioning to inactivate MAPK signaling through removal of phosphates from TXY motif. Active ERK2 docking to the C-terminal DEF motif of MKP-1 initiates phosphorylation at Ser296/Ser323, which is essential for ubiquitination and degradation of MKP-1. Unactive ERK2 also binds to the N-terminal KIM domain of MKP-1 enhancing phosphatase activity. This thesis investigated whether active ERK2 docking to KIM initiates MKP-1 phosphorylation in which specific SP sites are involved to affect MKP-1 stability. By using a variety of mutant proteins carrying defects in DEF, KIM and SP sites generated from site-directed mutagenesis, we found that active ERK2 also docks to KIM with lower affinity than unactive ERK2 does in vitro. In comparison with DEF, KIM has lower binding efficiency for active ERK2. Through active ERK2 docking to KIM, MKP-1 is phosphorylated in which two SP sites at Ser359/Ser364 are indispensable. Furthermore, phospho-mimetic Ser359/Ser364 MKP-1 mutant (S359D/S364D) is more stable than MKP-1(WT) under serum deprivation or stimulation during forced expression in H293 cells. Intriguingly, S359D/S364D decreases the docking affinity of active ERK2 to DEF of MKP-1 and phosphorylation at Ser296. These results suggest that active ERK2 docking to KIM triggers phosphorylation at Ser359/Ser364 of MKP-1, by which reduces DEF docking and Ser296 phosphorylation and thus indirectly stabilizes MKP-1. Taken together, active ERK2 directs site-specific phosphorylation through docking to KIM or DEF to positively and negatively regulate MKP-1 stability that provides a fine-tune mechanism for self-control of signaling strength and duration.
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