The essential function of eIF4E-binding protein 1 (4E-BP1) in translation initiation has been well established; however, the role of 4E-BP1 in normal cell cycle progression is coming to attention. Here, we revealed the role of 4E-BP1 on mitotic regulation and chromosomal DNA dynamics during mitosis. First, we have observed the co-localization of the phosphorylated 4E-BP1 at T37/46 with Polo-like kinase 1 (PLK1) at the centrosomes during. Depression of 4E-BP1 by small interfering RNA in HepG2 or HeLa cells resulted in an increased outcome of polyploidy and aberrant mitosis, including chromosomal DNA misaligned and multi-polar spindles or multiple centrosomes. We observed that 4E-BP1 interacted with PLK1 directly in vitro and in vivo in mitotic cells, and the C-terminal aa 77-118 of 4E-BP1 mediates its interaction with PLK1. PLK1 can phosphorylate 4E-BP1 in vitro. Furthermore, the depletion of 4E-BP1 sensitized HepG2 and HeLa cells to the microtubule disruption agent paclitaxel. These results demonstrate that 4E-BP1, beyond its role in translation regulation, can function as a regulator of mitosis via interacting with PLK1, and possibly plays a role in genomic stability maintaining.

Accurate mitotic regulation is as important as intrinsic DNA repair for maintaining genomic stability. It is believed that these two cellular mechanisms are interconnected with DNA damage. DNA-PKcs is a critical component of the non-homologous end-joining pathway of DNA double-stranded break repair, and it was recently discovered to be involved in mitotic processing. However, the underlying mechanism of DNA-PKcs action in mitotic control is unknown. Here, we demonstrated that depletion of DNA-PKcs led to the dysregulation of mitotic progression in response to DNA damage, which eventually resulted in multiple failures, including failure to segregate sister chromatids and failure to complete cytokinesis, with daughter cells becoming fused again. The depletion of DNA-PKcs resulted in a notable failure of cytokinesis, with a high incidence of multinucleated cells. There were also cytoplasmic bridges containing DNA that continuously connected the daughter cells after DNA damage was induced. Phosphorylated DNA-PKcs (T2609) colocalizes with PLK1 throughout mitosis, including at the centrosomes from prophase to anaphase and at the kinetochores from prometaphase to metaphase, with accumulation at the midbody during cytokinesis. Importantly, DNA-PKcs was found to associate with PLK1 in the mitotic phase, and the depletion of DNA-PKcs resulted in the overexpression of PLK1 due to increased protein stability. However, deficiency in DNA-PKcs attenuated the recruitment of phosphorylated PLK1 to the midbody but not to the kinetochores and centrosomes. Our results demonstrate the functional association of DNA-PKcs with PLK1, especially in chromosomal segregation and cytokinesis control.

Previous investigations have indicated that rutaecarpine activates the vanilloid receptor to evoke calcitonin gene-related peptide (CGRP) release. CGRP has been shown to alleviate cardiac anaphylactic injury. In the present study, the effect of rutaecarpine on cardiac anaphylaxis was examined. Challenge of presensitized guinea-pig hearts with a specific antigen caused marked decreases in coronary flow (CF), left ventricular pressure (LVP) and its derivatives (+/- dp/dt(max)), an increase in heart rate, and prolongation of the P-R interval. Rutaecarpine (0.3 or 1 microM) markedly increased the content of calcitonin gene-related peptide (CGRP) in the coronary effluent and decreased the content of tumor necrosis factor-alpha (TNF-alpha) in myocardial tissues concomitantly with a significant improvement of cardiac function and alleviation of the extension of the P-R interval. Rutaecarpine at the concentration of 1 microM also inhibited the sinus tachycardia. The protective effects of rutaecarpine on cardiac anaphylaxis were abolished by CGRP (8-37), a selective CGRP receptor antagonist. These results suggest that the protective effects of rutaecarpine on cardiac anaphylactic injury are related to inhibition of TNF-alpha production by stimulation of CGRP release.