Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain

2008 ◽  
Vol 415 (1) ◽  
pp. 155-163 ◽  
Author(s):  
M. Christine Bruce ◽  
Voula Kanelis ◽  
Fatemeh Fouladkou ◽  
Anne Debonneville ◽  
Olivier Staub ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Down Regulation ◽  
Hect Domain ◽  
Ww Domains ◽  
Protein Protein Interaction ◽  
C Terminus ◽  
Subsequent Degradation ◽  
Interaction Domains ◽  
The Stability ◽  
Py Motif

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein–protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse–chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain–HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.

scholarly journals WW domain–mediated regulation and activation of E3 ubiquitin ligase Suppressor of Deltex

2018 ◽  
Vol 293 (43) ◽  
pp. 16697-16708 ◽  
Author(s):  
Weiyi Yao ◽  
Zelin Shan ◽  
Aihong Gu ◽  
Minjie Fu ◽  
Zhifeng Shi ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Cell Cycle Progression ◽  
Regulatory Mechanism ◽  
Cycle Progression ◽  
Hect Domain ◽  
Ww Domain ◽  
Ww Domains ◽  
E3 Ligases ◽  
Regulation Of Cell Cycle ◽  
Py Motif

The Nedd4 family E3 ligases Itch and WWP1/2 play crucial roles in the regulation of cell cycle progression and apoptosis and are closely correlated with cancer development and metastasis. It has been recently shown that the ligase activities of Itch and WWP1/2 are tightly regulated, with the HECT domain sequestered intramolecularly by a linker region connecting WW2 and WW3. Here, we show that a similar autoinhibitory mechanism is utilized by the Drosophila ortholog of Itch and WWP1/2, Suppressor of Deltex (Su(dx)). We show that Su(dx) adopts an inactive steady state with the WW domain region interacting with the HECT domain. We demonstrate that both the linker and preceding WW2 are required for the efficient binding and regulation of Su(dx) HECT. Recruiting the multiple-PY motif–containing adaptor dNdfip via WW domains relieves the inhibitory state of Su(dx) and leads to substrate (e.g. Notch) ubiquitination. Our study demonstrates an evolutionarily conservative mechanism governing the regulation and activation of some Nedd4 family E3 ligases. Our results also suggest a dual regulatory mechanism for specific Notch down-regulation via dNdfip–Su(dx)–mediated Notch ubiquitination.

Crucial role of the C-terminus of PTEN in antagonizing NEDD4-1-mediated PTEN ubiquitination and degradation

2008 ◽  
Vol 414 (2) ◽  
pp. 221-229 ◽  
Author(s):  
Xinjiang Wang ◽  
Yuji Shi ◽  
Junru Wang ◽  
Guochang Huang ◽  
Xuejun Jiang
Keyword(s):  
Protein Interaction ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Ww Domains ◽  
Protein Protein Interaction ◽  
Cellular Analysis ◽  
C Terminus ◽  
Tryptophan Residues ◽  
Interaction Domains ◽  
Phosphatase And Tensin Homologue

PTEN (phosphatase and tensin homologue deleted on chromosome 10), a potent tumour suppressor and multifunctional signalling protein, is under intricate regulation. In the present study, we have investigated the mechanism and regulation of PTEN ubiquitination catalysed by NEDD4-1 (neural-precursor-cell-expressed, developmentally down-regulated 4-1), a ubiquitin ligase for PTEN we identified recently. Using the reconstituted assay and cellular analysis, we demonstrated that NEDD4-1-mediated PTEN ubiquitination depends on its intact HECT (homologous to E6-associated protein C-terminus) domain. Instead of using its WW domains (protein–protein interaction domains containing two conserved tryptophan residues) as a protein interaction module, NEDD4-1 interacts with PTEN through its N-terminal region containing a C2 domain as well as the HECT domain. Strikingly, we found that a C-terminal truncated PTEN fragment binds to NEDD4-1 with higher affinity than the full-length PTEN, suggesting an intrinsic inhibitory effect of the PTEN C-terminus on PTEN–NEDD4-1 interaction. Moreover, the C-terminal truncated PTEN is more sensitive to NEDD4-1-mediated ubiquitination and degradation. Therefore the present study reveals that the C-terminus of PTEN plays a critical role in stabilizing PTEN via antagonizing NEDD4-1-induced PTEN protein decay; conversely, truncation of the PTEN C-terminus results in rapid NEDD4-1-mediated PTEN degradation, a possible mechanism accounting for attenuation of PTEN function by certain PTEN mutations in human cancers.

