Both nuclear-cytoplasmic shuttling of the dual specificity phosphatase MKP-3 and its ability to anchor MAP kinase in the cytoplasm are mediated by a conserved nuclear export signal
Both nuclear-cytoplasmic shuttling of the dual specificity phosphatase MKP-3 and its ability to anchor MAP kinase in the cytoplasm are mediated by a conserved nuclear export signal
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DOI：
10.1074/jbc.m406720200
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发表时间：
2004-10-01
影响因子：
4.8
通讯作者：
Keyse, SM
中科院分区：
文献类型：
--
作者：
Karlsson, M;Mathers, J;Keyse, SM
MKP-3是一种胞质双特异性蛋白磷酸酶，能与哺乳动物细胞中的ERK1/2 MAP激酶特异性结合并使其失活。然而，MKP-3的细胞质定位及其生理意义的分子基础尚不清楚。我们将MKP-3-绿色荧光蛋白与软霉素B结合使用，表明MKP-3的细胞质定位是由依赖于染色体区域维持-1(CRM1)的核输出途径介导的。此外，在软霉素B存在的情况下，MKP-3的核转位是由一个活跃的过程介导的，表明MKP-3在细胞核和细胞质之间穿梭。MKP-3的氨基末端非催化结构域是磷酸酶核输出的必要条件和充分条件，并且含有单一的功能丰富的亮氨酸核输出信号(NES)。尽管该蛋白的这个结构域也介导了MKP-3与MAP激酶的结合，但我们发现取消ERK2结合的激酶相互作用基序的突变并不影响MKP-3的定位。相反，NES的突变并不影响MKP-3与ERK2的结合或磷酸酶活性，表明KI相互作用基序和NES独立发挥作用。最后，我们证明了MKP-3导致ERK2胞质滞留的能力需要一个功能激酶相互作用基序和NES。我们的结论是，MKP-3除了在调节MAP激酶的去磷酸化和失活方面已确立的功能外，还可能在决定其底物的亚细胞定位方面发挥作用。我们的结果强化了这样的观点，即MKP-3等调节蛋白可能在MAP激酶活性的时空调节中发挥关键作用。
MAP kinase phosphatase (MKP)-3 is a cytoplasmic dual specificity protein phosphatase that specifically binds to and inactivates the ERK1/2 MAP kinases in mammalian cells. However, the molecular basis of the cytoplasmic localization of MKP-3 or its physiological significance is unknown. We have used MKP-3-green fluorescent protein fusions in conjunction with leptomycin B to show that the cytoplasmic localization of MKP-3 is mediated by a chromosome region maintenance-1 (CRM1)-dependent nuclear export pathway. Furthermore, the nuclear translocation of MKP-3 seen in the presence of leptomycin B is mediated by an active process, indicating that MKP-3 shuttles between the nucleus and cytoplasm. The amino-terminal noncatalytic domain of MKP-3 is both necessary and sufficient for nuclear export of the phosphatase and contains a single functional leucine-rich nuclear export signal (NES). Even though this domain of the protein also mediates the binding of MKP-3 to MAP kinase, we show that mutations of the kinase interaction motif which abrogate ERK2 binding do not affect MKP-3 localization. Conversely, mutation of the NES does not affect either the binding or phosphatase activity of MKP-3 toward ERK2, indicating that the kinase interaction motif and NES function independently. Finally, we demonstrate that the ability of MKP-3 to cause the cytoplasmic retention of ERK2 requires both a functional kinase interaction motif and NES. We conclude that in addition to its established function in the regulated dephosphorylation and inactivation of MAP kinase, MKP-3 may also play a role in determining the subcellular localization of its substrate. Our results reinforce the idea that regulatory proteins such as MKP-3 may play a key role in the spatio-temporal regulation of MAP kinase activity.