Post-translational Regulation of Mitogen-activated Protein Kinase Phosphatase (MKP)-1 and MKP-2 in Macrophages Following Lipopolysaccharide Stimulation THE ROLE OF THE C TERMINI OF THE PHOSPHATASES IN DETERMINING THEIR STABILITY
Post-translational Regulation of Mitogen-activated Protein Kinase Phosphatase (MKP)-1 and MKP-2 in Macrophages Following Lipopolysaccharide Stimulation THE ROLE OF THE C TERMINI OF THE PHOSPHATASES IN DETERMINING THEIR STABILITY
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DOI：
10.1074/jbc.m114.591925
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发表时间：
2014-10-17
影响因子：
4.8
通讯作者：
Liu, Yusen
中科院分区：
文献类型：
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作者：
Crowell, Sara;Wancket, Lyn M.;Liu, Yusen
MAPK磷酸酶（MKPs）是先天免疫应答的关键调节剂，但调节其积累的机制仍知之甚少。在本研究中，我们研究了翻译后修饰在LPS刺激后巨噬细胞中MKP-1和MKP-2积累中的作用。我们发现，在LPS刺激后，MKP-1和MKP-2以不同的动力学积累：MKP-1水平在1 h达到峰值，而MKP-2水平持续升高至少6 h。通过抑制ERK级联反应减弱MKP-1和MKP-2的蓄积。有趣的是，在LPS刺激之前抑制p38对MKP-1和MKP-2蛋白水平几乎没有影响，但阻碍了M-18 MKP-1抗体对它们的检测。对M-18 MKP-1抗体识别的表位序列的研究表明，ERK途径对MKP-1和MKP-2的C末端的两个丝氨酸残基进行了广泛的磷酸化。值得注意的是，在存在ERK通路抑制剂的情况下，巨噬细胞中MKP-1和MKP-2的稳定性均显著降低。将MKP-1和MKP-2中的两个C-末端丝氨酸残基突变为丙氨酸会降低其半衰期，而将这些残基突变为天冬氨酸会显著增加其半衰期。从MKP-1和MKP-2中删除C末端也大大增加了它们的稳定性。令人惊讶的是，MKP-1和MKP-2突变体的稳定性增强与泛素化降低无关。蛋白酶体抑制剂可减弱MKP-1和MKP-2的降解。我们的研究表明，MKP-1和MKP-2的稳定性是由ERK介导的磷酸化通过一个独立的多聚泛素化的降解途径。
MAPK phosphatases (MKPs) are critical modulators of the innate immune response, and yet the mechanisms regulating their accumulation remain poorly understood. In the present studies, we investigated the role of post-translational modification in the accumulation of MKP-1 and MKP-2 in macrophages following LPS stimulation. We found that upon LPS stimulation, MKP-1 and MKP-2 accumulated with different kinetics: MKP-1 level peaked at similar to 1 h, while MKP-2 levels continued to rise for at least 6 h. Accumulation of both MKP-1 and MKP-2 were attenuated by inhibition of the ERK cascade. Interestingly, p38 inhibition prior to LPS stimulation had little effect on MKP-1 and MKP-2 protein levels, but hindered their detection by an M-18 MKP-1 antibody. Studies of the epitope sequence recognized by the M-18 MKP-1 antibody revealed extensive phosphorylation of two serine residues in the C terminus of both MKP-1 and MKP-2 by the ERK pathway. Remarkably, the stability of both MKP-1 and MKP-2 was markedly decreased in macrophages in the presence of an ERK pathway inhibitor. Mutation of the two C-terminal serine residues in MKP-1 and MKP-2 to alanine decreased their half-lives, while mutating these residues to aspartate dramatically increased their half-lives. Deletion of the C terminus from MKP-1 and MKP-2 also considerably increased their stabilities. Surprisingly, enhanced stabilities of the MKP-1 and MKP-2 mutants were not associated with decreased ubiquitination. Degradation of both MKP-1 and MKP-2 was attenuated by proteasomal inhibitors. Our studies suggest that MKP-1 and MKP-2 stability is regulated by ERK-mediated phosphorylation through a degradation pathway independent of polyubiquitination.