scholarly journals Regulation of FLIP(L) and TRAIL-R2 signalling by the SCFSkp2 Ubiquitin Ligase Complex

2019 ◽  
Author(s):  
JZ Roberts ◽  
C Holohan ◽  
T Sessler ◽  
J Fox ◽  
C. Higgins ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Large Subunit ◽  
Caspase 8 ◽  
Apoptosis Induction ◽  
Splice Form ◽  
Expression Levels ◽  
Ubiquitin Ligase Complex ◽  
Direct Binding ◽  
The Stability ◽  
Ring E3

AbstractDepending on its expression levels, the long splice form of the pseudo-caspase FLIP (FLIP(L)) can act as an inhibitor (high expression) or activator (low expression) of apoptosis induction by the TRAIL-R2 death-inducing signalling complex (DISC); its expression levels are therefore tightly regulated. Here, we demonstrate that the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2) regulates the stability of FLIP(L) (but not the short splice form FLIP(S)), and, unusually, this is mediated by direct binding of FLIP(L) to Cullin-1 rather than via Skp2. By fine mapping the interaction of FLIP(L) with Cullin-1 to the large subunit of its pseudo-caspase domain, we found that the interaction is significantly stronger with FLIP(L)’s DISC-processed p43-form. Importantly, this interaction disrupts the ability of p43-FLIP to interact with FADD, caspase-8 and another DISC component, TRAF2. Moreover, we find that SCFSkp2 associates with TRAIL-R2 constitutively and does so independently of FLIP(L) and other canonical DISC components. Inhibition of Cullin-1 expression (using siRNA) or activity (using a NEDDylation inhibitor, MLN4924) enhanced FLIP(L) and TRAF2 levels at the TRAIL-R2 DISC and enhanced caspase-8 processing. This suggests that processing of FLIP(L) to p43-FLIP at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP’s association with the DISC thereby altering caspase-8 processing. These findings provide important new insights into how FLIP(L) expression and TRAIL-R2 signaling is controlled.

scholarly journals Degradation of the Transcription Factor Gcn4 Requires the Kinase Pho85 and the SCFCDC4 Ubiquitin–Ligase Complex

2000 ◽  
Vol 11 (3) ◽  
pp. 915-927 ◽  
Author(s):  
Ariella Meimoun ◽  
Tsvi Holtzman ◽  
Ziva Weissman ◽  
Helen J. McBride ◽  
David J. Stillman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Ubiquitin Ligase ◽  
Protein A ◽  
Purine Biosynthesis ◽  
Cell Cycle Regulators ◽  
Rich Medium ◽  
Ubiquitin Ligase Complex ◽  
Ubiquitin Conjugating Enzyme ◽  
The Stability

Gcn4, a yeast transcriptional activator that promotes the expression of amino acid and purine biosynthesis genes, is rapidly degraded in rich medium. Here we report that SCFCDC4, a recently characterized protein complex that acts in conjunction with the ubiquitin-conjugating enzyme Cdc34 to degrade cell cycle regulators, is also necessary for the degradation of the transcription factor Gcn4. Degradation of Gcn4 occurs throughout the cell cycle, whereas degradation of the known cell cycle substrates of Cdc34/SCFCDC4 is cell cycle regulated. Gcn4 ubiquitination and degradation are regulated by starvation for amino acids, whereas the degradation of the cell cycle substrates of Cdc34/SCFCDC4 is unaffected by starvation. We further show that unlike the cell cycle substrates of Cdc34/SCFCDC4, which require phosphorylation by the kinase Cdc28, Gcn4 degradation requires the kinase Pho85. We identify the critical target site of Pho85 on Gcn4; a mutation of this site stabilizes the protein. A specific Pho85-Pcl complex that is able to phosphorylate Gcn4 on that site is inactive under conditions under which Gcn4 is stable. Thus, Cdc34/SCFCDC4 activity is constitutive, and regulation of the stability of its various substrates occurs at the level of their phosphorylation.

scholarly journals Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs

2022 ◽  
Author(s):  
Antonio Cuevas-Navarro ◽  
Laura Rodriguez-Muñoz ◽  
Joaquim Grego-Bessa ◽  
Alice Cheng ◽  
Katherine A Rauen ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Null Allele ◽  
E3 Ubiquitin Ligase ◽  
Adaptor Protein ◽  
Loss Of Function ◽  
Ras Gtpases ◽  
Ubiquitin Ligase Complex ◽  
Species Analysis ◽  
Cullin 3 ◽  
Ring E3

RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of canonical RAS GTPases. Here, we have analyzed the phenotypes of LZTR1 loss-of-function mutants in both fruit flies and mice and have demonstrated biochemical dependency on their RIT1 orthologs. Moreover, we show that LZTR1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of RIT1.

scholarly journals Distinct Mechanisms Control the Stability of the Related S-Phase Cyclins Clb5 and Clb6

