Abstract
Distinct cellular responses to environmental factors are crucial for maintaining cells. Signal transduction, which coordinates a cell stress signal and the corresponding response, is predominantly mediated through post-translational modifications. MEKK1 stands out as a unique signaling protein that performs two types of post-translational modifications— phosphorylation and ubiquitination—to regulate downstream pathways and modulate cellular responses. Microtubule stress is fundamental to MEKK1 signaling, which can result in either cell survival or apoptosis depending on the nature and strength of the stressor. Auto-regulation between the kinase and RING domains modulates MEKK1 in the activation and inhibition of downstream MAPK pathways and ultimately in determining cellular outcomes. Since the role of the SWIM domain was relatively uncharacterized, understanding how the SWIM domain plays into MEKK1 auto-regulation is important in understanding wider MEKK1 regulation.
The experimental structure of the N-terminal SWIM and RING domains was solved by X-ray crystallography to a resolution of 3.05 Å and was found to form a concentration- dependent dimer. The SWIM-RING interaction was a novel finding, so investigating how the interaction played into MEKK1 auto-regulation presented an interesting avenue to explore, as structural overlays revealed the SWIM domain and E2 ubiquitin-conjugating enzymes occupy the same RING domain interface.
Ubiquitin assays confirmed that the SWIM domain regulates the formation of poly- ubiquitin chains. Upon mutating a key residue in the SWIM domain, the SWIM-RING interaction was disrupted, leading to reduced dimerization and increased poly-ubiquitin chain formation. When the kinase domain was added into assays as a substrate, the kinase domain was auto-ubiquitinated within in-vitro assays. Other findings suggested interplay between the SWIM domain and the UIM were important in the ubiquitination of the kinase domain.
Overexpression of MEKK1 variants within mammalian cells could not be achieved to validate the in vitro assay discoveries. Alternatively, AlphaFold pulldowns did not identify novel interactions that may modulate the SIWM-RING interaction, four endosomal proteins were identified as putative MEKK1 SWIM-RING interactors.
The findings surrounding SWIM-RING auto-regulation dynamics were found to significantly contribute to overall MEKK1 regulation and proposals could be made encompassing MEKK1-dependent responses to distinct types of microtubule stress. The SWIM domain presents a core function to MEKK1 signaling, however, there is still much to be explored regarding how it plays into the wider role of MEKK1.