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.

In the interleukin 3-dependent hematopoietic cell line Ba/F3, inhibition of mitogen-activated protein kinase, a member of the MAPK/c-Jun N-terminal kinase/stressactivated protein kinase kinase family that plays an important role in cell growth and death control, rapidly leads to severe apoptosis. However, most of the antiapoptotic substrates of MAPK remain to be identified. Here we report that, upon interleukin-3 stimulation of Ba/F3 cells, the transcription factor GATA-1 is strongly phosphorylated at residue serine 26 by a MAPK-dependent pathway. Phosphorylation of GATA-1 increases GATA-1-mediated transcription of the E4bp4 survival gene without significantly changing the DNA-binding affinity of GATA-1. Further characterization of GATA-1 phosphorylation site mutants revealed that the antiapoptotic function of GATA-1 is strongly dependent upon its phosphorylation at the Ser-26 position and is probably mediated through its up-regulation of Bcl-X L expression. Taken together, our data demonstrate that MAPK-dependent GATA-1 phosphorylation is important for its transactivation of the E4bp4 gene, Bcl-X L expression and cell survival. Therefore, GATA-1 may represent a novel MAPK substrate that plays an essential role in a cytokine-mediated antiapoptotic response.