2006 ◽  
Vol 26 (6) ◽  
pp. 2456-2466 ◽  
Author(s):  
Leisa P. Jackson ◽  
Steven I. Reed ◽  
Steven B. Haase
Keyword(s):  
Ubiquitin Ligase ◽  
S Phase ◽  
Phosphorylation Sites ◽  
Anaphase Promoting Complex ◽  
Functional Differences ◽  
Ubiquitin Ligase Complex ◽  
N Terminus ◽  
Phase Progression ◽  
The Stability ◽  
Related Proteins

ABSTRACT The yeast S-phase cyclins Clb5 and Clb6 are closely related proteins that are synthesized late in G1. Although often grouped together with respect to function, Clb5 and Clb6 exhibit differences in their ability to promote S-phase progression. DNA replication is significantly slowed in clb5Δ mutants but not in clb6Δ mutants. We have examined the basis for the differential functions of Clb5 and Clb6 and determined that unlike Clb5, which is stable until mitosis, Clb6 is degraded rapidly at the G1/S border. N-terminal deletions of CLB6 were hyperstabilized, suggesting that the sequences responsible for directing the destruction of Clb6 reside in the N terminus. Clb6 lacks the destruction box motif responsible for the anaphase promoting complex-mediated destruction of Clb5 but contains putative Cdc4 degron motifs in the N terminus. Clb6 was hyperstabilized in cdc34-3 and cdc4-3 mutants at restrictive temperatures and when S/T-P phosphorylation sites in the N terminus were mutated to nonphosphorylatable residues. Efficient degradation of Clb6 requires the activities of both Cdc28 and Pho85. Finally, hyperstabilized Clb6 expressed from the CLB6 promoter rescued the slow S-phase defect exhibited by clb5Δ cells. Taken together, these findings suggest that the SCFCdc4 ubiquitin ligase complex regulates Clb6 turnover and that the functional differences exhibited by Clb5 and Clb6 arise from the distinct mechanisms controlling their stability.

scholarly journals Direct phosphorylation of HY5 by SPA1 kinase to regulate photomorphogenesis in Arabidopsis

2020 ◽  
Author(s):  
Wenli Wang ◽  
Inyup Paik ◽  
Junghyun Kim ◽  
Xilin Hou ◽  
Sibum Sung ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Active Form ◽  
E3 Ubiquitin Ligase ◽  
Kinase Domain ◽  
Ubiquitin Ligase Complex ◽  
The Stability ◽  
Target Promoters ◽  
Spa Proteins

SUMMARYELONGATED HYPOCOTYL5 (HY5) is a key transcription factor which promotes photomorphogenesis by regulating complex downstream growth programs. Previous studies suggest that the regulation of HY5 mainly depends on the CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) - SUPPRESSOR OF PHYTOCHROME A-105 (SPA) E3 ubiquitin ligase complex, which degrades positively acting transcription factors of light signaling to repress photomorphogenesis in the dark. SPA proteins function not only as a component of the E3 ubiquitin ligase complex but also as a kinase of PHYTOCHROME INTERACTING FACTOR1 (PIF1) through its N-terminal kinase domain. Here, we show that HY5 is a new substrate of SPA1 kinase. SPA1 can directly phosphorylate HY5 in vitro and in vivo. We also demonstrate that unphosphorylated HY5 strongly interacts with both COP1 and SPA1 than phosphorylated HY5, is the preferred substrate for degradation, whereas phosphorylated HY5 is more stable in the dark. In addition, unphosphorylated HY5 actively binds to the target promoters, and is physiologically more active form. Consistently, the transgenic plants expressing unphosphorylated mutant of HY5 displays enhanced photomorphogenesis. Collectively, our study revealed that SPA1 fine-tunes the stability and the activity of HY5 to regulate photomorphogenesis.

scholarly journals The Cullin-4 Complex DCDC Does Not Require E3 Ubiquitin Ligase Elements To Control Heterochromatin in Neurospora crassa

2014 ◽  
Vol 14 (1) ◽  
pp. 25-28 ◽  
Author(s):  
Keyur K. Adhvaryu ◽  
Jordan D. Gessaman ◽  
Shinji Honda ◽  
Zachary A. Lewis ◽  
Paula L. Grisafi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Neurospora Crassa ◽  
Ubiquitin Ligase ◽  
Covalent Attachment ◽  
Content Type ◽  
Heterochromatin Formation ◽  
C Terminus ◽  
Cullin 4 ◽  
The Stability ◽  
Ring E3

ABSTRACT The cullin-4 (CUL4) complex DCDC ( D IM-5/-7/-9/ C UL4/ D DB1 c omplex) is essential for DNA methylation and heterochromatin formation in Neurospora crassa . Cullins form the scaffold of cullin-RING E3 ubiquitin ligases (CRLs) and are modified by the covalent attachment of NEDD8, a ubiquitin-like protein that regulates the stability and activity of CRLs. We report that neddylation is not required for CUL4-dependent DNA methylation or heterochromatin formation but is required for the DNA repair functions. Moreover, the RING domain protein RBX1 and a segment of the CUL4 C terminus that normally interacts with RBX1, the E2 ligase, CAND1, and CSN are dispensable for DNA methylation and heterochromatin formation by DCDC. Our study provides evidence for the noncanonical functions of core CRL components.

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