PDLIM7 is a novel target of the ubiquitin ligase Nedd4-1 in skeletal muscle

2016 ◽  
Vol 473 (3) ◽  
pp. 267-276 ◽  
Author(s):  
Robert D'Cruz ◽  
Pamela J. Plant ◽  
Lesley A. Pablo ◽  
Shouzhe Lin ◽  
Joshua Chackowicz ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Ubiquitin Ligase ◽  
Muscle Wasting ◽  
Precursor Cell ◽  
Skeletal Muscle Atrophy ◽  
Neural Precursor ◽  
Neural Precursor Cell ◽  
Ww Domains ◽  
Novel Target ◽  
Py Motif

Nedd4-1 (neural precursor cell-expressed developmentally down-regulated 4-1) mediates skeletal muscle atrophy. We identify PDLIM7 as a novel Nedd4-1 substrate, binding Nedd4-1 WW domains via its PY motif and co-localizing with Nedd4-1 in myotubes. The interaction results in PDLIM7 ubiquitination and may underlie Nedd4-1-mediated muscle wasting.

14-3-3 proteins are promising LRRK2 interactors

2010 ◽  
Vol 430 (3) ◽  
pp. e5-e6 ◽  
Author(s):  
Iakov N. Rudenko ◽  
Mark R. Cookson
Keyword(s):  
Regulatory Mechanism ◽  
Cell Signalling ◽  
Regulatory Proteins ◽  
Protein Protein Interaction ◽  
Catalytic Domains ◽  
C Terminus ◽  
Disease Mutations ◽  
Biochemical Journal ◽  
Complex Protein ◽  
Interaction Domains

Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common cause of familial PD (Parkinson’s disease). Mutations that cause PD are found in either the GTPase or kinase domains of LRRK2 or an intervening sequence called the COR [C-terminus of ROC (Ras of complex proteins)] domain. As well as the two catalytic domains, LRRK2 possesses several protein–protein interaction domains, but their function and the proteins with which they interact are poorly understood. In this issue of the Biochemical Journal, Nichols et al. study the interaction of the N-terminal region of LRRK2 with 14-3-3 proteins, regulatory proteins that often bind to phosphorylated regions of components of cell signalling pathways. Using a combination of techniques, Nichols et al. have identified two residues (Ser910 and Ser935) that are critically responsible for 14-3-3 binding. The interaction of LRRK2 with 14-3-3 proteins can prevent dephosphorylation of Ser910/Ser935 and stabilize LRRK2 structure, perhaps by influencing the dimerization of LRRK2. The ability to interact with 14-3-3 correlates with the pattern of intracellular LRRK2 distribution. Collectively, these new results identify a potentially important regulatory mechanism of this complex protein and might provide ways to think about therapeutic opportunities for PD.

scholarly journals Rhabdoviruses and the Cellular Ubiquitin-Proteasome System: a Budding Interaction

2001 ◽  
Vol 75 (22) ◽  
pp. 10623-10629 ◽  
Author(s):  
Ronald N. Harty ◽  
Melissa E. Brown ◽  
James P. McGettigan ◽  
Guangli Wang ◽  
Himangi R. Jayakar ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
M Protein ◽  
Dependent Manner ◽  
Virus Budding ◽  
Ww Domains ◽  
M Proteins ◽  
Ubiquitin Proteasome ◽  
Py Motif ◽  
Free Ubiquitin

ABSTRACT The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies virus (RV) play a key role in both assembly and budding of progeny virions. A PPPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for mediating interactions with specific cellular proteins containing WW domains. The PY motif and flanking sequences of the M protein of VSV were used as bait to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple WW domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeast homolog of Nedd4, was able to interact both physically and functionally with full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M protein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding process of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrease the level of free ubiquitin in VSV- and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10- to 20-fold lower than those measured from untreated control cells, suggesting that free ubiquitin is important for efficient virus budding. Last, release of a VSV PY mutant was not inhibited in the presence of MG132, signifying that the functional L domain of VSV is required for the inhibitory effect exhibited by MG132. These data suggest that the cellular ubiquitin-proteasome machinery is involved in the budding process of VSV and RV.

scholarly journals Direct Binding to Rsp5p Regulates Ubiquitination-independent Vacuolar Transport of Sna3p

2007 ◽  
Vol 18 (5) ◽  
pp. 1781-1789 ◽  
Author(s):  
Hadiya Watson ◽  
Juan S. Bonifacino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ubiquitin Ligase ◽  
Adaptor Protein ◽  
Direct Interaction ◽  
Integral Membrane Proteins ◽  
Multivesicular Bodies ◽  
Hect Domain ◽  
Ww Domains ◽  
Cytosolic Domains ◽  
Direct Binding

The sorting of integral membrane proteins such as carboxypeptidase S (Cps1p) into the luminal vesicles of multivesicular bodies (MVBs) in Saccharomyces cerevisiae requires ubiquitination of their cytosolic domains by the ubiquitin ligases Rsp5p and/or Tul1p. An exception is Sna3p, which does not require ubiquitination for entry into MVBs. The mechanism underlying this ubiquitination-independent MVB sorting pathway has not yet been characterized. Here, we show that Sna3p sorting into the MVB pathway depends on a direct interaction between a PPAY motif within its C-terminal cytosolic tail and the WW domains of Rsp5p. Disruption of this interaction inhibits vacuolar targeting of Sna3p and causes its accumulation in a compartment that overlaps only partially with MVBs. Surprisingly, Sna3p does require a functional ubiquitin-ligase HECT domain within Rsp5p; however, the dependence of Sna3p on HECT domain activity is distinct from that of Cps1p. Last, we show that Sna3p requires neither Tul1p nor the transmembrane adaptor protein Bsd2p for its MVB sorting. Our data demonstrate that Sna3p follows a novel ubiquitination-independent, but Rsp5p-mediated, sorting pathway to the vacuole.

