The adapter protein FADD provides an alternate pathway for entry into the cell cycle by regulating APC/C-Cdh1 E3 ubiquitin ligase activity

The E3 ubiquitin ligase APC/C-Cdh1 maintains the G0/G1 state, and its inactivation is required for cell cycle entry. We reveal a novel role for Fas-associated protein with death domain (FADD) in the cell cycle through its function as an inhibitor of APC/C-Cdh1. Using real-time, single-cell imaging of live cells combined with biochemical analysis, we demonstrate that APC/C-Cdh1 hyperactivity in FADD- deficient cells leads to a G1 arrest despite persistent mitogenic signaling through oncogenic EGFR/KRAS. We further show that FADD_WT interacts with Cdh1, while a mutant lacking a consensus KEN-box motif (FADD_KEN) fails to interact with Cdh1 and results in a G1 arrest due to it its inability to inhibit APC/C-Cdh1. Additionally, enhanced expression of FADD_WT but not FADD_KEN, in cells arrested in G1 upon CDK4/6 inhibition, leads to APC/C-Cdh1 inactivation and entry into the cell cycle in the absence of retinoblastoma protein (Rb)- phosphorylation. FADD's function in the cell cycle requires its phosphorylation by CK1α at Ser-194 which promotes its nuclear translocation. Overall, FADD provides a CDK4/6-Rb-E2F independent "bypass" mechanism for cell cycle entry and thus a therapeutic opportunity for CDK4/6 inhibitor resistance.