E3 ubiquitin ligases and abscisic acid signaling

The ubiquitin proteasome system is involved in the regulation of nearly every aspect of plant growth and development. Protein ubiquitination involves the covalent attachment of ubiquitin to target proteins through a cascade catalyzed by three enzymes known as E1, E2 and E3. E3s are of particular interest as they confer substrate specificity during ubiquitination through their diverse substrate recognition domains. Recently, a number of E3s have been identified that actively participate in abscisic acid hormone biology, including regulation of biosynthesis, de-repression or activation of abscisic acid response and degradation of signaling components. In this review, we summarize recent exciting studies of the different types of E3s that target specific mediators of abscisic acid signaling or affect the plants response to the hormone.Key words: abscisic acid, E3 ubiquitin ligase, proteasome, ubiquitinationPost-translational control of protein degradation by the ubiquitin proteasome system (UPS) is a highly regulated process essential for the proper growth and development of all eukaryotes through removing abnormal proteins and most short-lived regulatory proteins.^1,2 Plants utilize the UPS to alter their proteome to mediate cellular changes required for growth, development and responses to biotic and abiotic stress. Plants also rely a great deal on hormones to induce changes in growth and development in response to a wide range of environmental stimuli. Hormone biosynthesis, perception, signaling and response can be exquisitely regulated through modulating protein levels via the UPS. Regulation of the abscisic acid (ABA) signaling pathway, like auxin, gibberellin, jasmonate and ethylene, have been linked to UPS components with the application of biochemical, genetic and genomic approaches.^3–5 Although some aspects of ABA signaling have been elucidated, the involvement of the UPS, especially E3 ubiquitin ligases, help us gain further insight into the entire network of ABA signal transduction. In this review we focus on recently identified E3s that play a variety of roles in ABA signaling. A number of articles are available that provide a comprehensive review of the role of E3 ligases in the biosynthesis, perception and signaling by other hormones such as auxin and ethylene.^3–5     

Cullin-containing E3 ubiquitin ligases in plant development

In eukaryotes, the ubiquitin-proteasome system participates in the control of signal transduction events by selectively eliminating regulatory proteins. E3 ubiquitin ligases specifically bind degradation substrates and mediate their poly-ubiquitylation, a prerequisite for their degradation by the 26S proteasome. On the basis of the analysis of the Arabidopsis genome sequence, it is predicted that there are more than 1000 E3 ubiquitin ligases in plants. Several types of E3 ubiquitin ligases have already been characterized in eukaryotes. Recently, some of these E3 enzymes have been implicated in specific plant signaling pathways.     

E3 ligases and deubiquitinating enzymes regulating the MAPK signaling pathway in cancers

The mitogen-activated protein kinase (MAPK) signaling pathway is the primary regulatory module of various cellular processes such as cell proliferation, differentiation, and stress responses. This pathway converts external stimuli to cellular responses via three major kinases: mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase (MAPKK), and mitogen-activated protein kinase kinase kinase (MAPKKK). Ubiquitination is a post-translational modification of proteins with ubiquitin, which results in the formation of mono- or poly-ubiquitin chains of substrate proteins. Conversely, removal of the ubiquitin by deubiquitinating enzymes (DUBs) is known as deubiquitination. This review summarizes mechanisms of the MAPK signaling pathways (ERK1/2, ERK5, p38, and JNK1/2/3 signaling pathway) in cancers, and of E3 ligases and DUBs that target the MAPK signaling components such as Raf, MEK1/2, ERK1/2, MEKK2/3, MEKK1-4, TAK1, DLK1, MLK1-4, ASK1/2, and MKK3-7.     

Dma/RNF8 proteins are evolutionarily conserved E3 ubiquitin ligases that target septins

The budding yeast proteins Dma1 and Dma2 are members of the unique FHA-RING domain protein family and are linked to mitotic regulation and septin organization by ill-defined mechanisms. We show that Dma2 has ubiquitin ligase activity, and that septins Shs1 and Cdc11 are likely direct in vivo targets. We further propose that human RNF8, rather than Chfr, is the mammalian Dma homolog. As in yeast, RNF8 localizes to the centrosomes and cell division sites and promotes ubiquitylation of the septin SEPT7, whose depletion increases cell division anomalies. Together, these findings reveal evolutionary and functional conservation of Dma proteins, and suggest that RNF8 maintains genome stability through independent, yet analogous, nuclear and cytoplasmic ubiquitylation activities.     

E3 ubiquitin ligases and mitosis: embracing the complexity

Faithful division of eukaryotic cells requires temporal and spatial coordination of morphological transitions, which ensures that the newly replicated copies of the genome are equally distributed into the two daughter cells during mitosis. One of the mechanisms ensuring the fidelity of mitotic progression is targeted, ubiquitin-dependent proteolysis of key regulators. E3-ubiquitin ligase complexes are crucial components in this pathway because they specifically select the relevant ubiquitination substrates. Cullin-based E3-ligases, such as Cul3, have recently emerged as crucial regulators of mitosis.     

Regulation of apoptosis by E3 ubiquitin ligases in ubiquitin proteasome system

Apoptosis is an organised ATP‐dependent programmed cell death that organisms have evolved to maintain homoeostatic cell numbers and eliminate unnecessary or unhealthy cells from the system. Dysregulation of apoptosis can have serious manifestations culminating into various diseases, especially cancer. Accurate control of apoptosis requires regulation of a wide range of growth enhancing as well as anti‐oncogenic factors. Appropriate regulation of magnitude and temporal expression of key proteins is vital to maintain functional apoptotic signalling. Controlled protein turnover is thus critical to the unhindered operation of the apoptotic machinery, disruption of which can have severe consequences, foremost being oncogenic transformation of cells. The ubiquitin proteasome system (UPS) is one such major cellular pathway that maintains homoeostatic protein levels. Recent studies have found interesting links between these two fundamental cellular processes, wherein UPS depending on the cue can either inhibit or promote apoptosis. A diverse range of E3 ligases are involved in regulating the turnover of key proteins of the apoptotic pathway. This review summarises an overview of key E3 ubiquitin ligases involved in the regulation of the fundamental proteins involved in apoptosis, linking UPS to apoptosis and attempts to emphasize the significance of this relationship in context of cancer.     

