Fanger G R, Widmann C, Porter A C, Sather S, Johnson G L, Vaillancourt R R
Program in Molecular Signal Transduction, Division of Basic Sciences, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
J Biol Chem. 1998 Feb 6;273(6):3476-83. doi: 10.1074/jbc.273.6.3476.
MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) kinases (MEKKs) regulate c-Jun N-terminal kinase and extracellular response kinase pathways. The 14-3-3zeta and 14-3-3epsilon isoforms were isolated in a two-hybrid screen for proteins interacting with the N-terminal regulatory domain of MEKK3. 14-3-3 proteins bound both the N-terminal regulatory and C-terminal kinase domains of MEKK3. The binding affinity of 14-3-3 for the MEKK3 N terminus was 90 nM, demonstrating a high affinity interaction. 14-3-3 proteins also interacted with MEKK1 and MEKK2, but not MEKK4. Endogenous 14-3-3 protein and MEKK1 and MEKK2 were similarly distributed in the cell, consistent with their in vitro interactions. MEKK1 and 14-3-3 proteins colocalized using two-color digital confocal immunofluorescence. Binding of 14-3-3 proteins mapped to the N-terminal 393 residues of 196-kDa MEKK1. Unlike MEKK2 and MEKK3, the C-terminal kinase domain of MEKK1 demonstrated little or no ability to interact with 14-3-3 proteins. MEKK1, but not MEKK2, -3 or -4, is a caspase-3 substrate that when cleaved releases the kinase domain from the N-terminal regulatory domain. Functionally, caspase-3 cleavage of MEKK1 releases the kinase domain from the N-terminal 14-3-3-binding region, demonstrating that caspases can selectively alter protein kinase interactions with regulatory proteins. With regard to MEKK1, -2 and -3, 14-3-3 proteins do not appear to directly influence activity, but rather function as "scaffolds" for protein-protein interactions.
丝裂原活化蛋白激酶/细胞外信号调节激酶激酶（MEK）激酶（MEKKs）调控c-Jun氨基末端激酶和细胞外应急激酶信号通路。在一个双杂交筛选中，分离出了14-3-3ζ和14-3-3ε亚型，它们是与MEKK3的N末端调节结构域相互作用的蛋白质。14-3-3蛋白与MEKK3的N末端调节结构域和C末端激酶结构域均结合。14-3-3对MEKK3 N末端的结合亲和力为90 nM，表明存在高亲和力相互作用。14-3-3蛋白也与MEKK1和MEKK2相互作用，但不与MEKK4相互作用。内源性14-3-3蛋白以及MEKK1和MEKK2在细胞中的分布相似，这与它们在体外的相互作用一致。利用双色数字共聚焦免疫荧光技术，发现MEKK1和14-3-3蛋白共定位。14-3-3蛋白的结合位点位于196-kDa MEKK1的N末端393个残基处。与MEKK2和MEKK3不同，MEKK1的C末端激酶结构域与14-3-3蛋白的相互作用很弱或几乎没有。MEKK1是caspase-3的底物，而MEKK2、MEKK3和MEKK4不是，MEKK1被切割后会从N末端调节结构域释放出激酶结构域。在功能上，MEKK1被caspase-3切割后会从N末端14-3-3结合区域释放出激酶结构域，这表明半胱天冬酶可以选择性地改变蛋白激酶与调节蛋白的相互作用。对于MEKK1、MEKK2和MEKK3而言，14-3-3蛋白似乎并不直接影响其活性，而是作为蛋白质-蛋白质相互作用的“支架”发挥作用。
