CRL4BDCAF11 E3 ligase targets p21 for degradation to control cell cycle progression in human osteosarcoma cells

Zhi Chen,Kaibiao Jiang,Canglong Hou,Zude Liu,Lie Qian,Hongxing Shen,Kun Wang,Guibin Zhong,Caiguo Zhang,Lifeng Lao,Jianwei Chen,Bin Chen

doi:10.1038/s41598-017-01344-9

Abstract

Cell cycle progression in mammals is strictly controlled by a number of cyclin-dependent kinases (CDKs) and CDK inhibitors (CKIs), the expression of which is often dysregulated in cancer cells. Our previous work revealed that Cullin 4B (CUL4B), a critical component of the Cullin4B-RING E3 ligase complex (CRL4B), is overexpressed in human osteosarcoma cells through an unknown mechanism. Here, we demonstrated that CUL4B forms an E3 ligase with RBX1 (RING-box 1), DDB1 (DNA damage binding protein 1), and DCAF11 (DDB1 and CUL4 associated factor 11) in human osteosarcoma cells. In vitro and in vivo ubiquitination analyses indicated that CRL4BDCAF11 E3 ligase was able to specifically ubiquitinate a CDK inhibitor—p21Cip1 at K16, K154, K161 and K163 but not at K75 and K141. Knocking down any component of the CRL4BDCAF11 complex, including CUL4B, DDB1 or DCAF11, using short hairpin RNAs (shRNAs) attenuated the ubiquitination level of p21Cip1, inhibited osteosarcoma cell proliferation, led to cell cycle arrest at S phase, and decreased colony formation rate. Taken together, our data suggest that the CRL4BDCAF11 complex represents a unique E3 ligase that promotes the ubiquitination of p21Cip1 and regulates cell cycle progression in human osteosarcoma cells.

Highlights

Both prokaryotic and eukaryotic cells are controlled by an ordered series of events known as the cell cycle, which includes the G0, G1, S, G2 and M phases[1, 2]
We discovered that Cullin 4B (CUL4B) interacted with damaged DNA binding protein 1 (DDB1), RING-box protein 1 (RBX1) and DCAF11 to form an E3 ligase known as CRL4BDCAF11
We determined that CRL4BDCAF11 directly targeted p21 for multi-monoubiquitination at K16, K154, K161 and K163 through in vitro and in vivo studies

Summary

Introduction

Both prokaryotic and eukaryotic cells are controlled by an ordered series of events known as the cell cycle, which includes the G0, G1, S, G2 and M phases[1, 2]. Two families of CKIs, including CDK interacting protein/kinase inhibitory protein (Cip/Kip) and inhibitor of kinase 4/alternative reading frame (INK4a/ARF), are able to disrupt cell cycle progression by affecting different CDKs8, 9. Dysregulation of either CDKs or CKIs can disrupt cell cycle progression, thereby resulting in the pathogenesis of a number of diseases, including cancer[10] Expression of these CDKs and CKIs can be regulated at both the transcriptional and post-transcriptional levels. All of the CRL4s in different organisms share a similar core architecture, in which E3 ligase activity is determined by CUL4-RBX1 and substrate specificity is controlled by DCAFs12, 14–17. To illuminate the molecular function of CUL4B, especially to determine interacting proteins and to identify the substrates of CRL4B E3 ligase in osteosarcoma cells, we first confirmed interactions between CUL4B and RBX1 or DDB1 in vivo and in vitro. Our in vivo and in vitro studies support a model in which CRL4BDCAF11 E3 targets p21Cip[1] for ubiquitination to control cell cycle progression in human osteosarcoma cells

Methods

Results

Conclusion