CUL4A-DDB1-DCAF10 is an N-recognin for N-terminally acetylated Src kinases

Co-translational N-terminal modifications such as methionine excision, acetylation, and myristoylation govern protein stability, localization, and folding. Disruption can expose N-terminal degrons that trigger ubiquitin-mediated degradation, safeguarding the proteome. N-terminal acetylation usually protects proteins from degradation, but can also promote it through the Ac/N-degron pathway. Src-family kinases (SFKs), signaling enzymes implicated in tumorigenesis, require N-terminal myristoylation for function. Using peptide pull-downs, mass spectrometry, and AlphaFold 3 predictions, we identify DCAF10 as the E3 ligase substrate receptor for alternatively N-terminally acetylated SFKs. Combining siRNA-mediated knockdown and CRISPR/Cas9-mediated knockout of endogenous Lyn with inducible Lyn-GFP variants confirms that DCAF10 regulates SFK levels by recognizing an N-terminal acetylated glycine residue. In vitro, a CUL4A-DDB1-DCAF10 complex ubiquitinates N-terminally acetylated SFKs. Thus, we define a novel N-degron pathway that monitors replacement of myristoylation by acetylation and activates degradation of SFKs upon acetylation. This mechanism may extend to other N-terminally myristoylated proteins beyond SFKs.