The proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication and damage repair. How its function is controlled remains an important question. Here, we show that the chromatin-bound PCNA protein is phosphorylated on Tyr 211, which is required for maintaining its function on chromatin and is dependent on the tyrosine kinase activity of EGF receptor (EGFR) in the nucleus. Phosphorylation on Tyr 211 by EGFR stabilizes chromatin-bound PCNA protein and associated functions. Consistently, increased PCNA Tyr 211 phosphorylation coincides with pronounced cell proliferation, and is better correlated with poor survival of breast cancer patients, as well as nuclear EGFR in tumours, than is the total PCNA level. These results identify a novel nuclear mechanism linking tyrosine kinase receptor function with the regulation of the PCNA sliding clamp.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is frequently over-expressed in human cancers and is associated with tumorigenesis, and increased tumor proliferation and progression. Also found in breast tumors with high levels is B-Myb, a transcription factor whose expression is activated by E2F1/3 at the late G1 phase and the level is sustained through the S phase. Recent reports suggest a casual correlation between EGFR and B-Myb expression in primary breast carcinomas. However, the mechanism for such co-expression remains un-investigated. Here, we report that EGFR is important for B-Myb expression and the underlying mechanism involves cooperated effects from EGFR and E2F1. EGF stimulation and forced expression of EGFR significantly increase B-Myb gene activity and such increase occurs in the G1 phase. EGF-induced B-Myb expression was not significantly suppressed following inhibition of PI-3K and ERK, two major EGFR downstream pathways. In contrast, we observed EGF-induced in vivo association of nuclear EGFR to the B-Myb promoter and the association is only detected at the G1/S phase and is abolished by EGFR kinase inhibitor. As EGFR lacks DNA-binding domain but contains transactivational activity and E2F1 activates B-Myb expression in the G1/S phase, we further reasoned that nuclear EGFR might cooperate with E2F1 leading to activation of B-Myb. Indeed, we found that EGFR co-immunoprecipitated with E2F1 in an EGF-dependent manner and that EGF activated in vivo binding of E2F1 to the B-Myb promoter. Consistently, forced expression of both EGFR and E2F1 in EGFR-null CHO cells greatly enhanced B-Myb promoter activity, compared to the vector control and expression of EGFR or E2F1 alone. Promoter mutagenesis studies showed that EGF-induced activation of B-Myb promoter required both E2F and EGFR target sites. In summary, our data suggest that deregulated EGFR signaling pathway facilitate tumor cell proliferation partly via EGFR interaction with E2F1 and subsequent activation of B-Myb gene expression.

The activity of E2F transcription factors plays a crucial role in mammalian cell-cycle progression and is controlled by physical association with the pocket proteins (pRb and its related p107 and p130). The E2F1 promoter, which contains two overlapping E2F-binding sites, is activated at the G1/S transition in an E2F-dependent manner. Mutational experiments have shown that the distal E2F-binding site on the E2F1 promoter is required for transcriptional repression in the G0 phase, whereas the proximal E2F-binding site contributes to transcriptional activation at the G1/S boundary. Consistent with these results, chromatin immunoprecipitation assays have revealed that the E2F4/p130 repressor complex specifically binds to the distal E2F-binding site, whereas E2F1 and E2F3 activators preferentially bind to the proximal E2F-binding site. The assays also showed that the specific binding of E2F4/p130 complex to the distal site was dramatically impaired by a mutation introduced into the contiguous repression site (cell Cycle gene Homology Region; CHR). Taken together, these findings indicate that the two E2F-binding sites play distinct roles in the regulation of E2F1 transcription by interacting with different sets of E2F members and cooperating with the contiguous repressor element.

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