scholarly journals Multisite Phosphorylation of the Saccharomyces cerevisiae Filamentous Growth Regulator Tec1 Is Required for its Recognition by the E3 Ubiquitin Ligase Adaptor Cdc4 and Its Subsequent Destruction In Vivo

2010 ◽  
Vol 9 (1) ◽  
pp. 31-36 ◽  
Author(s):  
Marie Z. Bao ◽  
Teresa R. Shock ◽  
Hiten D. Madhani
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Filamentous Growth ◽  
Mitogen Activated Protein Kinase ◽  
Ubiquitin Ligase Complex ◽  
Mitogen Activated Protein ◽  
Substrate Phosphorylation

ABSTRACT In Saccharomyces cerevisiae, the pheromone-induced ubiquitylation and degradation of the filamentation pathway-specific activator, Tec1, suppresses cross talk between the mating and filamentous growth mitogen-activated protein kinase (MAPK) pathways. The mating pathway MAPK, Fus3, phosphorylates Tec1, resulting in its recognition by the SCF (for Skp1, Cullin, F-box containing) E3 ubiquitin ligase complex, leading to its proteolysis. Previously, it was found that Tec1 destruction requires phosphorylation on threonine 273 (T273). T273 is embedded in the sequence LLpTP, which is identical to the canonical binding site for Cdc4, a conserved F-box substrate adaptor for the SCF complex. However, recent work on both Cdc4 and the human Cdc4 ortholog Fbw7 has shown that a second substrate phosphorylation can be required for optimal Cdc4 binding in vitro. We report here that high-affinity binding of recombinant Cdc4 to Tec1 phosphopeptides requires phosphorylation of not only T273 but also a second site, T276. Significantly, both phospho-sites on Tec1 and a conserved basic pocket on Cdc4 are critical for Tec1 proteolysis in response to pheromone treatment of cells, establishing a role for two-phosphate recognition by yeast Cdc4 in substrate targeting in vivo.

scholarly journals CAND1-Mediated Substrate Adaptor Recycling Is Required for Efficient Repression of Nrf2 by Keap1

2006 ◽  
Vol 26 (4) ◽  
pp. 1235-1244 ◽  
Author(s):  
Shih-Ching Lo ◽  
Mark Hannink
Keyword(s):  
Ubiquitin Ligase ◽  
Redox Homeostasis ◽  
Ectopic Expression ◽  
E3 Ubiquitin Ligase ◽  
Adaptor Protein ◽  
Bzip Transcription Factor ◽  
Ubiquitin Ligase Complex ◽  
Steady State Levels

ABSTRACT The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2. Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex to repress steady-state levels of Nrf2 and Nrf2-dependent transcription. Cullin-dependent ubiquitin ligase complexes have been proposed to undergo dynamic cycles of assembly and disassembly that enable substrate adaptor exchange or recycling. In this report, we have characterized the importance of substrate adaptor recycling for regulation of Keap1-mediated repression of Nrf2. Association of Keap1 with Cul3 was decreased by ectopic expression of CAND1 and was increased by small interfering RNA (siRNA)-mediated knockdown of CAND1. However, both ectopic overexpression and siRNA-mediated knockdown of CAND1 decreased the ability of Keap1 to target Nrf2 for ubiquitin-dependent degradation, resulting in stabilization of Nrf2 and activation of Nrf2-dependent gene expression. Neddylation of Cul3 on Lys 712 is required for Keap1-dependent ubiquitination of Nrf2 in vivo. However, the K712R mutant Cul3 molecule, which is not neddylated, can still assemble with Keap1 into a functional ubiquitin ligase complex in vitro. These results provide support for a model in which substrate adaptor recycling is required for efficient substrate ubiquitination by cullin-dependent E3 ubiquitin ligase complexes.

