Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport.

The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network

Early endosome-to-trans-Golgi network (TGN) transport is organized by the retromer complex. Consisting of cargo-selective and membrane-bound subcomplexes, retromer coordinates sorting with membrane deformation and carrier formation. Here, we describe four mammalian retromers whose membrane-bound subcomplexes contain specific combinations of the sorting nexins (SNX), SNX1, SNX2, SNX5, and SNX6. We establish that retromer requires a dynamic spatial organization of the endosomal network, which is regulated through association of SNX5/SNX6 with the p150(glued) component of dynactin, an activator of the minus-end directed microtubule motor dynein; an association further defined through genetic studies in C. elegans. Finally, we also establish that the spatial organization of the retromer pathway is mediated through the association of SNX1 with the proposed TGN-localized tether Rab6-interacting protein-1. These interactions describe fundamental steps in retromer-mediated transport and establish that the spatial organization of the retromer network is a critical element required for efficient retromer-mediated sorting.

CART: an Hrs/actinin-4/BERP/myosin V protein complex required for efficient receptor recycling.

Altering the number of surface receptors can rapidly modulate cellular responses to extracellular signals. Some receptors, like the transferrin receptor (TfR), are constitutively internalized and recycled to the plasma membrane. Other receptors, like the epidermal growth factor receptor (EGFR), are internalized after ligand binding and then ultimately degraded in the lysosome. Routing internalized receptors to different destinations suggests that distinct molecular mechanisms may direct their movement. Here, we report that the endosome-associated protein hrs is a subunit of a protein complex containing actinin-4, BERP, and myosin V that is necessary for efficient TfR recycling but not for EGFR degradation. The hrs/actinin-4/BERP/myosin V (CART [cytoskeleton-associated recycling or transport]) complex assembles in a linear manner and interrupting binding of any member to its neighbor produces an inhibition of transferrin recycling rate. Disrupting the CART complex results in shunting receptors to a slower recycling pathway that involves the recycling endosome. The novel CART complex may provide a molecular mechanism for the actin-dependence of rapid recycling of constitutively recycled plasma membrane receptors.

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

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.

Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism.

Activation of the mitogen-activated protein (MAP) kinase cascade by progesterone in Xenopus oocytes leads to a marked down-regulation of activity of the amiloride-sensitive epithelial sodium channel (ENaC). Here we have studied the signaling pathways involved in progesterone effect on ENaC activity. We demonstrate that: (i) the truncation of the C termini of the alphabetagammaENaC subunits results in the loss of the progesterone effect on ENaC; (ii) the effect of progesterone was also suppressed by mutating conserved tyrosine residues in the Pro-X-X-Tyr (PY) motif of the C termini of the beta and gamma ENaC subunits (beta(Y618A) and gamma(Y628A)); (iii) the down-regulation of ENaC activity by progesterone was also suppressed by co-expression ENaC subunits with a catalytically inactive mutant of Nedd4-2, a ubiquitin ligase that has been previously demonstrated to decrease ENaC cell-surface expression via a ubiquitin-dependent internalization/degradation mechanism; (iv) the effect of progesterone was significantly reduced by suppression of consensus sites (beta(T613A) and gamma(T623A)) for ENaC phosphorylation by the extracellular-regulated kinase (ERK), a MAP kinase previously shown to facilitate the binding of Nedd4 ubiquitin ligases to ENaC; (v) the quantification of cell-surface-expressed ENaC subunits revealed that progesterone decreases ENaC open probability (whole cell P(o), wcP(o)) and not its cell-surface expression. Collectively, these results demonstrate that the binding of active Nedd4-2 to ENaC is a crucial step in the mechanism of ENaC inhibition by progesterone. Upon activation of ERK, the effect of Nedd4-2 on ENaC open probability can become more important than its effect on ENaC cell-surface expression.

Renal tubular NEDD4-2 deficiency causes NCC-mediated salt-dependent hypertension.

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na+ channel (ENaC/SCNN1) to mediate Na+ homeostasis. Mutations in the human β/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6-8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high-Na+ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na+/K+ balance. Under a high-Na+ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of βENaC, γENaC, the renal outer medullary K+ channel (ROMK), and total and phosphorylated thiazide-sensitive Na+Cl- cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of β/γENaC, but not αENaC, and a normal Na+/K+ balance maintained by downregulation of ENaC activity and upregulation of ROMK.

Ubiquitylation of voltage-gated sodium channels.

