Structural Bases And Mechanism For Axin-Mediated JNK MAP Kinase Activation

Axin is a multidomain protein that plays a critical role in Wnt signaling, serving as a scaffold for down-regulation of p-catenin. It also activates the JNK mitogen-activated protein kinase by binding to MEKK1. However, it is intriguing that Axin required several additional elements for JNK activation, including a requirement for homodimerization, sumoylation at the extreme C-terminal sites, and a region in the protein phosphatase 2A binding domain. In my doctoral studies, I showed that another MEKK family member, MEKK4, also binds to Axin in vivo and mediates Axin-induced JNK activation. Surprisingly, MEKK4 binds to a region distinct from the MEKK 1-binding site. Dominant negative mutant of MEKK4 attenuates the JNK activation by Axin. Activation of JNK by Axin in MEKK1"7" mouse embryonic fibroblast cells supports the idea that another MEKK can mediate Axin-induced JNK activation. Expression of specific small interfering RNA against MEKK4 effectively attenuates JNK activation by the MEKK1 binding-defective Axin mutant in 293T cells and inhibits JNK activation by wild-type Axin in MEKKl cells, confirming that MEKK4 is indeed another mitogen-activated protein kinase kinase kinase that is specifically involved in Axin-mediated JNK activation independently of MEKK1. I also identified an additional domain between MEKK1- and MEKK4- binding sites as being required for JNK activation by Axin. MEKK1 and MEKK4 compete for Axin binding even though they bind to sites far apart, suggesting that Axin may selectively bind to MEKK1 or MEKK4 depending on distinct signals or cellular context. Furthermore, components of Wnt signaling pathway, GSK-3P and casein kinase I a/e could inhibit MEKK4-mediated JNK activation by Axin by competing against MEKK4 binding to Axin.Upon completion of the studies on MEKK4 function in the Axin/JNK pathway, I studied another Wnt regulator, Coiled-coil-DIXl(Ccdl). Ccdl is a novel protein that contains a DIX domain and plays an important role in Wnt signaling. It has been shown that Dishevelled, another positive regulator of Wnt signaling contains the DIX domain, could attenuate Axin-activated JNK activity by disrupting Axin homodimerization. I asked if the new DIX-domain protein also exerted a regulatory role in the Axin-mediated JNK activation. It was shown that Ccdl itself could not activate JNK, furthermore it drastically inhibited JNK activation by Axin. Ccdl required its DIX domain and coiled-coil domain for inhibition of JNK activation by Axin. Interestingly, Axin utilizes a novel domain upstream of DIX domain to interact with Ccdl. Consistently, Ccdl could not interfere with the heterodimerization of Axin and Dishevelled. I also found that Axin, when complexed with Ccdl, did not bind to MEKK1. Furthermore, Ccdl physically interacted with MEKK4 in the physiological concentration, preventing its binding to Axin. I have therefore demonstrated that Ccdl inhibits Axin-mediated JNK activation by simultaneously adopting two distinct mechanisms, one through conformational changes that disallow MEKK1 binding, and the other via direct sequestration of MEKK4.In sum, my studies have revealed several aspects of structural and biochemical basis for the Axin/JNK pathway. My findings will provide new insights into how the scaffold protein Axin mediates ultimate activation of different mitogen-activated protein kinase kinase kinases.