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 E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

scholarly journals Role for the Ran Binding Protein, Mog1p, in Saccharomyces cerevisiae SLN1-SKN7 Signal Transduction

2004 ◽  
Vol 3 (6) ◽  
pp. 1544-1556 ◽  
Author(s):  
Jade Mei-Yeh Lu ◽  
Robert J. Deschenes ◽  
Jan S. Fassler
Keyword(s):  
Signal Transduction ◽  
Transcription Factor ◽  
Osmotic Stress ◽  
Kinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
Osmotic Response ◽  
Mitogen Activated Protein ◽  
Kinase Pathway

ABSTRACT Yeast Sln1p is an osmotic stress sensor with histidine kinase activity. Modulation of Sln1 kinase activity in response to changes in the osmotic environment regulates the activity of the osmotic response mitogen-activated protein kinase pathway and the activity of the Skn7p transcription factor, both important for adaptation to changing osmotic stress conditions. Many aspects of Sln1 function, such as how kinase activity is regulated to allow a rapid response to the continually changing osmotic environment, are not understood. To gain insight into Sln1p function, we conducted a two-hybrid screen to identify interactors. Mog1p, a protein that interacts with the yeast Ran1 homolog, Gsp1p, was identified in this screen. The interaction with Mog1p was characterized in vitro, and its importance was assessed in vivo. mog1 mutants exhibit defects in SLN1-SKN7 signal transduction and mislocalization of the Skn7p transcription factor. The requirement for Mog1p in normal localization of Skn7p to the nucleus does not fully account for the mog1-related defects in SLN1-SKN7 signal transduction, raising the possibility that Mog1p may play a role in Skn7 binding and activation of osmotic response genes.

scholarly journals Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation

2005 ◽  
Vol 25 (2) ◽  
pp. 854-864 ◽  
Author(s):  
Sandrine Marchetti ◽  
Clotilde Gimond ◽  
Jean-Claude Chambard ◽  
Thomas Touboul ◽  
Danièle Roux ◽  
...  
Keyword(s):  
Map Kinases ◽  
Fluorescent Protein ◽  
Proteasomal Degradation ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Double Mutation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.

Amentoflavone Inhibits Osteoclastogenesis and Wear Debris-Induced Osteolysis via Suppressing NF-κB and MAPKs Signaling Pathways

Planta Medica ◽  
2018 ◽  
Vol 84 (11) ◽  
pp. 759-767 ◽  
Author(s):  
Zhen Zhang ◽  
Shuai Zhao ◽  
Xiaolei Li ◽  
Xiaoqi Zhuo ◽  
Wu Zhang ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Aseptic Loosening ◽  
Wear Debris ◽  
Mitogen Activated Protein Kinase ◽  
Micro Computed Tomography ◽  
Computed Tomography Scanning ◽  
Mitogen Activated Protein ◽  
And Function

AbstractWear debris-induced osteolysis is one of the major reasons for subsequent aseptic loosening after cementless hip arthroplasty. Increasing evidence suggests that receptor activator of nuclear factor kappa-B (NF-κB) ligand-mediated osteoclastogenesis and osteolysis are responsible for wear debris-induced aseptic loosening. In the present study, we explored the effect of amentoflavone (AMF) on inhibiting osteoclast generation and wear debris-induced osteolysis in vitro and in vivo. Twenty-four male C57BL/J6 mice were randomly divided into four groups: a sham group and groups with titanium wear debris treatment followed by intraperitoneal injection of various concentrations of AMF (0, 20, and 40 mg/kg/day). The micro computed tomography scanning and histological analysis were performed. Bone marrow-derived macrophages were cultured to investigate the effect of AMF on osteoclast generation and function. The results showed that AMF suppressed osteoclastogenesis, F-actin ring formation, and bone absorption without cytotoxicity. AMF prevented titanium wear debris-induced osteolysis in mice. AMF suppressed the relative proteins of NF-κB and mitogen-activated protein kinase (MAPKs) signaling pathways. Thus, the present study suggests that AMF derived from plants could inhibit osteoclastogenesis and titanium wear debris-induced osteolysis via suppressing NF-κB and MAPKs signaling pathways.

CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1

Blood ◽  
2021 ◽  
Author(s):  
Roger Belizaire ◽  
Sebastian Hassan John Koochaki ◽  
Namrata D. Udeshi ◽  
Alexis Vedder ◽  
Lei Sun ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Therapeutic Potential ◽  
Mutant Cell ◽  
E3 Ubiquitin Ligase ◽  
Akt Signaling ◽  
Allelic Series ◽  
Genetic Ablation

CBL encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and non-receptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10-20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, PIK3R1 recruitment, and downstream PI3K/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine-kinase binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

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