Ion channel proteins are regulated by different types of posttranslational modifications. The focus of this review is the regulation of voltage-gated sodium channels (Navs) upon their ubiquitylation. The amiloride-sensitive epithelial sodium channel (ENaC) was the first ion channel shown to be regulated upon ubiquitylation. This modification results from the binding of ubiquitin ligase from the Nedd4 family to a protein-protein interaction domain, known as the PY motif, in the ENaC subunits. Many of the Navs have similar PY motifs, which have been demonstrated to be targets of Nedd4-dependent ubiquitylation, tagging them for internalization from the cell surface. The role of Nedd4-dependent regulation of the Nav membrane density in physiology and disease remains poorly understood. Two recent studies have provided evidence that Nedd4-2 is downregulated in dorsal root ganglion (DRG) neurons in both rat and mouse models of nerve injury-induced neuropathic pain. Using two different mouse models, one with a specific knockout of Nedd4-2 in sensory neurons and another where Nedd4-2 was overexpressed with the use of viral vectors, it was demonstrated that the neuropathy-linked neuronal hyperexcitability was the result of Nav1.7 and Nav1.8 overexpression due to Nedd4-2 downregulation. These studies provided the first in vivo evidence of the role of Nedd4-2-dependent regulation of Nav channels in a disease state. This ubiquitylation pathway may be involved in the development of symptoms and diseases linked to Nav-dependent hyperexcitability, such as pain, cardiac arrhythmias, epilepsy, migraine, and myotonias.

Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2.

Nedd4-2 has been proposed to play a critical role in regulating epithelial Na+ channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgk1) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in Na+ homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other Na+ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaCl-sensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder.

Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.

Ubiquitin-specific protease 2-45 (Usp2-45) binds to epithelial Na+ channel (ENaC)-ubiquitylating enzyme Nedd4-2.

Regulation of the epithelial Na(+) channel (ENaC) by ubiquitylation is controlled by the activity of two counteracting enzymes, the E3 ubiquitin-protein ligase Nedd4-2 (mouse ortholog of human Nedd4L) and the ubiquitin-specific protease Usp2-45. Previously, Usp2-45 was shown to decrease ubiquitylation and to increase surface function of ENaC in Xenopus laevis oocytes, whereas the splice variant Usp2-69, which has a different N-terminal domain, was inactive toward ENaC. It is shown here that the catalytic core of Usp2 lacking the N-terminal domain has a reduced ability relative to Usp2-45 to enhance ENaC activity in Xenopus oocytes. In contrast, its catalytic activity toward the artificial substrate ubiquitin-AMC is fully maintained. The interaction of Usp2-45 with ENaC exogenously expressed in HEK293 cells was tested by coimmunoprecipitation. The data indicate that different combinations of ENaC subunits, as well as the α-ENaC cytoplasmic N-terminal but not C-terminal domain, coprecipitate with Usp2-45. This interaction is decreased but not abolished when the cytoplasmic ubiquitylation sites of ENaC are mutated. Importantly, coimmunoprecipitation in HEK293 cells and GST pull-down of purified recombinant proteins show that both the catalytic domain and the N-terminal tail of Usp2-45 physically interact with the HECT domain of Nedd4-2. Taken together, the data support the conclusion that Usp2-45 action on ENaC is promoted by various interactions, including through binding to Nedd4-2 that is suggested to position Usp2-45 favorably for ENaC deubiquitylation.

Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.

The epithelial Na(+) channel ENaC is a key player in the maintenance of whole body Na(+) balance, and consequently of blood pressure. It is tightly regulated by numerous signaling pathways including ubiquitylation via the ubiquitin-protein ligase Nedd4-2. This mechanism is itself under the control of several kinases, which phosphorylate Nedd4-2, thereby interfering with ENaC/Nedd4-2 interaction, or by Usp2-45, which binds to and deubiquitylates ENaC. Another, different regulatory mechanism concerns the proteolytic activation of ENaC, during which the channel is cleaved on its luminal side by intracellular convertases such as furin, and further activated by extracellular proteases such as CAP-1. This process is regulated as well but the underlying mechanisms are not understood. Previously, evidence was provided that the ubiquitylation status of ENaC may affect the cleavage of the channel. When ubiquitylation of ENaC was reduced, either by co-expressing Usp2-45, or mutating either the ENaC PY-motifs (i.e. the binding sites for Nedd4-2) or intracellular lysines (i.e. ubiquitylation sites), the level of channel cleavage was increased. Here we demonstrate that lysine-mutated ENaC channels are not ubiquitylated at the cell surface, are preferentially cleaved, and Usp2-45 does not affect their cleavage efficiency. We further show by limited proteolysis that the intracellular ubiquitylation status of ENaC affects the extracellular conformation of αENaC, by demonstrating that non-ubiquitylated channels are more efficiently cleaved when treated with extracellularly added trypsin or chymotrypsin. These results present a new paradigm in which an intracellular, post-translational modification (e.g. ubiquitylation) of a transmembrane protein can affect its extracellular conformation.

Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

FoxM1B regulates NEDD4-1 expression, leading to cellular transformation and full malignant phenotype in immortalized human astrocytes.

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms.

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.