Abstract
The ability to sense and respond to the environment is one of the defining features of life. Cells use a variety of signalling systems that carry information about environmental stimuli to the nucleus in order to generate an appropriate response. Phosphorylation and ubiquitination signalling are two of the most important post-translational modifications that control virtually all cellular processes, from proliferation, through stress responses, to apoptosis. Mitogen-activated protein 3-kinase 1 (MEKK1) is a key upstream regulator in the NF-κB and MAPK functional pathways—major cellular pathways that control stress responses and cell fate. MEKK1 is also the only known member of the mitogen-activated protein 3 kinase (MAP3K) family that contains both a kinase domain and a RING E3 ubiquitin ligase domain. The N-terminal half of MEKK1, including the RING domain, is involved in controlling the C- terminal kinase domain and downstream activation of MEKK1-driven pathways.
A range of biophysical methods were used to characterize N-terminal functional and regulatory domains of MEKK1. This work presents the crystal structure of a central domain of MEKK1 at 2.1 Å resolution, and identifies it as a tubulin-interacting TOG domain. The discovery and structural characterization of this domain of MEKK1 reveals a new direction in the research of MEKK1-mediated signalling.
As the function of TOG domains is highly dependent on their conformational selectivity towards specific conformations of tubulin, a characterization of MEKK1 TOG in terms of its interaction with the straight and curved conformation of tubulin was undertaken. The data presented herein indicate that MEKK1 TOG domain preferentially binds to the curved conformation of tubulin dimers, found in soluble tubulin and at depolymerizing microtubule ends.
The functional significance of the MEKK1 TOG domain is further underscored by the observation that mutations in this domain of MEKK1 reduce tubulin binding in vitro, and occur in human tumours. An analysis of the role of MEKK1 TOG in terms of its effects on MEKK1 ubiquitin ligase and kinase activities revealed a potential role in controlling MEKK1 ubiquitin ligase activity.
Based on these findings, a model is proposed whereby MEKK1 kinase activity is induced at the sites of microtubule catastrophes via clustering of MEKK1. Moreover, the interaction with tubulin affects the activity of the MEKK1 RING E3 ubiquitin ligase domain. Accordingly, MEKK1’s interaction with tubulin and microtubules could be relevant to both cancer development and efficacy of microtubule-targeting chemotherapies.