Interacting JNK-docking sites in MKK7 promote binding and activation of JNK mitogen-activated protein kinases
Interacting JNK-docking sites in MKK7 promote binding and activation of JNK mitogen-activated protein kinases
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DOI：
10.1074/jbc.m601010200
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发表时间：
2006-05-12
影响因子：
4.8
通讯作者：
Bardwell, L
中科院分区：
文献类型：
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作者：
Ho, DT;Bardwell, AJ;Bardwell, L
D-位点是一类存在于许多MAPK调节子和底物中的MAPK对接位点。在MAPK激酶MEK1、MEK2、MKK3、MKK4和MKK6的N端附近发现了一个功能强大的高亲和力D-位点。在这里，我们证明了MKK7通过一种新的机制识别其靶标JNK，该机制涉及MKK7 N-末端结构域中三个低亲和力D-位点的部分协同作用。三个对接位点中任何一个(D1、D2和D3)上的保守残基的突变使MKK7β的N末端结构域与JNK1结合的能力中断了约50%-70%。此外，三个D-位点中任何两个的突变使结合减少了约80%-90%，所有三个D-位点的突变使结合减少了95%。与野生型MKK7相比，含有组合D1/D2突变的全长MKK7与JNK1结合受到损害，并显示出JNK1激酶活性降低。来自MKK4或JIP-1支架蛋白的D-位点的多肽版本抑制了MKK7-JNK的结合，表明所有三个JNK调节因子都与JNK的同一区域结合。此外，MKK7的三个D-位点中的任何一个的多肽版本都抑制了JNK1和JNK2对其转录因子底物c-jun和ATF2的磷酸化能力，这表明含有D-位点的底物也与MKK7竞争与JNK的对接。最后，MKK7衍生的D位点肽对JNK1和ERK2具有选择性抑制作用。我们得出结论，MKK7包含三个JNK-对接位点，它们相互作用，选择性地结合JNK，并有助于JNK信号的传递和特异性。
D-sites are a class of MAPK-docking sites that have been found in many MAPK regulators and substrates. A single functional, high affinity D-site has been identified near the N terminus of each of the MAPK kinases ( MKKs orMEKs) MEK1, MEK2, MKK3, MKK4, and MKK6. Here we demonstrated that MKK7 recognizes its target JNK by a novel mechanism involving a partially cooperative interaction of three low affinity D-sites in the N-terminal domain of MKK7. Mutations of the conserved residues within any one of the three docking sites ( D1, D2, and D3) disrupted the ability of the N-terminal domain of MKK7 beta to bind JNK1 by about 50 - 70%. Moreover, mutation of any two of the three D-sites reduced binding by about 80 - 90%, and mutation of all three reduced binding by 95%. Full-length MKK7 containing combined D1/D2 mutations was compromised for binding to JNK1 and exhibited reduced JNK1 kinase activity when compared with wild-type MKK7. Peptide versions of the D-sites from MKK4 or the JIP-1 scaffold protein inhibited MKK7-JNK binding, suggesting that all three JNK regulators bind to the same region of JNK. Moreover, peptide versions of any of the three D-sites of MKK7 inhibited the ability of JNK1 and JNK2 to phosphorylate their transcription factor substrates c-Jun and ATF2, suggesting that D-site-containing substrates also compete with MKK7 for docking to JNK. Finally, MKK7-derived D-site peptides exhibited selective inhibition of JNK1 versus ERK2. We conclude that MKK7 contains three JNK-docking sites that interact to selectively bind JNK and contribute to JNK signal transmission and specificity.