SelO functions as a tumor suppressor through AMPylating Cdk5rap3

Cell fate is governed by signaling pathways involving diverse protein modifications. However, how protein AMPylation mediates signal transduction in mammalian cells remains largely unexplored. We demonstrate that the protein adenylyltransferase, SELENOO (SelO), is induced by chemotherapy, and has clinical relevance in cancer. In mice, SelO knockout facilitates primary tumor development and metastasis. In cancer cells, the cytosolic fraction of SelO suppresses cell proliferation and migration. Mechanistically, SelO AMPylates CDK5 regulatory subunit-associated protein 3 (Cdk5rap3) at T328, enhancing its interaction with ARF. The interaction competitively reduces ARF associated with the SUMOylation enzyme UBC9, reducing ARF-mediated SUMO1 modifications of Mdm2 and Slug. Concurrently, the strengthened Cdk5rap3-ARF affinity decreases Cdk5rap3 associated with PPM1D, impairing the ability of PPM1D to dephosphorylate Mdm2. These effects collectively lead to destabilization of Mdm2 and Slug proteins, suppressing their downstream pathways relating to cell proliferation and TGF-β-mediated migration. These findings reveal a role for protein AMPylation in cell signaling and cancer progression.