Nedd4 family-interacting protein 1 (Ndfip1) is required for the exosomal secretion of Nedd4 family proteins

The ability to remove unwanted proteins is an important cellular feature. Classically, this involves the enzymatic addition of ubiquitin moieties followed by degradation in the proteasome. Nedd4 proteins are ubiquitin ligases important not only for protein degradation, but also for protein trafficking. Nedd4 proteins can bind to target proteins either by themselves or through adaptor protein Ndfip1 (Nedd4 family-interacting protein 1). An alternative mechanism for protein removal and trafficking is provided by exosomes, which are small vesicles (50-90-nm diameter) originating from late endosomes and multivesicular bodies (MVBs). Exosomes provide a rapid means of shedding obsolete proteins and also for cell to cell communication. In the present work, we show that Ndfip1 is detectable in exosomes secreted from transfected cells and also from primary neurons. Compared with control, Ndfip1 increases exosome secretion from transfected cells. Furthermore, while Nedd4, Nedd4-2, and Itch are normally absent from exosomes, expression of Ndfip1 results in recruitment of all three Nedd4 proteins into exosomes. Together, these results suggest that Ndfip1 is important for protein trafficking via exosomes, and provides a mechanism for cargoing passenger proteins such as Nedd4 family proteins. Given the positive roles of Ndfip1/Nedd4 in improving neuronal survival during brain injury, it is possible that exosome secretion provides a novel route for rapid sequestration and removal of proteins during stress.     

p18 Ink4c and Pten constrain a positive regulatory loop between cell growth and cell cycle control

Inactivation of the Rb-mediated G1 control pathway is a common event found in many types of human tumors. To test how the Rb pathway interacts with other pathways in tumor suppression, we characterized mice with mutations in both the cyclin-dependent kinase (CDK) inhibitor p18 Ink4c and the lipid phosphatase Pten, which regulates cell growth. The double mutant mice develop a wider spectrum of tumors, including prostate cancer in the anterior and dorsolateral lobes, with nearly complete penetrance and at an accelerated rate. The remaining wild-type allele of Pten was lost at a high frequency in Pten+/- cells but not in p18+/- Pten+/- or p18-/- Pten+/- prostate tumor cells, nor in other Pten+/- tumor cells, suggesting a tissue- and genetic background-dependent haploinsufficiency of Pten in tumor suppression. p18 deletion, CDK4 overexpression, or oncoviral inactivation of Rb family proteins caused activation of Akt/PKB that was recessive to the reduction of PTEN activity. We suggest that p18 and Pten cooperate in tumor suppression by constraining a positive regulatory loop between cell growth and cell cycle control pathways.     

Iron status and lipopolysaccharide regulate Ndfip1 by activation of nuclear factor-kappa B

Nedd4 family interacting protein 1 (Ndfip1) is an adaptor protein for the Nedd4 family of ubiquitin ligases that target proteins for degradation. Recent studies confirmed the role of Ndfip1 as a regulator of iron metabolism and pointed out that Ndfip1 was involved in iron homeostasis by regulating the degradation of iron importer divalent metal transporter 1 (DMT1). However, little is known about how Ndfip1 is regulated. The aim of this article was to investigate the regulation of Ndfip1 levels and the possible mechanisms. In this study, we investigated the effect of various stimuli, including iron status and lipopolysaccharide (LPS) on Ndfip1 expression in MES23.5 dopaminergic cell lines. Results showed that Ndfip1 expression in these cells was enhanced by ferrous iron overload, but not ferric iron overload, and decreased after iron deprivation by deferoxamine. In addition, LPS could significantly increase the expression of Ndfip1. Furthermore, we demonstrated that the regulation of Ndfip1 expression by these various stimuli was achieving by activation of nuclear factor-kappa B. We speculate that iron status and LPS may contribute to the changes of Ndfip1 expression by activation of nuclear factor-kappa B.     

