Phosphorylation-dependent Lys⁶³-linked polyubiquitination of Daxx is essential for sustained TNF-α-induced ASK1 activation

Apoptosis signal-regulating kinase 1 (ASK1) is a key regulatory kinase in the proapoptotic response to various stresses. ASK1 phosphorylation of Daxx, an ASK1 activator protein, increases Daxx accumulation in cells and further enhances ASK1 activity through a positive feedback mechanism. Here, we show that ASK1-dependent phosphorylation of Daxx induces Lys⁶³ (K63)-linked polyubiquitination on Lys¹²² of Daxx. Polyubiquitination is dispensable for Daxx accumulation or Daxx interaction with ASK1 because mutant Daxx deficient in polyubiquitin still exhibits ASK1-dependent accumulation and interaction with cellular ASK1. However, K63-linked Daxx polyubiquitination is required for tumor necrosis factor-α (TNF-α)-induced activation of ASK1. Therefore, K63-linked polyubiquitination of Daxx functions as a molecular switch to initiate and amplify the stress kinase response in the TNF-α signaling pathway.