Characterization of intrinsic MKP-1 sequences for ERK-directed proteolysis

ERK激酶引導MKP-1蛋白降解之必要序列分析

郭又慈(Yu-Tzu Kuo)

指導教授 ： 楊嘉鈴

國立清華大學/生命科學院/生物科技研究所/碩士(2008年)

摘要

The dual-specificity MAPK phosphatase-1 (MKP-1) is a mitogen/stress-inducible nuclear protein that can dephosphorylate and inactivate MAPK family of protein kinases. MAPK binding to the MKP-1 N-terminal KIM domain increases phosphatase activity, while ERK docking to the MKP-1 C-terminal DEF motif triggers MKP-1 proteolysis via direct phosphorylation of the Ser296/Ser323 motif. It has also been proposed that ERK-induced phosphorylation of MKP-1 at the Ser359/Ser364 sites may increase its stabilization. To reveal how intrinsic MKP-1 amino acid residues impact on its destruction or stabilization controlled by ERK, we generated KIM- (R3A), DEF- (ANA), R3A/S359D/S364D (R3A/DD) and R3A/ANA mutants from the wild-type MKP-1 (WT) tagged with GST or Flag at the N-terminus. In vitro assay showed that the efficiency of active ERK binding to MKP-1 mutants was WT > R3A > ANA > R3A/DD or R3A/ANA. Active ERK elicited MKP-1 S359 phosphorylation in vitro independent of the KIM or DEF docking sites. Expression of Flag-MKP-1 mutants in H293 cells showed that the stability of exogenous proteins was R3A/DD or R3A/ANA > ANA or R3A > WT in chase experiments using cycloheximide or 26S proteasome inhibitors. Whereas phosphorylation of MKP-1 at S359 in vivo was dependent on ERK signaling, the KIM and DEF were not prerequisite for. Together, results shown here suggest that (1) KIM in addition to DEF is an essential ERK docking site for MKP-1 proteolysis; (2) the phospho-mimetic S359/S364 sequence interferes with ERK binding to the DEF motif, thereby facilitates MKP-1 stabilization; and (3) S359 phosphorylation is associated with ERK activity, yet, the event is independent of KIM/DEF and is not obligatory for MKP-1 degradation/stabilization.

關鍵字

雙特異性磷酸酶 ； ERK激酶 ； 磷酸化

並列摘要

MKP-1是一種可受分裂素或壓力誘發之位於細胞核的雙特異性磷酸酶，其主要功能是去除MAPK活性以終止激酶訊號。MAPK可與MKP-1胺基端之KIM區域嵌合，藉此加強去磷酸酶的活性。另外ERK2嵌合至MKP-1羧基端之DEF區位可直接磷酸化Ser296及Ser323二個SP序列以加快MKP-1降解。先前亦有研究推測ERK引起之MKP-1上Ser359/Ser364位置磷酸化可能會增加其穩定性。我們利用一系列在胺基端接上含有GST或Flag 標示及DEF、KIM或SP序列缺陷之MKP-1點突變蛋白質，包括KIM- (R3A)、DEF- (ANA)、R3A/S359A/S364A (R3A/AA)、R3A/S359D/S364D (R3A/DD)以及 R3A/ANA，進行探討MKP-1的胺基酸序列對於它受到ERK激酶媒介之降解或穩定作用之影響。由活體外實驗發現，MKP-1與活化態ERK嵌合效能依序為WT > R3A > ANA > R3A/DD、R3A/ANA，而活化態ERK促使MKP-1 Ser359的磷酸化是不需要KIM和DEF二區位參與。強制表現MKP-1點突變蛋白質於人類胚胎腎細胞株H293並利用蛋白質合成抑制劑cycloheximide或26S蛋白酶體抑制劑進行chase實驗以比較蛋白質穩定性，發現R3A/DD、R3A/ANA > ANA、R3A > WT。在活體內MKP-1 Ser359的磷酸化也是不需KIM和DEF二區位，且隨著ERK訊號活化而增加。總結本篇研究顯示三項發現；一.DEF和KIM區域是ERK激酶引導MKP-1蛋白降解必要之ERK嵌合序列。二.S359/S364之模擬磷酸化可能干擾活化態ERK2嵌合於DEF區位，進而間接增加MKP-1的穩定性。三. S359磷酸化雖可經由ERK訊號誘發，但卻不需KIM和DEF這兩個嵌合區域且與MKP-1的降解或穩定無直接關係。

並列關鍵字

HASH(0x1c600770) ； HASH(0x1c600810) ； HASH(0x1c6008b0)

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