Arabidopsis COP1 SUPPRESSOR 2 Represses COP1 E3 Ubiquitin Ligase Activity through Their Coiled-Coil Domains Association

CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) functions as an E3 ubiquitin ligase and mediates a variety of developmental processes in Arabidopsis by targeting a number of key regulators for ubiquitination and degradation. Here, we identify a novel COP1 interacting protein, COP1 SUPPRESSOR 2 (CSU2). Loss of function mutations in CSU2 suppress the constitutive photomorphogenic phenotype of cop1-6 in darkness. CSU2 directly interacts with COP1 via their coiled-coil domains and is recruited by COP1 into nuclear speckles in living plant cells. Furthermore, CSU2 inhibits COP1 E3 ubiquitin ligase activity in vitro, and represses COP1 mediated turnover of HY5 in cell-free extracts. We propose that in csu2 cop1-6 mutants, the lack of CSU2’s repression of COP1 allows the low level of COP1 to exhibit higher activity that is sufficient to prevent accumulation of HY5 in the dark, thus restoring the etiolated phenotype. In addition, CSU2 is required for primary root development under normal light growth condition.     

Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1 interacting protein 8 (CIP8)

Arabidopsis COP1 is a negative regulator of photomorphogenesis, which targets HY5, a positive regulator of photomorphogenesis, for degradation via the proteasome pathway in the absence of light. COP1 and its interactive partner CIP8 both possess RING finger motifs, characteristic of some E3 ubiquitin ligases. Here we show that CIP8 promotes ubiquitin attachment to HY5 in E2-dependent fashion in vitro. CIP8 exhibits a strong interaction with the E2 enzyme AtUBC8 through its N-terminal domain. Phosphorylation of HY5 by casein kinase II requires the beta subunit 2, but does not affect HY5's susceptibility to ubiquitination. The RING domain of CIP8 is required but is not sufficient for ubiquitin ligase activity. Although the RING domain of CIP8 interacts with the RING domain of COP1, addition of recombinant COP1 fails to affect CIP8's ubiquitin ligase activity towards HY5 in vitro. However, recombinant COP1 can pull-down native CIP8 from the extract of dark-grown seedlings, but not from the extract of light-grown seedlings in a column-binding assay, implying a requirement for light-regulated modification in vivo. Our data suggest that CIP8 can form a minimal ubiquitin ligase in co-operation with the E2 enzyme AtUBC8. It is possible that the AtUBC8-CIP8 module might interact with COP1 in vivo, thereby participating in proteasome-mediated degradation of HY5.     

An Arabidopsis SUMO E3 Ligase,SIZ1, Negatively Regulates Photomorphogenesis by Promoting COP1 Activity

COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a ubiquitin E3 ligase, is a central negative regulator of photomorphogenesis. However, how COP1 activity is regulated by post-translational modifications remains largely unknown. Here we show that SUMO (small ubiquitin-like modifier) modification enhances COP1 activity. Loss-of-function siz1 mutant seedlings exhibit a weak constitutive photomorphogenic phenotype. SIZ1 physically interacts with COP1 and mediates the sumoylation of COP1. A K193R substitution in COP1 blocks its SUMO modification and reduces COP1 activity in vitro and in planta. Consistently, COP1 activity is reduced in siz1 and the level of HY5, a COP1 target protein, is increased in siz1. Sumoylated COP1 may exhibits higher transubiquitination activity than does non-sumoylated COP1, but SIZ1-mediated SUMO modification does not affect COP1 dimerization, COP1-HY5 interaction, and nuclear accumulation of COP1. Interestingly, prolonged light exposure reduces the sumoylation level of COP1, and COP1 mediates the ubiquitination and degradation of SIZ1. These regulatory mechanisms may maintain the homeostasis of COP1 activity, ensuing proper photomorphogenic development in changing light environment. Our genetic and biochemical studies identify a function for SIZ1 in photomorphogenesis and reveal a novel SUMO-regulated ubiquitin ligase, COP1, in plants.     

COP1 functions as a FoxO1 ubiquitin E3 ligase to regulate FoxO1-mediated gene expression

COP1 is a Ring-Finger E3 ubiquitin ligase that is involved in plant development, mammalian cell survival, growth, and metabolism. Here we report that COP1, whose expression is enhanced by insulin, regulates FoxO1 protein stability. We found that in Fao hepatoma cells, ectopic expression of COP1 decreased, whereas knockdown of COP1 expression increased the level of endogenous FoxO1 protein without impacting other factors such as C/EBPalpha and CREB (cAMP-response element-binding protein). We further showed that COP1 binds FoxO1, enhances its ubiquitination, and promotes its degradation via the ubiquitin-proteasome pathway. To determine the biological significance of COP1-mediated FoxO1 protein degradation, we have examined the impact of COP1 on FoxO1-mediated gene expression and found that COP1 suppressed FoxO1 reporter gene as well as FoxO1 target genes such as glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two key targets for FoxO1 in the regulation of gluconeogenesis, with corresponding changes of hepatic glucose production in Fao cells. We suggest that by functioning as a FoxO1 E3 ligase, COP1 may play a role in the regulation of hepatic glucose metabolism.     

