Stimulus-induced uncoupling of extracellular signal-regulated kinase phosphorylation from nuclear localization is dependent on docking domain interactions
Stimulus-induced uncoupling of extracellular signal-regulated kinase phosphorylation from nuclear localization is dependent on docking domain interactions
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DOI：
10.1242/jcs.076349
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发表时间：
2010-12-15
影响因子：
4
通讯作者：
McArdle, Craig A.
中科院分区：
文献类型：
--
作者：
Caunt, Christopher J.;McArdle, Craig A.
许多刺激通过 TEY 基序的磷酸化来激活细胞外信号调节激酶 (ERK)。激活的 ERK 典型地在细胞核中积累，但所涉及的潜在机制尚不清楚。使用自动显微镜探索单个完整细胞中的 ERK 调节，我们发现，当蛋白激酶 C 或表皮生长因子受体被激活时，ERK 核定位反应的很大一部分与 TEY 磷酸化脱钩。这种磷酸化不可归因的核定位反应在酪氨酸磷酸酶和蛋白质合成抑制剂存在的情况下发生。对于催化失活的 ERK2-GFP 突变体以及无法结合许多 ERK 结合伴侣中发现的 DEF（ERK 对接位点，F/Y-X-F/Y-P）结构域的突变体也很明显。然而，MEK 抑制和阻止 TEY 磷酸化或 D（对接）结构域依赖性 ERK 结合 (D319N) 的突变会降低该值。因此，我们表明 MEK 催化的 ERK 磷酸化对于完整的核定位反应是必要的，但还不够：反应中存在一个额外的磷酸化不可归因的成分，它不反映核锚定的诱导表达，并且独立于 ERK 催化活性或 DEF 结构域结合。然而，它依赖于 D 结构域结合，突出了 ERK 基序在核靶向过程中的独特作用。
Many stimuli activate the extracellular signal-regulated kinase (ERK) by phosphorylation on the TEY motif. Activated ERK characteristically accumulates in the nucleus, but the underlying mechanisms involved are unclear. Using automated microscopy to explore ERK regulation in single intact cells, we find that, when protein kinase C or epidermal growth factor receptors are activated, a substantial fraction of the ERK nuclear localization response is uncoupled from TEY phosphorylation. This phosphorylation-unattributable nuclear localization response occurs in the presence of inhibitors of tyrosine phosphatases and protein synthesis. It was also evident with a catalytically inactive ERK2-GFP mutant, and with a mutant incapable of binding the DEF (docking site for ERK, F/Y-X-F/Y-P) domains found in many ERK binding partners. It was, however, reduced by MEK inhibition and by mutations preventing either TEY phosphorylation or D (docking)-domain-dependent ERK binding (D319N). Thus, we show that MEK-catalysed ERK phosphorylation is necessary but not sufficient for the full nuclear localization response: there is an additional phosphorylation-unattributable component of the response that does not reflect induced expression of nuclear anchors and is independent of ERK catalytic activity or DEF-domain binding. It is, however, dependent upon D-domain binding, highlighting distinct roles of ERK motifs during nuclear targeting.