Regulation of cell signalling by iRhoms
Keywords: EGFR
iRhom
ADAM17/TACE
metalloprotease

My first project focuses upon the regulation of TACE by oncogenic stimulation. TACE (for TNFα-activating enzyme) is a major regulator of numerous cell signalling pathways, including epidermal growth factor receptor (EGFR) signalling. TACE acts at the plasma membrane to release signalling factors into the extracellular medium. iRhom1 and iRhom2 are multi-pass membrane proteins that belong to the rhomboid-like superfamily, and they are the ultimate regulators of TACE activity. It has been recently discovered that the phosphorylation of iRhom2 is required for the stimulated activity of TACE at the plasma membrane. However, whether iRhom phosphorylation is crucial in other pathological contexts is unknown. In addition, the role of iRhom1 phosphorylation in TACE activation remains unstudied.
I have demonstrated that the phosphorylation of iRhom1 is required for TACE activity upon pharmacological stimulation. Therefore, the mechanism of TACE activation is conserved between iRhom1 and iRhom2. To study the role of iRhom phosphorylation in cancer, the role of the central proto-oncogenes Ras was investigated. Oncogenic KRas and HRas stimulate TACE activity, which demonstrates the conserved role of Ras proteins in stimulating the release of EGFR ligands by TACE. The reconstitution of double knockout (DKO) cells with iRhom2, but not with iRhom1 supports KRas-induced shedding. The specificity towards iRhom2 therefore constitutes a characteristic feature of oncogenic stimulation of TACE activity. Importantly, I have shown KRas-induced shedding requires iRhom2 phosphorylation and recruitment of the phospho-binding proteins 14-3-3. To assess the importance of these findings in lung cancer, the expression of iRhoms was genetically removed in non-small-cell lung cancer cells, thereby allowing me to test the requirement of iRhom2 phosphorylation for in vivo tumourigenesis.
My second project concerns another regulatory function of iRhoms: the degradation of EGFR ligands. Before being cleaved off at the plasma membrane, EGFR ligands are produced as single-pass transmembrane proteins in the endoplasmic reticulum. It was discovered that iRhoms induce the degradation of EGFR ligands by the proteasome. The role of iRhoms as negative regulators of EGFR ligands is conserved in Drosophila and in mammalian cells. However, the molecular mechanism by which iRhoms destabilise EGFR ligands was unknown.
I established a system in human cells and confirmed that ectopic iRhom expression triggers dose-dependent degradation of EGF. I found that the two degradation machineries of the cell support iRhom-driven degradation: the proteasome and the lysosome. Focussing on lysosomal degradation, I found that iRhoms drive EGF to lysosomal degradation directly from the ER, suggesting that this constitutes an ER-phagy route. Importantly, I found a requirement for the autophagy core protein Beclin-1 in this process. Finally, I attempted to determine the requirement of endogenous iRhoms in the degradation of EGF. Although I found an upregulation in EGF protein in iRhom-depleted cells, further validation with iRhom depletion and genetic ablation of iRhom1/2 by CRISPR/Cas9 highlighted technical issues with my approach. Therefore, further work is required to establish the involvement of endogenous iRhoms in EGF degradation.