CNOT4 Suppresses Bladder Cancer Progression by Promoting Ubiquitination‐Mediated Degradation of EIF2AK2 and Inhibiting Autophagy

EIF2AK2, also known as PKR, is a stress-responsive kinase that regulates multiple cellular processes, including autophagy, apoptosis, and tumor progression. However, the mechanisms underlying EIF2AK2 stability and function in bladder cancer (BLCA) remain underexplored. In this study, we investigated the mechanisms governing post-translational EIF2AK2 expression and its impact on BLCA progression. Through a luciferase-based screening of candidate E3 ubiquitin ligases, we identified CNOT4 as a potential regulator of EIF2AK2. Bioinformatics analysis of public BLCA transcriptomic datasets showed that CNOT4 expression is significantly decreased in BLCA tissues relative to that in normal bladder tissues, and reduced CNOT4 levels are associated with unfavorable clinical outcomes. Functional assays demonstrated that CNOT4 overexpression suppresses viability and motility, while promoting apoptosis in BLCA cells. Moreover, CNOT4 inhibited autophagy and related signaling pathways. Mechanistic analyses revealed that CNOT4 interacted with EIF2AK2 and promoted its K48-linked ubiquitination and proteasomal degradation. Restoration of EIF2AK2 expression largely reversed the tumor-suppressive effects of CNOT4, indicating that EIF2AK2 acts as a key downstream effector. Consistent with these findings, CNOT4 overexpression significantly suppressed tumor growth in a BLCA xenograft model. Collectively, our observations identify CNOT4 as a previously unrecognized EIF2AK2 stability regulator and reveal that CNOT4 suppresses BLCA progression by promoting EIF2AK2 degradation and thereby suppressing autophagy. These results reveal a novel mechanism for the post-translational modulation of EIF2AK2 and suggest a potential therapeutic target for BLCA.