Generation of free ubiquitin chains is up-regulated in stress and facilitated by the HECT domain ubiquitin ligases UFD4 and HUL5

2012 ◽  
Vol 444 (3) ◽  
pp. 611-617 ◽  
Author(s):  
Ori Braten ◽  
Nitzan Shabek ◽  
Yelena Kravtsova-Ivantsiv ◽  
Aaron Ciechanover
Keyword(s):  
Signal Transduction ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
26S Proteasome ◽  
Specific Substrate ◽  
Polyubiquitin Chains ◽  
Hect Domain ◽  
Protein Substrate ◽  
C Terminus ◽  
Free Ubiquitin

Polyubiquitin chains serve a variety of physiological roles. Typically the chains are bound covalently to a protein substrate and in many cases target it for degradation by the 26S proteasome. However, several studies have demonstrated the existence of free polyubiquitin chains which are not linked to a specific substrate. Several physiological functions have been attributed to these chains, among them playing a role in signal transduction and serving as storage of ubiquitin for utilization under stress. In the present study, we have established a system for the detection of free ubiquitin chains and monitoring their level under changing conditions. Using this system, we show that UFD4 (ubiquitin fusion degradation 4), a HECT (homologous with E6-AP C-terminus) domain ubiquitin ligase, is involved in free chain generation. We also show that generation of these chains is stimulated in response to a variety of stresses, particularly those caused by DNA damage. However, it appears that the stress-induced synthesis of free chains is catalysed by a different ligase, HUL5 (HECT ubiquitin ligase 5), which is also a HECT domain E3.

scholarly journals Critical Role for a Central Part of Mdm2 in the Ubiquitylation of p53

2003 ◽  
Vol 23 (14) ◽  
pp. 4929-4938 ◽  
Author(s):  
Erik Meulmeester ◽  
Ruth Frenk ◽  
Robert Stad ◽  
Petra de Graaf ◽  
Jean-Christophe Marine ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
P53 Protein ◽  
Critical Role ◽  
Ring Finger ◽  
26S Proteasome ◽  
Functional Difference ◽  
Subsequent Degradation ◽  
The Stability ◽  
Internal Domain

ABSTRACT The stability of the p53 protein is regulated by Mdm2. By acting as an E3 ubiquitin ligase, Mdm2 directs the ubiquitylation of p53 and its subsequent degradation by the 26S proteasome. In contrast, the Mdmx protein, although structurally similar to Mdm2, cannot ubiquitylate or degrade p53 in vivo. To ascertain which domains determine this functional difference between Mdm2 and Mdmx and consequently are essential for p53 ubiquitylation and degradation, we generated Mdm2-Mdmx chimeric constructs. Here we show that, in addition to a fully functional Mdm2 RING finger, an internal domain of Mdm2 (residues 202 to 302) is essential for p53 ubiquitylation. Strikingly, the function of this domain can be fulfilled in trans, indicating that the RING domain and this internal region perform distinct activities in the ubiquitylation of p53.

Distinct characteristics of two human Nedd4 proteins with respect to epithelial Na+ channel regulation

2001 ◽  
Vol 281 (3) ◽  
pp. F469-F477 ◽  
Author(s):  
Elena Kamynina ◽  
Caroline Tauxe ◽  
Olivier Staub
Keyword(s):  
Close Relative ◽  
Na Channel ◽  
Domain Interaction ◽  
Hect Domain ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Activity Structure ◽  
Py Motif ◽  
Epithelial Na Channel ◽  
Salt Homeostasis

The epithelial Na+ channel (ENaC) is regulated via PY motif-WW domain interaction by the mouse (m) ubiquitin-protein ligase mNedd4-2 but not by its close relative mNedd4-1. Whereas mNedd4-1 is composed of one C2, three WW, and one HECT domain, mNedd4-2 comprises four WW domains and one HECT domain. Both proteins have human (h) homologs, hNedd4-1 and hNedd4-2; however, both of them include four WW domains. Therefore, we characterized hNedd4-1 and hNedd4-2 in Xenopus laevisoocytes with respect to ENaC binding and interaction. We found that hNedd4-2 binds to and abrogates ENaC activity, whereas hNedd4-1 does not coimmunoprecipitate with ENaC and has only modest effects on ENaC activity. Structure-function studies revealed that the C2 domain of hNedd4-1 prevents this protein from downregulating ENaC and that WW domains 3 and 4, involved in interaction with ENaC, do not by themselves provide specificity for ENaC recognition. Taken together, our data demonstrate that hNedd4-2 inhibits ENaC, implying that this protein is a modulator of salt homeostasis, whereas hNedd4-1 is not primarily involved in ENaC regulation.

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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