Identification of ZNF313 as a novel ubiquitin E3 ligase targeting CDK inhibitors

다국어 초록 (Multilingual Abstract)

ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here I report that ZNF313 is a ubiquitin E3 ligase that plays a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XAF1-interacting protein, which up-regulates the stability and proapoptotic effect of XAF1. Intriguingly, it was found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21WAF1. ZNF313 ubiquitinates p21WAF1 and also destabilizes p27KIP1 and p57KIP2, three members of the CIP/KIP family of CDK inhibitors whereas it does not affect the stability of the INK4 family members, such as p16INK4A and p15INK4B. ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21WAF1-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is down-regulated during cellular differentiation process in vitro and in vivo while it is commonly up-regulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21WAF1, whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.

목차 (Table of Contents)

• ABSTRACT I
• TABLE OF CONTENTS II
• LIST OF FIGURES IV
• ABBREVIATIONS VI
• INTRODUCTION 1
• MATERIALS AND METHODS 4
• Cell culture and construction of stable cell lines 4
• Yeast two-hybrid assay 4
• Immunoprecipitation and GST pull-down assay 5
• Immunoblotting, immunofluorescence and immunohistochemistry assay 5
• Semi-quantitative RT-PCR assay 6
• Expression plasmids, siRNA and transfection 6
• Cell cycle, apoptosis and colony formation assay 7
• Senescence associated-β-galactosidase activity assay 7
• Ubiquitination assay 7
• Animal studies 8
• Statistical analysis 8
• RESULTS 9
• Identification of ZNF313 as a novel XAF1-binding protein 9
• ZNF313 increases XAF1 protein stability 9
• ZNF313 is a novel cell cycle activator with proapoptotic potential 10
• ZNF313 is a negative regulator of cellular senescence 10
• ZNF313 is a specific antagonist of the CIP/KIP family members of CDK inhibitors 11
• ZNF313 promotes ubiquitination and degradation of p21WAF1 12
• ZNF313 stimulates a G1 to S phase transition of the cell cycle 13
• ZNF313 is reduced in differentiation process 14
• ZNF313 is elevated in cancer cells 14
• DISCUSSION 49
• REFERENCES 53
• ABSTRACT IN KOREAN 58