E3 Ubiquitin Ligase Nedd4 Research Articles

WNK3 Prevents the Nedd4‐2 Inhibition of the Renal Na‐Cl Cotransporter (NCC)

NCC and ENaC are inhibited by the E3 ubiquitin ligase Nedd4‐2 (JASN, 2011) and elimination of two canonical SGK1 phosphorylation sites on Nedd4‐2 (S222A and S328A), renders the Nedd4‐2 mediated inhibition of NCC and ENaC irrecoverable by SGK1. In search of new regulatory proteins, we found that the NH2 terminal domain of WNK3 interacts with Nedd4‐2. In Xenopus laevis oocytes inhibition of NCC by Nedd4‐2 is completely reversed in the presence of WNK3. In oocytes injected with NCC alone, WNK3 increases NCC activity by 264 ± 22 %, while in the presence of NCC and Nedd4‐2, the WNK3‐induced increase was of 353 ± 44 %. Additionally, in contrast to SGK1, WNK3 further activates NCC in the presence of double mutant Nedd4‐2 S222A/S328A to 501 ± 48 %. Thus, in the presence of wild type or mutant Nedd4‐2, WNK3 is a more potent stimulator of NCC. In the absence or presence of Nedd4‐2, NCC activation by WNK3 is prevented by elimination of WNK3 histidine 1497 and glycine 1508 that have been shown to be critical for interaction between WNKs (TBiochem J, 2011). In order to test for non‐specific WNK interactions we tested WNK4 and saw no difference in the Nedd4‐2 mediated inhibition of NCC. Thus, we propose that WNK3 is a regulator of the Nedd4‐2‐NCC pathway.

Quantitative Proteomics Identifies Vasopressin-Responsive Nuclear Proteins in Collecting Duct Cells

Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (β-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 5'-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaCβ), and Scnn1g (ENaCγ), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in β-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics (http://helixweb.nih.gov/ESBL/Database/mNPD/) and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct.

Repeated Stress Causes Cognitive Impairment by Suppressing Glutamate Receptor Expression and Function in Prefrontal Cortex

Chronic stress could trigger maladaptive changes associated with stress-related mental disorders; however, the underlying mechanisms remain elusive. In this study, we found that exposing juvenile male rats to repeated stress significantly impaired the temporal order recognition memory, a cognitive process controlled by the prefrontal cortex (PFC). Concomitantly, significantly reduced AMPAR- and NMDAR-mediated synaptic transmission and glutamate receptor expression were found in PFC pyramidal neurons from repeatedly stressed animals. All these effects relied on activation of glucocorticoid receptors and the subsequent enhancement of ubiquitin/proteasome-mediated degradation of GluR1 and NR1 subunits, which was controlled by the E3 ubiquitin ligase Nedd4-1 and Fbx2, respectively. Inhibition of proteasomes or knockdown of Nedd4-1 and Fbx2 in PFC prevented the loss of glutamatergic responses and recognition memory in stressed animals. Our results suggest that repeated stress dampens PFC glutamatergic transmission by facilitating glutamate receptor turnover, which causes the detrimental effect on PFC-dependent cognitive processes.

Abstract A4: Androgen receptor (AR) degradation is controlled by the co-operation of PMEPA1 and the E3 ubiquitin ligase NEDD4-1