Physiological functions of Pten in mouse tissues

PTEN is a tumor suppressor gene mutated in many human sporadic cancers and in hereditary cancer syndromes such as Cowden disease, Bannayan-Zonana syndrome and Lhermitte-Duclos disease. The major substrate of PTEN is PIP3, a second messenger molecule produced following PI3K activation induced by variety of stimuli. PIP3 activates the serine-threonine kinase PKB/Akt which is involved in anti-apoptosis, proliferation and oncogenesis. In mice, heterozygosity for a null mutation of Pten (Pten(+/-) mice) frequently leads to the development of a variety of cancers and autoimmune disease. Homozygosity for the null mutation (Pten (-/-) mice) results in early embryonic lethality, precluding the functional analysis of Pten in various organs. To investigate the physiological functions of Pten in viable mice, various tissue-specific Pten mutations have been generated using the Cre-loxP system. This review will summarize the phenotypes of conditional mutant mice lacking Pten function in specific tissues, and discuss how these phenotypes relate to the physiological roles of Pten in various organ systems.     

Increased Ndfip1 in the Substantia Nigra of Parkinsonian Brains Is Associated with Elevated Iron Levels

Iron misregulation is a central component in the neuropathology of Parkinson''s disease. The iron transport protein DMT1 is known to be increased in Parkinson''s brains linking functional transport mechanisms with iron accumulation. The regulation of DMT1 is therefore critical to the management of iron uptake in the disease setting. We previously identified post-translational control of DMT1 levels through a ubiquitin-mediated pathway led by Ndfip1, an adaptor for Nedd4 family of E3 ligases. Here we show that loss of Ndfip1 from mouse dopaminergic neurons resulted in misregulation of DMT1 levels and increased susceptibility to iron induced death. We report that in human Parkinson''s brains increased iron concentrations in the substantia nigra are associated with upregulated levels of Ndfip1 in dopaminergic neurons containing α-synuclein deposits. Additionally, Ndfip1 was also found to be misexpressed in astrocytes, a cell type normally devoid of this protein. We suggest that in Parkinson''s disease, increased iron levels are associated with increased Ndfip1 expression for the regulation of DMT1, including abnormal Ndfip1 activation in non-neuronal cell types such as astrocytes.     

Rab5 and Ndfip1 Are Involved in Pten Ubiquitination and Nuclear Trafficking

The spatial regulation of Pten is critical for its role as a tumour suppressor with both nuclear and cytoplasmic locations being implicated with distinct functions. In the cytoplasm, Pten plays a central role in opposing PI3K/Akt cell signalling, whereas in the nucleus, Pten is important for maintaining genome stability and enhancing the tumour suppressor activity of APC‐CDH1. Despite this diversity in protein function at different subcellular locations, there is limited knowledge on how Pten is able to find different cellular niches. Here, we report that Rab5 GTPase is required for efficient trafficking and ubiquitination of Pten on endosomes inside the cytosol. Using bimolecular fluorescence complementation (BiFC) for imaging protein interactions, we observed that ubiquitinated Pten is localized to peri‐nuclear and nuclear regions of the cell. Nuclear trafficking of Pten required both Rab5 as well as the E3 ligase adaptor protein Ndfip1. Rab5 colocalization with Pten was observed on endosomes and expression of a dominant negative form of Rab5 significantly reduced Pten ubiquitination and nuclear trafficking. Genomic deletion of Ndfip1 abrogated nuclear trafficking of ubiquitinated Pten, even in the presence of Rab5. Our findings show that endosomal trafficking and ubiquitination are important mechanisms for the subcellular distribution of Pten.      

Author Correction: Neddylation of PTEN regulates its nuclear import and promotes tumor development

PTEN tumor suppressor opposes the PI3K/Akt signaling pathway in the cytoplasm and maintains chromosomal integrity in the nucleus. Nucleus–cytoplasm shuttling of PTEN is regulated by ubiquitylation, SUMOylation and phosphorylation, and nuclear PTEN has been proposed to exhibit tumor-suppressive functions. Here we show that PTEN is conjugated by Nedd8 under high glucose conditions, which induces PTEN nuclear import without effects on PTEN stability. PTEN neddylation is promoted by the XIAP ligase and removed by the NEDP1 deneddylase. We identify Lys197 and Lys402 as major neddylation sites on PTEN. Neddylated PTEN accumulates predominantly in the nucleus and promotes rather than suppresses cell proliferation and metabolism. The nuclear neddylated PTEN dephosphorylates the fatty acid synthase (FASN) protein, inhibits the TRIM21-mediated ubiquitylation and degradation of FASN, and then promotes de novo fatty acid synthesis. In human breast cancer tissues, neddylated PTEN correlates with tumor progression and poor prognosis. Therefore, we demonstrate a previously unidentified pool of nuclear PTEN in the Nedd8-conjugated form and an unexpected tumor-promoting role of neddylated PTEN.Subject terms: Neddylation, Breast cancer     

