Cullin–RING complexes comprise the largest known class of ubiquitin ligases. Owing to the great diversity of their substrate-receptor subunits, it is possible that there are hundreds of distinct cullin–RING ubiquitin ligases in eukaryotic cells, which establishes these enzymes as key mediators of post-translational protein regulation. In this review, we focus on the composition, regulation and function of cullin–RING ligases, and describe how these enzymes can be characterized by a set of general principles.

Function and regulation of cullin-RING ubiquitin ligases.

The p53 pathway responds to stresses that can disrupt the fidelity of DNA replication and cell division. A stress signal is transmitted to the p53 protein by post-translational modifications. This results in the activation of the p53 protein as a transcription factor that initiates a program of cell cycle arrest, cellular senescence or apoptosis. The transcriptional network of p53-responsive genes produces proteins that interact with a large number of other signal transduction pathways in the cell and a number of positive and negative autoregulatory feedback loops act upon the p53 response. There are at least seven negative and three positive feedback loops described here, and of these, six act through the MDM-2 protein to regulate p53 activity. The p53 circuit communicates with the Wnt-beta-catenin, IGF-1-AKT, Rb-E2F, p38 MAP kinase, cyclin-cdk, p14/19 ARF pathways and the cyclin G-PP2A, and p73 gene products. There are at least three different ubiquitin ligases that can regulate p53 in an autoregulatory manner: MDM-2, Cop-1 and Pirh-2. The meaning of this redundancy and the relative activity of each of these feedback loops in different cell types or stages of development remains to be elucidated. The interconnections between signal transduction pathways will play a central role in our understanding of cancer.

The p53 pathway: positive and negative feedback loops

The p53 protein regulates the transcription of many different genes in response to a wide variety of stress signals. Following DNA damage, p53 regulates key processes, including DNA repair, cell-cycle arrest, senescence and apoptosis, in order to suppress cancer. This Analysis article provides an overview of the current knowledge of p53-regulated genes in these pathways and others, and the mechanisms of their regulation. In addition, we present the most comprehensive list so far of human p53-regulated genes and their experimentally validated, functional binding sites that confer p53 regulation.

Transcriptional control of human p53-regulated genes

Modes of p53 regulation.

Grain weight is one of the most important components of grain yield and is controlled by quantitative trait loci (QTLs) derived from natural variations in crops. However, the molecular roles of QTLs in the regulation of grain weight have not been fully elucidated. Here, we report the cloning and characterization of GW2, a new QTL that controls rice grain width and weight. Our data show that GW2 encodes a previously unknown RING-type protein with E3 ubiquitin ligase activity, which is known to function in the degradation by the ubiquitin-proteasome pathway. Loss of GW2 function increased cell numbers, resulting in a larger (wider) spikelet hull, and it accelerated the grain milk filling rate, resulting in enhanced grain width, weight and yield. Our results suggest that GW2 negatively regulates cell division by targeting its substrate(s) to proteasomes for regulated proteolysis. The functional characterization of GW2 provides insight into the mechanism of seed development and is a potential tool for improving grain yield in crops.

A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase

COP1 (constitutively photomorphogenic 1) is a RING-finger-containing protein that functions to repress plant photomorphogenesis, the light-mediated programme of plant development. Mutants of COP1 are constitutively photomorphogenic, and this has been attributed to their inability to negatively regulate the proteins LAF1 (ref. 1) and HY5 (ref. 2). The role of COP1 in mammalian cells is less well characterized. Here we identify the tumour-suppressor protein p53 as a COP1-interacting protein. COP1 increases p53 turnover by targeting it for degradation by the proteasome in a ubiquitin-dependent fashion, independently of MDM2 or Pirh2, which are known to interact with and negatively regulate p53. Moreover, COP1 serves as an E3 ubiquitin ligase for p53 in vitro and in vivo, and inhibits p53-dependent transcription and apoptosis. Depletion of COP1 by short interfering RNA (siRNA) stabilizes p53 and arrests cells in the G1 phase of the cell cycle. Furthermore, we identify COP1 as a p53-inducible gene, and show that the depletion of COP1 and MDM2 by siRNA cooperatively sensitizes U2-OS cells to ionizing-radiation-induced cell death. Overall, these results indicate that COP1 is a critical negative regulator of p53 and represents a new pathway for maintaining p53 at low levels in unstressed cells.

The ubiquitin ligase COP1 is a critical negative regulator of p53.

The ataxia telangiectasia mutated (ATM) protein kinase is a critical component of a DNA-damage response network configured to maintain genomic integrity. The abundance of an essential downstream effecter of this pathway, the tumor suppressor protein p53, is tightly regulated by controlled degradation through COP1 and other E3 ubiquitin ligases, such as MDM2 and Pirh2; however, the signal transduction pathway that regulates the COP1-p53 axis following DNA damage remains enigmatic. We observed that in response to DNA damage, ATM phosphorylated COP1 on Ser(387) and stimulated a rapid autodegradation mechanism. Ionizing radiation triggered an ATM-dependent movement of COP1 from the nucleus to the cytoplasm, and ATM-dependent phosphorylation of COP1 on Ser(387) was both necessary and sufficient to disrupt the COP1-p53 complex and subsequently to abrogate the ubiquitination and degradation of p53. Furthermore, phosphorylation of COP1 on Ser(387) was required to permit p53 to become stabilized and to exert its tumor suppressor properties in response to DNA damage.

https://science.sciencemag.org/content/sci/313/5790/1122.full.pdf

Harumi Shimizu

Papers

The ubiquitin ligase COP1 is a critical negative regulator of p53.

ATM Engages Autodegradation of the E3 Ubiquitin Ligase COP1 After DNA Damage