Abstract Introduction and Objective: Androgen receptor (AR) plays critical roles in prostate growth and differentiation and prostate tumorigenesis. Our recent investigations have discovered a novel mechanism of AR degradation in CaP cells involving PMEPA1 and NEDD4-1 which is independent of MDM2. Previously we have shown that PMEPA1 is a highly androgen-inducible gene in CaP cells that recruits the NEDD4-1, an E3 ubiquitin ligase, to degrade AR. In an independent study NEDD4-1 has been identified as an E3 ligase controlling the levels of PTEN. We hypothesize that decreased or loss of PMPEA1 expression that is frequently observed in human prostate tumor cells may lead to enhanced AR functions. Elevated AR levels may increase NEDD4-1 and in turn destabilize PTEN in prostate cancer cells. Here, we further investigate the co-operation between PMEPA1 and NEDD4-1 to alter AR and PTEN protein levels. Methods: PMEPA1 and NEDD4-1 were ectopically expressed in androgen responsive LNCaP and VCaP cells by pCMV-XL4 and pcDNA3.1 vectors, respectively. AR, PMEPA1, NEDD4-1, MDM2 and PTEN were silenced by using specific siRNAs. Cell growth was monitored in response modulation of PMEPA1, NEDD4 and AR. Protein levels of AR, PMEPA1, NEDD4-1, PTEN and PSA were assessed by immunoblot assays. Results: Decreases in AR protein levels, inhibited AR signaling and cell growth in response to heterologous expression of NEDD4-1. Consistent with this observation, inhibition of NEDD4-1 by siRNA resulted in elevated AR protein levels, AR signaling and increased cell growth. Reduced cell growth by AR knock-down or by heterologous expression of NEDD4-, were rescued by the knockdown of PMEPA1. It was also shown that AR up-regulates the expression of NEDD4-1 in hormone responsive CaP cells. In VCaP cells, overexpression of NEDD4-1 resulted in decreased PTEN protein levels. Remarkably, PMEPA1 knockdown resulted in elevated AR and NEDD4-1 levels and the downregulation of PTEN protein. Conclusions: NEDD4-1 mediated AR degradation in hormone responsive CaP cells requires PMEPA1. The frequently observed decreased expression of PMEPA1 results in elevated AR protein levels and may facilitate NEDD4-1 mediated degradation of PTEN in CaP. Our findings revealed a complex interplay of the AR, PMEPA1, NEDD4 and PTEN underscoring the impact of the loss of PMEPA1 to increased AR and decreased PTEN, both proteins critical in prostate tumorigenesis. Citation Format: Hua Li, Albert Dobi, Shiv Srivastava. Androgen receptor (AR)degradation is controlled by the cooperation of PMEPA1 and the E3 ubiquitin ligase NEDD4-1 [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A4.

Ndfip1 regulates nuclear Pten import in vivo to promote neuronal survival following cerebral ischemia

PTEN (phosphatase and tensin homologue deleted on chromosome TEN) is the major negative regulator of phosphatidylinositol 3-kinase signaling and has cell-specific functions including tumor suppression. Nuclear localization of PTEN is vital for tumor suppression; however, outside of cancer, the molecular and physiological events driving PTEN nuclear entry are unknown. In this paper, we demonstrate that cytoplasmic Pten was translocated into the nuclei of neurons after cerebral ischemia in mice. Critically, this transport event was dependent on a surge in the Nedd4 family-interacting protein 1 (Ndfip1), as neurons in Ndfip1-deficient mice failed to import Pten. Ndfip1 binds to Pten, resulting in enhanced ubiquitination by Nedd4 E3 ubiquitin ligases. In vitro, Ndfip1 overexpression increased the rate of Pten nuclear import detected by photobleaching experiments, whereas Ndfip1(-/-) fibroblasts showed negligible transport rates. In vivo, Ndfip1 mutant mice suffered larger infarct sizes associated with suppressed phosphorylated Akt activation. Our findings provide the first physiological example of when and why transient shuttling of nuclear Pten occurs and how this process is critical for neuron survival.

βPix is a New Player in Renal Physiology

GENERAL COMMENTARY article Front. Physiol., 20 July 2012Sec. Renal Physiology and Pathophysiology Volume 3 - 2012 | https://doi.org/10.3389/fphys.2012.00268

A Role for the Ubiquitin Ligase Nedd4 in Membrane Sorting of LAPTM4 Proteins

BackgroundThe Lysosome associated protein transmembrane (LAPTM) family is comprised of three members: LAPTM5, LAPTM4a and LAPTM4b, with the latter previously shown to be overexpressed in numerous cancers. While we had demonstrated earlier the requirement of the E3 ubiquitin ligase Nedd4 for LAPTM5 sorting to lysosomes, the regulation of sorting of LAPTM4 proteins is less clear.Methodology/Principal FindingsHere we show that LAPTM4a and LAPTM4b are localized to the lysosome, but unique to LAPTM4b, a fraction of it is present at the plasma membrane and its overexpression induces the formation of actin-based membrane protrusions. We demonstrate that LAPTM4s, like LAPTM5, are able to co-immunoprecipitate with the E3 ubiquitin ligase Nedd4, an interaction that is dependent on LAPTM4 PY motifs and plays a role in membrane sorting. Accordingly, in Nedd4 knockout mouse embryonic fibroblasts (MEFs), LAPTM4a and LAPTM4b show reduced lysosomal localization. Moreover, lack of PY motifs leads to enhanced missorting of LAPTM4b to the plasma membrane instead of the lysosome.Conclusions/SignificanceThese results suggest that while some requisites of LAPTM5 lysosomal sorting are conserved among LAPTM4 proteins, LAPTM4a and LAPTM4b have also developed distinct sorting requirements.

AMP‐Activated Protein Kinase Downregulates Kv7.1 Cell Surface Expression

The potassium channel Kv7.1 is expressed in the heart, where it contributes to the repolarization of the cardiac action potential. Additionally, Kv7.1 is expressed in epithelial tissues playing a role in salt and water transport. We recently demonstrated that surface-expressed Kv7.1 is internalized in response to polarization of the epithelial Madin-Darby canine kidney (MDCK) cell line and that this was mediated by activation of protein kinase C (PKC). In this study, the pathway downstream of PKC, which leads to internalization of Kv7.1 upon cell polarization, is elucidated. We show by confocal microscopy that Kv7.1 is endocytosed upon initiation of the polarization process and sent for degradation by the lysosomal pathway. The internalization could be mimicked by pharmacological activation of the AMP-activated protein kinase (AMPK) using three different AMPK activators. We demonstrate that the downstream effector of AMPK is the E3 ubiquitin ligase Nedd4-2. Additionally, we show that AMPK activation results in a downregulation of Kv7.1 currents in Xenopus oocytes through a Nedd4-2-dependent mechanism. In summary, surface-expressed Kv7.1 channels are endocytosed and sent for degradation in lysosomes by an AMPK-mediated activation of Nedd4-2 during the initial phase of the MDCK cell polarization process.

RETRACTED: The Role of Ubiquitination in Hepcidin-Independent and Hepcidin-Dependent Degradation of Ferroportin

The iron exporter ferroportin (Fpn) is essential to transfer iron from cells to plasma. Systemic iron homeostasis in vertebrates is regulated by the hepcidin-mediated internalization of Fpn. Here, we demonstrate a second route for Fpn internalization; when cytosolic iron levels are low, Fpn is internalized in a hepcidin-independent manner dependent upon the E3 ubiquitin ligase Nedd4-2 and the Nedd4-2 binding protein Nfdip-1. Retention of cell-surface Fpn through reductions in Nedd4-2 results in cell death through depletion of cytosolic iron. Nedd4-2 is also required for internalization of Fpn in the absence of ferroxidase activity as well as for the entryof hepcidin-induced Fpn into the multivesicularbody. C.elegans lacks hepcidin genes, and C.elegans Fpn expressed in mammalian cells is not internalized by hepcidin but is internalized in response to iron deprivation in a Nedd4-2-dependent manner, supporting the hypothesis that Nedd4-2-induced internalization of Fpn is evolutionarily conserved.

Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2) Regulation by 14-3-3 Protein Binding at Canonical Serum and Glucocorticoid Kinase 1 (SGK1) Phosphorylation Sites

Regulation of epithelial Na(+) channel (ENaC)-mediated transport in the distal nephron is a critical determinant of blood pressure in humans. Aldosterone via serum and glucocorticoid kinase 1 (SGK1) stimulates ENaC by phosphorylation of the E3 ubiquitin ligase Nedd4-2, which induces interaction with 14-3-3 proteins. However, the mechanisms of SGK1- and 14-3-3-mediated regulation of Nedd4-2 are unclear. There are three canonical SGK1 target sites on Nedd4-2 that overlap phosphorylation-dependent 14-3-3 interaction motifs. Two of these are termed "minor," and one is termed "major," based on weak or strong binding to 14-3-3 proteins, respectively. By mass spectrometry, we found that aldosterone significantly stimulates phosphorylation of a minor, relative to the major, 14-3-3 binding site on Nedd4-2. Phosphorylation-deficient minor site Nedd4-2 mutants bound less 14-3-3 than did wild-type (WT) Nedd4-2, and minor site Nedd4-2 mutations were sufficient to inhibit SGK1 stimulation of ENaC cell surface expression. As measured by pulse-chase and cycloheximide chase assays, a major binding site Nedd4-2 mutant had a shorter cellular half-life than WT Nedd4-2, but this property was not dependent on binding to 14-3-3. Additionally, a dimerization-deficient 14-3-3ε mutant failed to bind Nedd4-2. We conclude that whereas phosphorylation at the Nedd4-2 major site is important for interaction with 14-3-3 dimers, minor site phosphorylation by SGK1 may be the relevant molecular switch that stabilizes Nedd4-2 interaction with 14-3-3 and thus promotes ENaC cell surface expression. We also propose that major site phosphorylation promotes cellular Nedd4-2 protein stability, which potentially represents a novel form of regulation for turnover of E3 ubiquitin ligases.

How ubiquitination regulates the TGF‐β signalling pathway: New insights and new players

Ubiquitination of protein species in regulating signal transduction pathways is universally accepted as of fundamental importance for normal development, and defects in this process have been implicated in the progression of many human diseases. One pathway that has received much attention in this context is transforming growth factor-beta (TGF-β) signalling, particularly during the regulation of epithelial-mesenchymal transition (EMT) and tumour progression. While E3-ubiquitin ligases offer themselves as potential therapeutic targets, much remains to be unveiled regarding mechanisms that culminate in their regulation. With this in mind, the focus of this review highlights the regulation of the ubiquitination pathway and the significance of a recently described group of NEDD4 E3-ubiquitin ligase isoforms in the context of TGF-β pathway regulation. Moreover, we now broaden these observations to incorporate a growing number of protein isoforms within the ubiquitin ligase superfamily as a whole, and discuss their relevance in defining a new 'iso-ubiquitinome'.

Ubiquitination of TrkA by Nedd4-2 regulates receptor lysosomal targeting and mediates receptor signaling

The nerve growth factor receptor TrkA (tropomyosin-related kinase receptor) participates in the survival and differentiation of several neuronal populations. The C-terminal tail of TrkA contains a PPXY motif, the binding site of the E3 ubiquitin-ligase Nedd4-2 (neural precursor cell expressed, developmentally down-regulated 4-2). In order to analyze the role of Nedd4-2 ubiquitination on TrkA function, we generated three TrkA mutants, by introducing point mutations on conserved hydrophobic amino acids - Leu784 and Val790 switched to Ala. TrkA mutants co-localized and co-immunoprecipitated more efficiently with Nedd4-2 and consequently a strong increase in the basal multimonoubiquitination of the mutant receptors was observed. In addition, we found a decrease in TrkA abundance because of the preferential sorting of mutant receptors towards the late endosome/lysosome pathway instead of recycling back to the plasma membrane. Despite the reduction in the amount of membrane receptor caused by the C-terminal changes, TrkA mutants were able to activate signaling cascades and were even more efficient in promoting neurite outgrowth than the wild-type receptor. Our results demonstrate that the C-terminal tail hydrophobicity of TrkA regulates Nedd4-2 binding and activity and therefore controls receptor turnover. In addition, TrkA multimonoubiquitination does not interfere with the activation of signaling cascades, but rather potentiates receptor signaling leading to differentiation.

Nedd4-1 and β-Arrestin-1 Are Key Regulators of Na+/H+ Exchanger 1 Ubiquitylation, Endocytosis, and Function

The ubiquitously expressed mammalian Na(+)/H(+) exchanger 1 (NHE1) controls cell volume and pH but is also critically involved in complex biological processes like cell adhesion, cell migration, cell proliferation, and mechanosensation. Pathways controlling NHE1 turnover at the plasma membrane, however, are currently unclear. Here, we demonstrate that NHE1 undergoes ubiquitylation at the plasma membrane by a process that is unprecedented for a mammalian ion transport protein. This process requires the adapter protein β-arrestin-1 that interacts with both the E3 ubiquitin ligase Nedd4-1 and the NHE1 C terminus. Truncation of NHE1 C terminus to amino acid 550 abolishes binding to β-arrestin-1 and NHE1 ubiquitylation. Overexpression of β-arrestin-1 or of wild type but not ligase-dead Nedd4-1 increases NHE1 ubiquitylation. siRNA-mediated knock-down of Nedd4-1 or β-arrestin-1 reduces NHE1 ubiquitylation and endocytosis leading to increased NHE1 surface levels. Fibroblasts derived from β-arrestin-1 and Nedd4-1 knock-out mice show loss of NHE1 ubiquitylation, increased plasmalemmal NHE1 levels and greatly enhanced NHE1 transport compared with wild-type fibroblasts. These findings reveal Nedd4-1 and β-arrestin-1 as key regulators of NHE1 ubiquitylation, endocytosis, and function. Our data suggest a broader role for β-arrestins in the regulation of membrane ion transport proteins than currently known.

Structural Basis for the Interaction between the Growth Factor-binding Protein GRB10 and the E3 Ubiquitin Ligase NEDD4

In addition to inhibiting insulin receptor and IGF1R kinase activity by directly binding to the receptors, GRB10 can also negatively regulate insulin and IGF1 signaling by mediating insulin receptor and IGF1R degradation through ubiquitination. It has been shown that GRB10 can interact with the C2 domain of the E3 ubiquitin ligase NEDD4 through its Src homology 2 (SH2) domain. Therefore, GRB10 might act as a connector, bringing NEDD4 close to IGF1R to facilitate the ubiquitination of IGF1R by NEDD4. This is the first case in which it has been found that an SH2 domain could colocalize a ubiquitin ligase and its substrate. Here we report the crystal structure of the NEDD4 C2-GRB10 SH2 complex at 2.0 Å. The structure shows that there are three interaction interfaces between NEDD4 C2 and GRB10 SH2. The main interface centers on an antiparallel β-sheet composed of the F β-strand of GRB10 SH2 and the C β-strand of NEDD4 C2. NEDD4 C2 binds at nonclassical sites on the SH2 domain surface, far from the classical phosphotyrosine-binding pocket. Hence, this interaction is phosphotyrosine-independent, and GRB10 SH2 can bind the C2 domain of NEDD4 and the kinase domain of IGF1R simultaneously. Based on these results, a model of how NEDD4 interacts with IGF1R through GRB10 has been proposed. This report provides further evidence that SH2 domains can participate in important signaling interactions beyond the classical recognition of phosphotyrosine.

Purinergic Receptor-mediated Rapid Depletion of Nuclear Phosphorylated Akt Depends on Pleckstrin Homology Domain Leucine-rich Repeat Phosphatase, Calcineurin, Protein Phosphatase 2A, and PTEN Phosphatases

Akt is an important oncoprotein, and data suggest a critical role for nuclear Akt in cancer development. We have previously described a rapid (3-5 min) and P2X7-dependent depletion of nuclear phosphorylated Akt (pAkt) and effects on downstream targets, and here we studied mechanisms behind the pAkt depletion. We show that cholesterol-lowering drugs, statins, or extracellular ATP, induced a complex and coordinated response in insulin-stimulated A549 cells leading to depletion of nuclear pAkt. It involved protein/lipid phosphatases PTEN, pleckstrin homology domain leucine-rich repeat phosphatase (PHLPP1 and -2), protein phosphatase 2A (PP2A), and calcineurin. We employed immunocytology, immunoprecipitation, and proximity ligation assay techniques and show that PHLPP and calcineurin translocated to the nucleus and formed complexes with Akt within 3 min. Also PTEN translocated to the nucleus and then co-localized with pAkt close to the nuclear membrane. An inhibitor of the scaffolding immunophilin FK506-binding protein 51 (FKBP51) and calcineurin, FK506, prevented depletion of nuclear pAkt. Furthermore, okadaic acid, an inhibitor of PP2A, prevented the nuclear pAkt depletion. Chemical inhibition and siRNA indicated that PHLPP, PP2A, and PTEN were required for a robust depletion of nuclear pAkt, and in prostate cancer cells lacking PTEN, transfection of PTEN restored the statin-induced pAkt depletion. The activation of protein and lipid phosphatases was paralleled by a rapid proliferating cell nuclear antigen (PCNA) translocation to the nucleus, a PCNA-p21(cip1) complex formation, and cyclin D1 degradation. We conclude that these effects reflect a signaling pathway for rapid depletion of pAkt that may stop the cell cycle.

Phosphopeptide Screen Uncovers Novel Phosphorylation Sites of Nedd4-2 That Potentiate Its Inhibition of the Epithelial Na+ Channel

The E3 ubiquitin ligase Nedd4-2 regulates several ion transport proteins, including the epithelial Na(+) channel (ENaC). Nedd4-2 decreases apical membrane expression and activity of ENaC. Although it is subject to tight hormonal control, the mechanistic basis of Nedd4-2 regulation remains poorly understood. To characterize regulatory inputs to Nedd4-2 function, we screened for novel sites of Nedd4-2 phosphorylation using tandem mass spectrometry. Three of seven identified Xenopus Nedd4-2 Ser/Thr phosphorylation sites corresponded to previously identified target sites for SGK1, whereas four were novel, including Ser-293, which matched the consensus for a MAPK target sequence. Further in vitro and in vivo phosphorylation experiments revealed that Nedd4-2 serves as a target of JNK1, but not of p38 MAPK or ERK1/2. Additional rounds of tandem mass spectrometry identified two other phosphorylated residues within Nedd4-2, including Thr-899, which is present within the catalytic domain. Nedd4-2 with mutations at these sites had markedly inhibited JNK1-dependent phosphorylation, virtually no ENaC inhibitory activity, and significantly reduced ubiquitin ligase activity. These data identify phosphorylatable residues that activate Nedd4-2 and may work together with residues targeted by inhibitory kinases (e.g. SGK1 and protein kinase A) to govern Nedd4-2 regulation of epithelial ion transport.

Calcium Activates Nedd4 E3 Ubiquitin Ligases by Releasing the C2 Domain-mediated Auto-inhibition

Nedd4 E3 ligases are members of the HECT E3 ubiquitin ligase family and regulate ubiquitination-mediated protein degradation. In this report, we demonstrate that calcium releases the C2 domain-mediated auto-inhibition in both Nedd4-1 and Nedd4-2. Calcium disrupts binding of the C2 domain to the HECT domain. Consistent with this, calcium activates the E3 ubiquitin ligase activity of Nedd4. Elevation of intracellular calcium by ionomycin treatment, or activation of acetylcholine receptor or epidermal growth factor receptor by carbachol or epidermal growth factor stimulation induced activation of endogenous Nedd4 in vivo evaluated by assays of either Nedd4 E3 ligase activity or ubiquitination of Nedd4 substrate ENaC-beta. The activation effect of calcium on Nedd4 E3 ligase activity was dramatically enhanced by a membrane-rich fraction, suggesting that calcium-mediated membrane translocation through the C2 domain might be an activation mechanism of Nedd4 in vivo. Our studies have revealed an activation mechanism of Nedd4 E3 ubiquitin ligases and established a connection of intracellular calcium signaling to regulation of protein ubiquitination.