E3 ubiquitin ligase Cbl-b regulates Pten via Nedd4 in T cells independently of its ubiquitin ligase activity

E3 ubiquitin ligase Cbl-b plays a crucial role in T cell activation and tolerance induction. However, the molecular mechanism by which Cbl-b inhibits T cell activation remains unclear. Here, we report that Cbl-b does not inhibit PI3K but rather suppresses TCR/CD28-induced inactivation of Pten. The elevated Akt activity in Cbl-b(-/-) T cells is therefore due to heightened Pten inactivation. Suppression of Pten inactivation in T cells by Cbl-b is achieved by impeding the association of Pten with Nedd4, which targets Pten K13 for K63-linked polyubiquitination. Consistent with this finding, introducing Nedd4 deficiency into Cbl-b(-/-) mice abrogates hyper-T cell responses caused by the loss of Cbl-b. Hence, our data demonstrate that Cbl-b inhibits T cell activation by suppressing Pten inactivation independently of its ubiquitin ligase activity.     

Ubiquitination regulates PTEN nuclear import and tumor suppression

The PTEN tumor suppressor is frequently affected in cancer cells, and inherited PTEN mutation causes cancer-susceptibility conditions such as Cowden syndrome. PTEN acts as a plasma-membrane lipid-phosphatase antagonizing the phosphoinositide 3-kinase/AKT cell survival pathway. However, PTEN is also found in cell nuclei, but mechanism, function, and relevance of nuclear localization remain unclear. We show that nuclear PTEN is essential for tumor suppression and that PTEN nuclear import is mediated by its monoubiquitination. A lysine mutant of PTEN, K289E associated with Cowden syndrome, retains catalytic activity but fails to accumulate in nuclei of patient tissue due to an import defect. We identify this and another lysine residue as major monoubiquitination sites essential for PTEN import. While nuclear PTEN is stable, polyubiquitination leads to its degradation in the cytoplasm. Thus, we identify cancer-associated mutations of PTEN that target its posttranslational modification and demonstrate how a discrete molecular mechanism dictates tumor progression by differentiating between degradation and protection of PTEN.     

Aagab acts as a novel regulator of NEDD4-1-mediated Pten nuclear translocation to promote neurological recovery following hypoxic-ischemic brain damage

Hypoxic-ischemic encephalopathy (HIE) is a main cause of mortality and severe neurologic impairment in the perinatal and neonatal period. However, few satisfactory therapeutic strategies are available. Here, we reported that a rapid nuclear translocation of phosphatase and tensin homolog deleted on chromosome TEN (PTEN) is an essential step in hypoxic-ischemic brain damage (HIBD)- and oxygen-glucose deprivation (OGD)-induced neuronal injures both in vivo and in vitro. In addition, we found that OGD-induced nuclear translocation of PTEN is dependent on PTEN mono-ubiquitination at the lysine 13 residue (K13) that is mediated by neural precursor cell expressed developmentally downregulated protein 4-1 (NEDD4-1). Importantly, we for the first time identified α- and γ-adaptin binding protein (Aagab) as a novel NEDD4-1 regulator to regulate the level of NEDD4-1, subsequently mediating Pten nuclear translocation. Finally, we demonstrated that genetic upregulation of Aagab or application of Tat-K13 peptide (a short interference peptide that flanks K13 residue of PTEN) not only reduced Pten nuclear translocation, but also significantly alleviated the deficits of myodynamia, motor and spatial learning and memory in HIBD model rats. These results suggest that Aagab may serve as a regulator of NEDD4-1-mediated Pten nuclear translocation to promote functional recovery following HIBD in neonatal rats, and provide a new potential therapeutic target to guide the clinical treatment for HIE.Subject terms: Cognitive ageing, Neurological disorders     

