Investigation of the novel function of CHFR in DNA damage response and the novel kinases in mitotic progression regulation

Abstract

Checkpoint with FHA and RING finger domains (CHFR) processes a variety of biological functions. It is a prophase checkpoint protein that delays chromosome condensation in the presence of microtubule poisons, a tumor suppressor gene that frequently silenced in many epithelial cancers, and a ubiquitin ligase that can target proteins for degradation or signaling transduction. Poly(ADP-ribose) polymerase-1 (PARP-1) is the leading member of PARP family that functions in early DNA damage responses. PARP-1 is immediately activated and recruited to DNA damage sites and catalyzes poly(ADP-ribosyl)ation, thereby facilitating the recruitment of DNA damage repair and checkpoint proteins. Previous structural studies demonstrated that CHFR contains a poly(ADP-ribose) binding domain at its C-terminus. Biochemical analysis also supported CHFR as a potential poly(ADP-ribosyl)ated target of PARP-1. However, little is known about,:the precise relationship between CHFR and PARP-1 and their physiological functions. In this study, CHFR was found to co-localize with PARP-1 to the nucleus and bind to PARP-1 through the PBZ domain. In addition, a reversed expression pattern of CHFR and PARP-1 was found in several cell lines. CHFR and PARP-1 were able to regulate each other reciprocally. On the one hand, CHFR could downregulate PARP-1 through its E3 ligase activity, suggesting a novel mechanism for the tumor suppressive function of CHFR through regulation of PARP-1. On the other hand, the E3 ligase activity of CHFR was positively regulated by PARP-1, in particular after DNA damage-induced PAPR-1 activation. Consistent with these data, I found that CHFR could promote the activation of DNA damage responses through more efficient activation of ATM. Taken together, these data suggested a novel reciprocal regulation between CHFR and PARP-1, as well as a potential DNA damage response pathway involving the cooperation between CHFR and PARP-1. The main purpose of mitosis is to segregate sister chromatids into two nascent cells, such that each daughter cell inherits one complete set of chromosomes. Protein phosphorylation is one of the main after-translational regulation mechanisms of mitosis. A number of protein kinases such as cyclin-dependent kinases, Aurora kinases, Polo-like kinases (PLKs), NIMA-related kinases, and spindle assembly checkpoints kinases BUB1, BUBR1 and MPS1, are known to control various mitotic processes. However, the whole repertoire of mitotic kinases probably remains to be deciphered. In another study, I sought to identify novel mitotic kinases with a RNA interference approach. Mouse fibroblasts NIH3T3 expressing histone H2B-GFP were transfected with a mouse kinome siRNA library to deplete individual kinases. Based on the increase of mitotic index, 20 out of 588 kinases were identified and then verified by subsequent confirmatory experiments. Among these candidates, six were chosen for further study and their impacts on mitosis were consistently found in mammalian cell models. QSK and BRSK2, both of which belong to the same AMPK-related family, were selected for detailed analysis. Knockdown of QSK and BRSK2 resulted in pronounced increases of mitotic length, which is due to delayed mitotic exit but not mitotic entry. A spectrum of mitotic defects was found after QSK knockdown. Immunoblotting analysis revealed a remarkable mitosis-specific shift-up of QSK and BRSK2 bands, suggesting that QSK and BRSK2 were potential substrates for CDK1. Taken together, this study identified a number of novel mitotic kinase candidates and provides evidence to support QSK and BRSK2 as important mitotic kinases.

Date of Award	2012
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology