A new CULLIN 1 mutant has altered responses to hormones and light in Arabidopsis

Regulated protein degradation contributes to plant development by mediating signaling events in many hormone, light, and developmental pathways. Ubiquitin ligases recognize and ubiquitinate target proteins for subsequent degradation by the 26S proteasome. The multisubunit SCF is the best-studied class of ubiquitin ligases in Arabidopsis (Arabidopsis thaliana). However, the extent of SCF participation in signaling networks is unclear. SCFs are composed of four subunits: CULLIN 1 (CUL1), ASK, RBX1, and an F-box protein. Null mutations in CUL1 are embryo lethal, limiting insight into the role of CUL1 and SCFs in later stages of development. Here, we describe a viable and fertile weak allele of CUL1, called cul1-6. cul1-6 plants have defects in seedling and adult morphology. In addition to reduced auxin sensitivity, cul1-6 seedlings are hyposensitive to ethylene, red, and blue light conditions. An analysis of protein interactions with the cul1-6 gene product suggests that both RUB (related to ubiquitin) modification and interaction with the SCF regulatory protein CAND1 (cullin associated and neddylation dissociated) are disrupted. These findings suggest that the morphological defects observed in cul1-6 plants are caused by defective SCF complex formation. Characterization of weak cul1 mutants provides insight into the role of SCFs throughout plant growth and development.     

Molecular and functional characterization of Arabidopsis Cullin 3A

Cullin proteins, which belong to multigenic families in all eukaryotes, associate with other proteins to form ubiquitin protein ligases (E3s) that target substrates for proteolysis by the 26S proteasome. Here, we present the molecular and genetic characterization of a plant Cullin3. In contrast to fungi and animals, the genome of the model plant Arabidopsis thaliana contains two related CUL3 genes, called CUL3A and CUL3B. We found that CUL3A is ubiquitously expressed in plants and is able to interact with the ring-finger protein RBX1. A genomic search revealed the existence of at least 76 BTB-domain proteins in Arabidopsis belonging to 11 major families. Yeast two-hybrid experiments indicate that representative members of certain families are able to physically interact with both CUL3A and CUL3B, suggesting that Arabidopsis CUL3 forms E3 protein complexes with certain BTB domain proteins. In order to determine the function of CUL3A, we used a reverse genetic approach. The cul3a null mutant flowers slightly later than the control plants. Furthermore, this mutant exhibits a reduced sensitivity of the inhibition of hypocotyl growth in far-red light and miss-expresses COP1. The viability of the mutant plants suggests functional redundancy between the two CUL3 genes in Arabidopsis.     

CUL4 associates with DDB1 and DET1 and its downregulation affects diverse aspects of development in Arabidopsis thaliana

Cullins are central scaffolding subunits in eukaryotic E3 ligases that facilitate the ubiquitination of target proteins. Arabidopsis contains at least 11 cullin proteins but only a few of them have been assigned biological roles. In this work Arabidopsis cullin 4 is shown to assemble with DDB1, RBX1, DET1 and DDB2 in vitro and in planta. In addition, by using T-DNA insertion and CUL4 antisense lines we demonstrate that corresponding mutants are severely affected in different aspects of development. Reduced CUL4 expression leads to a reduced number of lateral roots, and to abnormal vascular tissue and stomatal development. Furthermore, cul4 mutants display a weak constitutive photomorphogenic phenotype. These results therefore assign an important function to CUL4 during plant development and provide strong evidence that CUL4 assembles together with RBX1 and DDB1 proteins to form a functional E3 ligase in Arabidopsis.     

E3 ubiquitin ligase Cullin4B mediated polyubiquitination of p53 for its degradation

Controlled protein ubiquitination through E3 ubiquitin ligases and degradation via 26S proteasome machinery is required for orderly progression through cell cycle, chromatin remodeling, DNA repair, and development. Each cullin-dependent ubiquitin ligase (E3) complex can recruit various substrates for their degradation. Cullin 4A (CUL4A) and Cullin 4B (CUL4B) are members of cullin family proteins that mediate ubiquitin dependent proteolysis. Though, these two cul4 genes are functionally redundant, Cullin 4B is not a substitute for all the Cullin 4A functions. Published report has shown that CUL4A interacts with p53 and induces its decay. Although, CUL4A has been known to control several cellular processes, little is known about CUL4B functions. Therefore, in this study, we analyzed the role of CUL4B on p53 polyubiquitination. Our stable cell line and transient transfection studies show that CUL4B indeed interacts with p53 and induces its polyubiquitination. Importantly, both CUL4A and CUL4B overexpressing cells show almost equal levels of p53 polyubiquitination. Moreover, we observed an increased level of polyubiquitination on p53 in CUL4B overexpressing stable cell line upon treatment with siRNA specific for CUL4A indicating that CUL4B plays a vital role in p53 stability. In addition, we have observed the differential expression of CUL4B in various eukaryotic cell lines and mouse tissues suggesting the important role of CUL4B in various tissues. Together, these observations establish an important negative regulatory role of CUL4B on p53 stability.     

Plant CULLIN-based E3s: phytohormones come first

CULLIN (CUL)-dependent ubiquitin ligases form a class of structurally related multi-subunit enzymes that control the rapid and selective degradation of important regulatory proteins involved in cell cycle progression and development, among others. Several classes of these E3s are also conserved in plants and genetic analyses, using Arabidopsis thaliana, indicate that they play an important function during plant development and responses to the environment. In this review, we will discuss the molecular composition and function of these enzymes in plants with a major emphasis on phytohormone signal transduction pathways.     

Characterization of Arabidopsis and rice DWD proteins and their roles as substrate receptors for CUL4-RING E3 ubiquitin ligases

A subset of WD40 proteins that contain a DWD motif (for DDB1 binding WD40) is reported to act as substrate receptors for DDB1-CUL4-ROC1 (for Damaged DNA Binding 1-Cullin 4-Regulator of Cullins 1) based E3 ubiquitin ligases in humans. Here, we report 85 Arabidopsis thaliana and 78 rice (Oryza sativa) proteins containing the conserved 16-amino acid DWD motif. We show by yeast two-hybrid and in vivo coimmunoprecipitation that 11 Arabidopsis DWD proteins directly interact with DDB1 and thus may serve as substrate receptors for the DDB1-CUL4 machinery. We further examine whether the DWD protein PRL1 (for Pleiotropic Regulatory Locus 1) may act as part of a CUL4-based E3 ligase. PRL1 directly interacts with DDB1, and prl1 and cul4cs mutants exhibited similar phenotypes, including altered responses to a variety of stimuli. Moreover, AKIN10 (for Arabidopsis SNF1 Kinase Homolog 10) was degraded more slowly in cell extracts of prl1 and cul4cs than in cell extracts of the wild type. Thus, both genetic and biochemical analyses support the conclusion that PRL1 is the substrate receptor of a CUL4-ROC1-DDB1-PRL1 E3 ligase involved in the degradation of AKIN10. This work adds a large new family to the current portfolio of plant E3 ubiquitin ligases.     

Arabidopsis AXR6 encodes CUL1 implicating SCF E3 ligases in auxin regulation of embryogenesis

The AXR6 gene is required for auxin signaling in the Arabidopsis embryo and during postembryonic development. One of the effects of auxin is to stimulate degradation of the Aux/IAA auxin response proteins through the action of the ubiquitin protein ligase SCF(TIR1). Here we show that AXR6 encodes the SCF subunit CUL1. The axr6 mutations affect the ability of mutant CUL1 to assemble into stable SCF complexes resulting in reduced degradation of the SCF(TIR1) substrate AXR2/IAA7. In addition, we show that CUL1 is required for lateral organ initiation in the shoot apical meristem and the inflorescence meristem. These results indicate that the embryonic axr6 phenotype is related to a defect in SCF function and accumulation of Aux/IAA proteins such as BDL/IAA12. In addition, we show that CUL1 has a role in auxin response throughout the life cycle of the plant.     

Cullins 3a and 3b assemble with members of the broad complex/tramtrack/bric-a-brac (BTB) protein family to form essential ubiquitin-protein ligases (E3s) in Arabidopsis

Selective modification of proteins by ubiquitination is directed by diverse families of ubiquitin-protein ligases (or E3s). A large collection of E3s use Cullins (CULs) as scaffolds to form multisubunit E3 complexes in which the CUL binds a target recognition subcomplex and the RBX1 docking protein, which delivers the activated ubiquitin moiety. Arabidopsis and rice contain a large collection of CUL isoforms, indicating that multiple CUL-based E3s exist in plants. Here we show that Arabidopsis CUL3a and CUL3b associate with RBX1 and members of the broad complex/tramtrack/bric-a-brac (BTB) protein family to form BTB E3s. Eighty genes encoding BTB domain-containing proteins were identified in the Arabidopsis genome, indicating that a diverse array of BTB E3s is possible. In addition to the BTB domain, the encoded proteins also contain various other interaction motifs that likely serve as target recognition elements. DNA microarray analyses show that BTB genes are expressed widely in the plant and that tissue-specific and isoform-specific patterns exist. Arabidopsis defective in both CUL3a and CUL3b are embryo-lethal, indicating that BTB E3s are essential for plant development.     

X-linked mental retardation gene CUL4B targets ubiquitylation of H3K4 methyltransferase component WDR5 and regulates neuronal gene expression

CUL4B, encoding a scaffold protein for the assembly of Cullin4B-Ring ubiquitin ligase (CRL4B) complexes, is frequently mutated in X-linked mental retardation (XLMR) patients. Here, we show that CUL4B, but not its paralog, CUL4A, targets WDR5, a core subunit of histone H3 lysine 4 (H3K4) methyltransferase complexes, for ubiquitylation and degradation in the nucleus. Knocking down CUL4B increases WDR5 and trimethylated H3K4 (H3K4me3) on the neuronal gene promoters and induces their expression. Furthermore, CUL4B depletion suppresses neurite outgrowth of PC12 neuroendocrine cells, which can be rescued by codepletion of WDR5. XLMR-linked mutations destabilize CUL4B and impair its ability to support neurite outgrowth of PC12 cells. Our results identify WDR5 as a critical substrate of CUL4B in regulating neuronal gene expression and suggest epigenetic change as a common pathogenic mechanism for CUL4B-associated XLMR.     

Arabidopsis CULLIN3 Genes Regulate Primary Root Growth and Patterning by Ethylene-Dependent and -Independent Mechanisms

CULLIN3 (CUL3) together with BTB-domain proteins form a class of Cullin-RING ubiquitin ligases (called CRL3s) that control the rapid and selective degradation of important regulatory proteins in all eukaryotes. Here, we report that in the model plant Arabidopsis thaliana, CUL3 regulates plant growth and development, not only during embryogenesis but also at post-embryonic stages. First, we show that CUL3 modulates the emission of ethylene, a gaseous plant hormone that is an important growth regulator. A CUL3 hypomorphic mutant accumulates ACS5, the rate-limiting enzyme in ethylene biosynthesis and as a consequence exhibits a constitutive ethylene response. Second, we provide evidence that CUL3 regulates primary root growth by a novel ethylene-dependant pathway. In particular, we show that CUL3 knockdown inhibits primary root growth by reducing root meristem size and cell number. This phenotype is suppressed by ethylene-insensitive or resistant mutations. Finally, we identify a function of CUL3 in distal root patterning, by a mechanism that is independent of ethylene. Thus, our work highlights that CUL3 is essential for the normal division and organisation of the root stem cell niche and columella root cap cells.     

Dimerization of CUL7 and PARC is not required for all CUL7 functions and mouse development

CUL7, a recently identified member of the cullin family of E3 ubiquitin ligases, forms a unique SCF-like complex and is required for mouse embryonic development. To further investigate CUL7 function, we sought to identify CUL7 binding proteins. The p53-associated, parkin-like cytoplasmic protein (PARC), a homolog of CUL7, was identified as a CUL7-interacting protein by mass spectrometry. The heterodimerization of PARC and CUL7, as well as homodimerization of PARC and CUL7, was confirmed in vivo. To determine the biological role of PARC by itself and in conjunction with CUL7, a targeted deletion of Parc was created in the mouse. In contrast to the neonatal lethality of the Cul7 knockout mice, Parc knockout mice were born at the expected Mendelian ratios and exhibited no apparent phenotype. Additionally, Parc deletion did not appear to affect the stability or function of p53. These results suggest that PARC and CUL7 form an endogenous complex and that PARC and CUL7 functions are at least partially nonoverlapping. In addition, although PARC and p53 form a complex, the absence of effect of Parc deletion on p53 stability, localization, and function suggests that p53 binding to PARC may serve to control PARC function.     

NEDD8 modification of CUL1 dissociates p120(CAND1), an inhibitor of CUL1-SKP1 binding and SCF ligases

Cullin proteins assemble a large number of RING E3 ubiquitin ligases and regulate various physiological processes. Covalent modification of cullins by the ubiquitin-like protein NEDD8 activates cullin ligases through an as yet undefined mechanism. We show here that p120(CAND1) selectively binds to unneddylated CUL1 and is dissociated by CUL1 neddylation. CAND1 formed a ternary complex with CUL1 and ROC1. CAND1 dissociated SKP1 from CUL1 and inhibited SCF ligase activity in vitro. Suppression of CAND1 in vivo increased the level of the CUL1-SKP1 complex. We suggest that by restricting SKP1-CUL1 interaction, CAND1 regulated the assembly of productive SCF ubiquitin ligases, allowing a common CUL1-ROC core to be utilized by a large number of SKP1-F box-substrate subcomplexes.     

Arabidopsis has two redundant Cullin3 proteins that are essential for embryo development and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase complexes in vivo

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.     

Arabidopsis CAND1, an unmodified CUL1-interacting protein, is involved in multiple developmental pathways controlled by ubiquitin/proteasome-mediated protein Degradation

Ubiquitin/proteasome-mediated protein degradation controls various developmental pathways in eukaryotes. Cullin-containing complexes are both versatile and abundant groups of RING family ubiquitin E3 ligases, whose activities are subject to control by RUB/Nedd8 (for related to ubiquitin/neural precursor cell-expressed developmentally downregulated 8) modification of their cullin subunits. Here, we report the identification of an Arabidopsis thaliana counterpart of human CAND1 (cullin-associated and neddylation-dissociated) and demonstrate that it can preferentially interact with unmodified CUL1. The Arabidopsis cand1-1 null mutant displays distinct phenotypes, including late flowering, aerial rosettes, floral organ defects, low fertility, dwarfism, loss of apical dominance, and altered responses to multiple plant hormones. Molecular analyses show that many of these defects are because of compromised activity of CUL1-containing ubiquitin E3 ligases, indicating that CAND1 is required for their optimal activity. Furthermore, the cand1-1 mutant displays a partial constitutive photomorphogenic phenotype and has defects in HY5 degradation in the absence of light, a process mediated by a different RING family E3, COP1. Thus, our data provides genetic support for a critical role of CAND1 in regulating various ubiquitin E3 ligases and their targeted cellular and developmental pathways.     

CUL3 and protein kinases: Insights from PLK1/KLHL22 interaction

Posttranslational mechanisms drive fidelity of cellular processes. Phosphorylation and ubiquitination of substrates represent very common, covalent, posttranslational modifications and are often co-regulated. Phosphorylation may play a critical role both by directly regulating E3-ubiquitin ligases and/or by ensuring specificity of the ubiquitination substrate. Importantly, many kinases are not only critical regulatory components of these pathways but also represent themselves the direct ubiquitination substrates. Recent data suggest the role of CUL3-based ligases in both proteolytic and non-proteolytic regulation of protein kinases. Our own recent study identified the mitotic kinase PLK1 as a direct target of the CUL3 E3-ligase complex containing BTB-KELCH adaptor protein KLHL22.¹ In this study, we aim at gaining mechanistic insights into CUL3-mediated regulation of the substrates, in particular protein kinases, by analyzing mechanisms of interaction between KLHL22 and PLK1. We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination. Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes. We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases.     

AXL and AXR1 have redundant functions in RUB conjugation and growth and development in Arabidopsis

Cullin-RING ubiquitin-protein ligases such as the Skp1, cullin, F-box protein (SCF) have been implicated in many growth and developmental processes in plants. Normal SCF function requires that the CUL1 subunit be post-translationally modified by related to ubiquitin (RUB), a protein related to ubiquitin. This process is mediated by two enzymes: the RUB-activating and RUB-conjugating enzymes. In Arabidopsis, the RUB-activating enzyme is a heterodimer consisting of AXR1 and ECR1. Mutations in the AXR1 gene result in a pleiotropic phenotype that includes resistance to the plant hormone auxin. Here we report that the AXL (AXR1-like) gene also functions in the RUB conjugation pathway. Overexpression of AXL in the axr1-3 background complements the axr1-3 phenotype. Biochemical analysis indicates that AXL overexpression restores CUL1 modification to the wild-type level, indicating that AXR1 and AXL have the same biochemical activity. Although the axl mutant resembles wild-type plants, the majority of axr1 axl-1 double mutants are embryo or seedling lethal. Furthermore, the axl-1 mutation reveals novel RUB-dependent processes in embryo development. We conclude that AXR1 and AXL function redundantly in the RUB conjugating pathway.