Author(s): Huajun Zhao 1 , Po-Chun Ho 1 , Yuan-Hung Lo 1 , Alexsandra Espejo 2 , Mark T. Bedford 2 , Mien-Chie Hung 3 , 4 , 5 , Shao-Chun Wang 1 , *
The c-Abl proto-oncogene is a multi-functional non-receptor tyrosine kinase that shuttles between the cytoplasm and the nucleus . A substantial body of knowledge has been established regarding the mechanisms of c-Abl in regulating cell migration, the response to oxidative stresses, and apoptosis . The c-Abl kinase was originally identified as a frequent target of oncogenic chromosomal translocation in hematopoietic neoplasia, but has been increasingly recognized for its involvement in solid tumors. In lung and breast cancers, deregulated c-Abl kinase contributes to tumor development -. In breast cancer, activated c-Abl kinase promotes cancer progression , , while inhibition of c-Abl blocks the transforming phenotypes by suppressing anchorage-independent growth, inducing apoptosis, and inhibiting cell proliferation . Consistent with this, our recent study showed that increased c-Abl expression is a frequent event in breast cancer (~40%) .
Proliferating cell nuclear antigen (PCNA) is the molecular coordinator in the core DNA synthesis machinery -. PCNA forms a homotrimeric ring encircling the DNA double helix and acts as a molecular platform to recruit proteins involved in DNA synthesis, cell-cycle control, and DNA-damage response and repair , , -. PCNA exists in two distinct forms: the replication-competent chromatin-bound form and the chromatin-unbound form, which is not involved in DNA synthesis . Not much is known about how the two populations of PCNA are regulated. We previously reported that the chromatin-bound PCNA, but not the unbound form, was phosphorylated at Y211 (phospho-Y211) by the EGF receptor . This phosphorylation event was upregulated during the S phase of the cell cycle. Further study demonstrated that this phosphorylation enhanced the stability of chromatin-associated PCNA and enhanced its activity in DNA replication.
In the current study, we identify c-Abl as a binding partner of PCNA and show that Y211 phosphorylation of PCNA serves as a binding signal for PCNA to associate with c-Abl under normal growth conditions and in cells responding to DNA-damage stresses. We further demonstrate that c-Abl promotes chromatin association of PCNA and that Y211 phosphorylation is an important cell growth-related event downstream of c-Abl.
We tested whether Y211 phosphorylation of PCNA can serve as a signaling event that, in turn, regulates its function. To do this, we screened a microarray of functional domains derived from different proteins and tested whether phospho-Y211 preferentially associated with these motifs. Synthetic peptides, encompassing the wild-type sequence surrounding the Y211 residue or the same peptide with phosphorylated Y211, were conjugated to a fluorescent dye (Cy3) and used for probing the microarray (data not shown). The Cy3-conjugated peptide with the non-phosphorylated wild-type sequence did not bind to any of the functional domains. In contrast, incubation with the phosphorylated peptide identified the SH2 domain of the non-receptor tyrosine kinase c-Abl as a phospho-Y211-binding motif. Further verification with co-immunoprecipitation (co-IP) using extracts of MDA-MB-231 and BT474 cells demonstrated...