Abstract
Substrates of the E3 ubiquitin ligase CRL4(Cdt2), including Cdt1 and p21, contain a PCNA-binding motif called a PIP box. Upon binding of the PIP box to PCNA on chromatin, CRL4(Cdt2) is recruited and the substrate is ubiquitylated. Importantly, a PIP box cannot be sufficient for destruction, as most PIP box proteins are stable. Using Xenopus egg extracts, we identify two sequence elements in CRL4(Cdt2) substrates that promote their proteolysis: a specialized PIP box that confers exceptionally efficient PCNA binding and a basic amino acid 4 residues downstream of the PIP box, which recruits CRL4(Cdt2) to the substrate-PCNA complex. We also identify two mechanisms that couple CRL4(Cdt2)-dependent proteolysis to the chromatin-bound form of PCNA, ensuring that this proteolysis pathway is active only in S phase or after DNA damage. Thus, CRL4(Cdt2) recognizes an unusual degron, which is assembled specifically on chromatin via the binding of a specialized PIP box to PCNA.
Publication types
-
Research Support, N.I.H., Extramural
MeSH terms
-
Amino Acid Sequence
-
Animals
-
Binding Sites / genetics
-
Cell Cycle Proteins / genetics
-
Cell Cycle Proteins / metabolism
-
Chromatin / metabolism*
-
Cyclin-Dependent Kinase Inhibitor p21 / chemistry
-
Cyclin-Dependent Kinase Inhibitor p21 / genetics
-
Cyclin-Dependent Kinase Inhibitor p21 / metabolism
-
DNA-Binding Proteins / genetics
-
DNA-Binding Proteins / metabolism
-
Flap Endonucleases / genetics
-
Flap Endonucleases / metabolism
-
Humans
-
Immunoprecipitation
-
Models, Molecular
-
Molecular Sequence Data
-
Multiprotein Complexes / genetics
-
Multiprotein Complexes / metabolism
-
Mutation
-
Proliferating Cell Nuclear Antigen / chemistry
-
Proliferating Cell Nuclear Antigen / genetics
-
Proliferating Cell Nuclear Antigen / metabolism*
-
Protein Binding
-
Protein Structure, Tertiary
-
Sequence Homology, Amino Acid
-
Ubiquitin-Protein Ligases / genetics
-
Ubiquitin-Protein Ligases / metabolism*
-
Xenopus
-
Xenopus Proteins / genetics
-
Xenopus Proteins / metabolism*
Substances
-
Cdt1 protein, Xenopus
-
Cell Cycle Proteins
-
Chromatin
-
Cyclin-Dependent Kinase Inhibitor p21
-
DNA-Binding Proteins
-
Multiprotein Complexes
-
Proliferating Cell Nuclear Antigen
-
Xenopus Proteins
-
Ubiquitin-Protein Ligases
-
Flap Endonucleases