COP1 re‐accumulates in the nucleus under shade

Shade‐avoider plants typically respond to shade‐light signals by increasing the rate of stem growth. CONSTITTUTIVE PHOTOMORPHOGENESIS 1 (COP1) is an E3 ligase involved in the ubiquitin labelling of proteins targeted for degradation. In dark‐grown seedlings, COP1 accumulates in the nucleus and light exposure causes COP1 migration to the cytosol. Here, we show that in Arabidopsis thaliana, COP1 accumulates in the nucleus under natural or simulated shade, despite the presence of far‐red light. In plants grown under white light, the transfer to shade‐light conditions triggers an unexpectedly rapid re‐accumulation of COP1 in the nucleus. The partial simulation of shade by lowering either blue or red light levels (maintaining far‐red light) caused COP1 nuclear re‐accumulation. Hypocotyl growth of wild‐type seedlings is more sensitive to afternoon shade than to morning shade. A residual response to shade was observed in the cop1 mutant background, but these seedlings showed inverted sensitivity as they responded to morning shade and not to afternoon shade. COP1 overexpression exaggerated the wild‐type pattern by enhancing afternoon sensitivity and making morning shade inhibitory of growth. COP1 nuclear re‐accumulation also responded more strongly to afternoon shade than to morning shade. These results are consistent with a signalling role of COP1 in shade avoidance. We propose a function of COP1 in setting the daily patterns of sensitivity to shade in the fluctuating light environments of plant canopies.     

Targeting Proteins for Degradation by Arabidopsis COP1： Teamwork Is What Matters

Arsbidopsis COP1 （Constitutive Photomorphogenic 1） defines a key repressor of photomorphogenesis in darkness by acting as an E3 ubiquitin Iigase in the nucleus, and is responsible for the targeted degradation of a number of photomorphogenesis-promoting factors, including phyA, HY5, LAF1, and HFR1. Light activation of multiple classes of photoreceptors （including both phytochromes and cryptochromes） inactivates COP1 and reduces its nuclear abundance, allowing the accumulation of these positively acting light signaling intermediates to promote photomorphogenic development. Recent studies suggest that Arabidopsis COP1 teams up with a family of SPA proteins （SPA1-SPA4） to form the physiologically active COP1-SPA E3 ubiquitin ligase complexes. These COP1-SPA complexes play overlapping and distinct functions in regulating seedling photomorphogenesis under different light conditions and adult plant growth. Further, the COP1-SPA complexes act In concert at a biochemical level with the CDD （COP10, DET1, and DDB1） complex and COP9 signalosome （CSN） to orchestrate the repression of photomorphogenesis.     

A COP1‐PIF‐HEC regulatory module fine‐tunes photomorphogenesis in Arabidopsis

Light responses mediated by the photoreceptors play crucial roles in regulating different aspects of plant growth and development. An E3 ubiquitin ligase complex CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)1/SUPPRESSOR OF PHYA (SPA), one of the central repressors of photomorphogenesis, is critical for maintaining skotomorphogenesis. It targets several positive regulators of photomorphogenesis for degradation in darkness. Recently, we revealed that basic helix‐loop‐helix factors, HECATEs (HECs), function as positive regulators of photomorphogenesis by directly interacting and antagonizing the activity of another group of repressors called PHYTOCHROME‐INTERACTING FACTORs (PIFs). It was also shown that HECs are partially degraded in the dark through the ubiquitin/26S proteasome pathway. However, the underlying mechanism of HEC degradation in the dark is still unclear. Here, we show that HECs also interact with both COP1 and SPA proteins in darkness, and that HEC2 is directly targeted by COP1 for degradation via the ubiquitin/26S proteasome pathway. Moreover, COP1‐mediated polyubiquitylation and degradation of HEC2 are enhanced by PIF1. Therefore, the ubiquitylation and subsequent degradation of HECs are significantly reduced in both cop1 and pif mutants. Consistent with this, the hec mutants partially suppress photomorphogenic phenotypes of both cop1 and pifQ mutants. Collectively, our work reveals that the COP1/SPA‐mediated ubiquitylation and degradation of HECs contributes to the coordination of skoto/photomorphogenic development in plants.     

