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

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Co-translational N-terminal modifications regulate protein fate.
• N-terminal acetylation can either stabilize or destabilize proteins via the Ac/N-degron pathway.
• Src-family kinases (SFKs) require N-terminal myristoylation for their signaling functions.

Purpose of the Study:

• To identify the E3 ligase substrate receptor for N-terminally acetylated SFKs.
• To elucidate the mechanism by which SFK levels are regulated.
• To define a novel N-degron pathway impacting SFK stability.

Main Methods:

• Peptide pull-downs and mass spectrometry were used to identify protein interactions.
• AlphaFold 3 predictions aided in structural analysis.
• siRNA knockdown, CRISPR/Cas9 knockout, and inducible protein variants were employed to study protein function in vivo.
• In vitro ubiquitination assays were performed.

Main Results:

• DCAF10 was identified as the E3 ligase substrate receptor for alternatively N-terminally acetylated SFKs.
• DCAF10 regulates SFK protein levels by recognizing an N-terminal acetylated glycine residue.
• The CUL4A-DDB1-DCAF10 complex ubiquitinates N-terminally acetylated SFKs in vitro.

Conclusions:

• A novel N-degron pathway is defined, targeting SFKs for degradation upon N-terminal acetylation.
• This pathway monitors the switch from myristoylation to acetylation at the N-terminus.
• The findings suggest this degradation mechanism may apply to other N-terminally myristoylated proteins.