We hypothesized that MEK inhibition activates AKT by inhibiting ERK activity, which blocks an inhibitory threonine phosphorylation on the juxtamembrane domains of EGFR and HER2, thereby increasing ERBB3 phosphorylation.
To test this hypothesis, we transiently transfected CHO-KI cells, which do not express ERBB receptors endogenously, with wild-type ERBB3 with either wild-type EGFR or EGFRT669A.
In cells transfected with wild-type EGFR, MEK inhibition led to feedback activation of phospho-ERBB3 and phosho-EGFR, recapitulating the results we had observed in our panel of cancer cell lines (Figure 6A).
In contrast, the EGFRT669A mutant increased both basal EGFR and ERBB3 tyrosine phosphorylation that was not augmented by MEK inhibition.
As a control, we treated CHO-KI cells expressing EGFRT669A with HRG ligand to induce maximal ERBB3 phosphorylation (Figure 6A), indicating that the lack of induction of phospho-ERBB3 in EGFRT669A expressing cells following MEK inhibition was not simply due to the saturation of the system with phospho-ERBB3.
We observed analogous results in CHO-KI cells expressing wild-type ERBB3 in combination with wild-type or T677A mutant HER2 (Figure 6B).
Together these results support the hypothesis that inhibition of ERK-mediated phosphorylation of a conserved juxtamembrane domain threonine residue leads to feedback activation of EGFR, HER2, and ERBB3 (Figure 7).