X-linked intellectual disability gene CUL4B targets Jab1/CSN5 for degradation and regulates bone morphogenetic protein signaling

Cullin 4B (CUL4B) is a scaffold protein involved in the assembly of cullin-RING ubiquitin ligase (E3) complexes. Contemporary reports have identified multiple mutations of CUL4B gene as being causally associated with X-linked intellectual disability (XLID). Identifying the specific protein substrates will help to better understand the physiological functions of CUL4B. The current study identified Jun activation domain-binding protein (Jab1/CSN5) in the COP9 signalosome (CSN) complex as a novel proteolytic target for the CUL4B ubiquitin ligase complex. The impaired degradation of Jab1 was observed in cells after RNAi-mediated CUL4B depletion. Integrity of DDB1-CUL4B-ROC1 was further demonstrated to be indispensable for the degradation of Jab1. In addition, the degradation of Jab1 is independent of CUL4A, a cullin family member closely related to CUL4B. In vitro and in vivo ubiquitination assays revealed that CUL4B promoted the polyubiquitination of Jab1. Interestingly, CUL4B-silenced cells were shown to exhibit abnormal upregulation of bone morphogenetic protein (BMP) signaling. Furthermore, in vivo studies of embryonic fibroblasts in Cul4b-deficient mice demonstrated Jab1 accumulation and increased activation of the BMP signaling pathway. Together, the current findings demonstrate the CUL4B E3 ubiquitin ligase plays a key role in targeting Jab1 for degradation, potentially revealing a previously undocumented mechanism for regulation of the BMP signaling pathway involved with the CUL4B-based E3 complex. This observation may provide novel insights into the molecular mechanisms underlying CUL4B-associated XLID pathogenesis.     

Identification and characterization of an E3 ubiquitin ligase Rbx1 in maize (Zea mays L.)

E3 ubiquitin ligases catalyze the ubiquitination of a variety of biologically significant protein substrates for targeted degradation through the 26S proteasome, as well as for nonproteolytic regulation of their functions or subcellular localizations. Here we report the identification and characterization of an E3 ubiquitin ligase, the Ring box1 (Rbx1) homologue in maize, which is designated as Zm-Rbx1. Analysis of the genomic organization showed that the gene of Zm-Rbx1 belonged to the chromosome 4 of maize and contained five exons and six introns. Amino acids sequence analysis revealed that Zm-Rbx1 contained conserved cysteine/histidine residues, which are the characteristics of Rbx proteins. Real-time PCR analysis revealed that the expression levels of Zm-Rbx1 increased quickly after salicylic acid, jasmonic acid and sugarcane mosaic virus challenge. Then we suggest that Zm-Rbx1 is involved in the defense response of maize, although detailed molecular mechanism needs to be further studied. After prokaryotic expression and purification of the recombinant Zm-Rbx1 protein from Escherichia coli BL21 (DE3) cells, the ubiquitination assay demonstrated that Zm-Rbx1 showed ubiquitin ligase activity.     

An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana

The ubiquitination proteasome pathway has been demonstrated to regulate all plant developmental and signaling processes. E3 ligase/substrate‐specific interactions and ubiquitination play important roles in this pathway. However, due to technical limitations only a few instances of E3 ligase–substrate binding and protein ubiquitination in plants have been directly evidenced. An efficient in vivo and in vitro ubiquitination assay was developed for analysis of protein ubiquitination reactions by agroinfiltration expression of both substrates and E3 ligases in Nicotiana benthamiana. Using a detailed analysis of the well‐known E3 ligase COP1 and its substrate HY5, we demonstrated that this assay allows for fast and reliable detection of the specific interaction between the substrate and the E3 ligase, as well as the effects of MG132 and substrate ubiquitination and degradation. We were able to differentiate between the original and ubiquitinated forms of the substrate in vivo with antibodies to ubiquitin or to the target protein. We also demonstrated that the substrate and E3 ligase proteins expressed by agroinfiltration can be applied to analyze ubiquitination in in vivo or in vitro reactions. In addition, we optimized the conditions for different types of substrate and E3 ligase expression by supplementation with the gene‐silencing suppressor p19 and by time‐courses of sample collection. Finally, by testing different protein extraction buffers, we found that different types of buffer should be used for different ubiquitination analyses. This method should be adaptable to other protein modification studies.     

The F-box Protein FBXO25 Promotes the Proteasome-dependent Degradation of ELK-1 Protein

FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens.     

Arabidopsis ANGUSTIFOLIA3 (AN3) is associated with the promoter of CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) to regulate light‐mediated stomatal development

Light signals are perceived by multiple photoreceptors that converge to suppress the RING E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) for the regulation of stomatal development. Thus, COP1 is a point of integration between light signaling and stomatal patterning. However, how light signaling is collected into COP1 for the production and spacing of stomata is still unknown. Here, we report that the loss‐of‐function mutant of ANGUSTIFOLIA3 (AN3) delays asymmetric cell division, which leads to decreased stomatal index. Furthermore, overexpression of AN3 accelerates asymmetric cell division, which results in clusters of stomata. In addition, the stomatal development through AN3 regulation is mediated by light signaling. Finally, we find that an3 is a light‐signaling mutant, and that AN3 protein is light regulated. Self‐activation by AN3 contributes to the control of AN3 expression. Thus, AN3 is a point of collection between light signaling and stomatal patterning. Target‐gene analysis indicates that AN3 is associated with COP1 promoter for the regulation of light‐controlling stomatal development. Together, these components for regulating stomatal development form an AN3–COP1–E3 ubiquitin ligase complex, allowing the integration of light signaling into the production and spacing of stomata.