The interplay between DSL proteins and ubiquitin ligases in Notch signaling

Lateral inhibition is a pattern refining process that generates single neural precursors from a field of equipotent cells and is mediated via Notch signaling. Of the two Notch ligands Delta and Serrate, only the former was thought to participate in this process. We now show that macrochaete lateral inhibition involves both Delta and Serrate. In this context, Serrate interacts with Neuralized, a ubiquitin ligase that was heretofore thought to act only on Delta. Neuralized physically associates with Serrate and stimulates its endocytosis and signaling activity. We also characterize a mutation in mib1, a Drosophila homolog of mind bomb, another Delta-targeting ubiquitin ligase from zebrafish. Mib1 affects the signaling activity of Delta and Serrate in both lateral inhibition and wing dorsoventral boundary formation. Simultaneous absence of neuralized and mib1 completely abolishes Notch signaling in both aforementioned contexts, making it likely that ubiquitination is a prerequisite for Delta/Serrate signaling.     

Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling

Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub) ligases. We have annotated approximately 617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein - two transmembrane domains mediate its localization to the organelle's outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-kappaB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.     

Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila

Signaling by the Notch ligands Delta (Dl) and Serrate (Ser) regulates a wide variety of essential cell-fate decisions during animal development. Two distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), have been shown to regulate Dl signaling in Drosophila melanogaster and Danio rerio, respectively. While the neur and mib genes are evolutionarily conserved, their respective roles in the context of a single organism have not yet been examined. We show here that the Drosophila mind bomb (D-mib) gene regulates a subset of Notch signaling events, including wing margin specification, leg segmentation, and vein determination, that are distinct from those events requiring neur activity. D-mib also modulates lateral inhibition, a neur- and Dl-dependent signaling event, suggesting that D-mib regulates Dl signaling. During wing development, expression of D-mib in dorsal cells appears to be necessary and sufficient for wing margin specification, indicating that D-mib also regulates Ser signaling. Moreover, the activity of the D-mib gene is required for the endocytosis of Ser in wing imaginal disc cells. Finally, ectopic expression of neur in D-mib mutant larvae rescues the wing D-mib phenotype, indicating that Neur can compensate for the lack of D-mib activity. We conclude that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila.     

PHD domains and E3 ubiquitin ligases: viruses make the connection

PHD domains constitute a widely distributed subfamily of zinc fingers whose biochemical functions have been unclear until now. Recently, several PHD-containing viral proteins have been identified that promote immune evasion by downregulating proteins that govern immune recognition. Studies show that these viral regulators lead to ubiquitination of their targets by functioning as E3 ubiquitin ligases -- an activity that requires the PHD motif. These are the first examples linking the PHD domain to E3 activity, but the recent discovery of PHD-dependent E3 activity in the cellular kinase MEKK1 and the close structural relation of PHD domains to RING fingers hint that many other PHD proteins might share this activity.     

Building and remodelling Cullin–RING E3 ubiquitin ligases

Cullin–RING E3 ubiquitin ligases (CRLs) control a plethora of biological pathways through targeted ubiquitylation of signalling proteins. These modular assemblies use substrate receptor modules to recruit specific targets. Recent efforts have focused on understanding the mechanisms that control the activity state of CRLs through dynamic alterations in CRL architecture. Central to these processes are cycles of cullin neddylation and deneddylation, as well as exchange of substrate receptor modules to re‐sculpt the CRL landscape, thereby responding to the cellular requirements to turn over distinct proteins in different contexts. This review is focused on how CRLs are dynamically controlled with an emphasis on how cullin neddylation cycles are integrated with receptor exchange.     

E3 ubiquitin ligases and their control of T cell autoreactivity

A loss of T cell tolerance underlies the development of most autoimmune diseases. The design of therapeutic strategies to reinstitute immune tolerance, however, is hampered by uncertainty regarding the molecular mechanisms involved in the inactivation of potentially autoreactive T cells. Recently, E3 ubiquitin ligases have been shown to mediate the development of a durable state of unresponsiveness in T cells called clonal anergy. In this review, we will discuss the mechanisms used by E3 ligases to control the activation of T cells and prevent the development of autoimmunity.     

RGLG3 and RGLG4, novel ubiquitin ligases modulating jasmonate signaling

JAs are important hormones for plant development and defense, and JA signaling is regulated by diverse mechanisms. We have recently identified two RING-type ubiquitin ligases, RGLG3 and RGLG4, as essential JA signaling regulators. In this addendum, we discuss some characters of RGLG3 and RGLG4, which further support their important roles in JA pathway. RGLG3 and RGLG4 didn’t interact with known key factors of the core JA pathway, rather, it might target on unknown protein that negatively regulated JA signaling. RGLG3 and RGLG4 expression was suppressed by SA treatment in an NPR1-independent manner, and rglg3 rglg4 moderated SA-inhibited JA-responsive PDF1.2 expression, suggesting RGLG3 and RGLG4 took roles in SA-JA antagonism. RGLG3 and RGLG4 could be important players of a regulatory network and coordinated diverse signals to modulate JA signaling.