Stability of c-Myc Protein in Early S Phase Is Regulated by the Interaction with PCNA

The transcription factor c-Myc is known to regulate DNA replication via a non-transcriptional
mechanism by interacting with proteins of the pre-replicative complex. In addition, c-Myc
localizes to DNA replication foci, similarly to Proliferating Cell Nuclear Antigen (PCNA);
however, the significance of this localization remains unclear. Here, we investigated
whether c-Myc interacts with PCNA and analyzed the possible function of this association.
We found a conserved interaction motif, the PCNA-interacting protein (PIP) box, in the
N-terminal region of c-Myc. Confocal microscopy analysis showed co-localization with
PCNA in early S-phase, but not in late S-phase cells. Co-immunoprecipitation from cell
extracts and pull-down of recombinant proteins indicated a direct physical association
between c-Myc and PCNA, which was confirmed in situ by the Proximity Ligation Assay
(PLA). Further experiments demonstrated that c-Myc interacts with CUL4A and DDB1,
components of the Cullin Ring E3 ubiquitin ligase 4 (CRL4) complex, in which PCNA
functions as a cofactor. Mutations in the PIP box of c-Myc, as well as depletion of CUL4A
by RNA interference, resulted in an increased stability of c-Myc protein. These results
suggest that the interaction with PCNA functionally contributes to the regulation of c-Myc
stability in early S phase via the CRL4 complex.