A Capsid-Encoded PPxY-Motif Facilitates Adenovirus Entry

Viruses use cellular machinery to enter and infect cells. In this study we address the cell entry mechanisms of nonenveloped adenoviruses (Ads). We show that protein VI, an internal capsid protein, is rapidly exposed after cell surface attachment and internalization and remains partially associated with the capsid during intracellular transport. We found that a PPxY motif within protein VI recruits Nedd4 E3 ubiquitin ligases to bind and ubiquitylate protein VI. We further show that this PPxY motif is involved in rapid, microtubule-dependent intracellular movement of protein VI. Ads with a mutated PPxY motif can efficiently escape endosomes but are defective in microtubule-dependent trafficking toward the nucleus. Likewise, depletion of Nedd4 ligases attenuates nuclear accumulation of incoming Ad particles and infection. Our data provide the first evidence that virus-encoded PPxY motifs are required during virus entry, which may be of significance for several other pathogens.

N4BP1 is a newly identified nucleolar protein that undergoes SUMO-regulated polyubiquitylation and proteasomal turnover at promyelocytic leukemia nuclear bodies

A number of proteins can be conjugated with both ubiquitin and the small ubiquitin-related modifier (SUMO), with crosstalk between these two post-translational modifications serving to regulate protein function and stability. We previously identified N4BP1 as a substrate for monoubiquitylation by the E3 ubiquitin ligase Nedd4. Here, we describe Nedd4-mediated polyubiquitylation and proteasomal degradation of N4BP1. In addition, we show that N4BP1 can be conjugated with SUMO1 and that this abrogates N4BP1 ubiquitylation. Consistent with this, endogenous N4BP1 is stabilized in primary embryonic fibroblasts from mutants of the desumoylating enzyme SENP1, which show increased steady-state sumoylation levels. We have localized endogenous N4BP1 predominantly to the nucleolus in primary cells. However, a small fraction is found at promyelocytic leukemia (PML) nuclear bodies (NBs). In cells deficient for SENP1 or in wild-type cells treated with the proteasome inhibitor MG132, there is considerable accumulation of N4BP1 at PML NBs. These findings suggest a dynamic interaction between subnuclear compartments, and a role for post-translational modification by ubiquitin and SUMO in the regulation of nucleolar protein turnover.

HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK

ACK (activated Cdc42-associated tyrosine kinase) (also Tnk2) is an ubiquitin-binding protein and plays an important role in ligand-induced and ubiquitination-mediated degradation of epidermal growth factor receptor (EGFR). Here we report that ACK is ubiquitinated by HECT E3 ubiquitin ligase Nedd4-1 and degraded along with EGFR in response to EGF stimulation. ACK interacts with Nedd4-1 through a conserved PPXY WW-binding motif. The WW3 domain in Nedd4-1 is critical for binding to ACK. Although ACK binds to both Nedd4-1 and Nedd4-2 (also Nedd4L), Nedd4-1 is the E3 ubiquitin ligase for ubiquitination of ACK in cells. Interestingly, deletion of the sterile alpha motif (SAM) domain at the N terminus dramatically reduced the ubiquitination of ACK by Nedd4-1, while deletion of the Uba domain dramatically enhanced the ubiquitination. Use of proteasomal and lysosomal inhibitors demonstrated that EGF-induced ACK degradation is processed by lysosomes, not proteasomes. RNA interference (RNAi) knockdown of Nedd4-1, not Nedd4-2, inhibited degradation of both EGFR and ACK, and overexpression of ACK mutants that are deficient in either binding to or ubiquitination by Nedd4-1 blocked EGF-induced degradation of EGFR. Our findings suggest an essential role of Nedd4-1 in regulation of EGFR degradation through interaction with and ubiquitination of ACK.