Gastrointestinal microbiota do not significantly contribute to T cell activation or GI inflammation in Ndfip1-cKO mice

The bacteria inhabiting the mammalian gastrointestinal (GI) tract play a vital role in normal digestion and immune function. In a healthy host, the immune system is tolerant to gut bacteria and does not mount an effector response to bacteria-derived antigens. Loss of tolerance to intestinal microflora has been associated with inflammatory bowel disease (IBD) in both mice and humans. Mice lacking Ndfip1, an adaptor protein for E3 ubiquitin ligases of the Nedd4-family, in T cells (Ndfip1-cKO) develop a disease resembling IBD. Inflammation in these mice is characterized by increased activation of peripheral T cells, infiltration of eosinophils into the GI tract, and epithelial hypertrophy in the esophagus. We hypothesized that this intestinal inflammation in Ndfip1-cKO mice is caused by a loss of T-cell tolerance to bacterial antigens. Here, we show that treatment of Ndfip1-cKO mice with broad-spectrum antibiotics drastically reduced bacterial load in stool but had little effect on T-cell activation and did not affect eosinophil infiltration into the GI tract or epithelial hypertrophy in the esophagus. Thus, inflammation in Ndfip1-cKO mice is not caused by a loss of tolerance to intestinal microbiota. Rather, T cell activation and eosinophilia may instead be triggered by other environmental antigens.     

Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation

Nedd4-family E3 ubiquitin ligases regulate an array of biologic processes. Autoinhibition maintains these catalytic ligases in an inactive state through several mechanisms. However, although some Nedd4 family members are activated by binding to Nedd4 family-interacting proteins (Ndfips), how binding activates E3 function remains unclear. Our data reveal how these two regulatory processes are linked functionally. In the absence of Ndfip1, the Nedd4 family member Itch can bind an E2 but cannot accept ubiquitin onto its catalytic cysteine. This is because Itch is autoinhibited by an intramolecular interaction between its HECT (homologous to the E6-AP carboxy terminus domain) and two central WW domains. Ndfip1 binds these WW domains to release the HECT, allowing trans-thiolation and Itch catalytic activity. This molecular switch also regulates the closely related family member WWP2. Importantly, multiple PY motifs are required for Ndfip1 to activate Itch, functionally distinguishing Ndfips from single PY-containing substrates. These data establish a novel mechanism for control of the function of a subfamily of Nedd4 E3 ligases at the level of E2-E3 trans-thiolation.     

Cellular up-regulation of Nedd4 family interacting protein 1 (Ndfip1) using low levels of bioactive cobalt complexes

The delivery of metal ions using cell membrane-permeable metal complexes represents a method for activating cellular pathways. Here, we report the synthesis and characterization of new [Co(III)(salen)(acac)] complexes capable of up-regulating the ubiquitin ligase adaptor protein Ndfip1. Ndfip1 is a neuroprotective protein that is up-regulated in the brain after injury and functions in combination with Nedd4 ligases to ubiquitinate harmful proteins for removal. We previously showed that Ndfip1 can be increased in human neurons using CoCl(2) that is toxic at high concentration. Here we demonstrate a similar effect can be achieved by low concentrations of synthetic Co(III) complexes that are non-toxic and designed to be activated following cellular entry. Activation is achieved by intracellular reduction of Co(III) to Co(II) leading to release of Co(II) ions for Ndfip1 up-regulation. The cellular benefit of Ndfip1 up-regulation by Co(III) complexes includes demonstrable protection against cell death in SH-SY5Y cells during stress. In vivo, focal delivery of Co(III) complexes into the adult mouse brain was observed to up-regulate Ndfip1 in neurons. These results demonstrate that a cellular response pathway can be advantageously manipulated by chemical modification of metal complexes, and represents a significant step of harnessing low concentration metal complexes for therapeutic benefit.     

PTEN deficiency causes dyschondroplasia in mice by enhanced hypoxia-inducible factor 1alpha signaling and endoplasmic reticulum stress

Chondrocytes within the growth plates acclimatize themselves to a variety of stresses that might otherwise disturb cell fate. The tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) has been implicated in the maintenance of cell homeostasis. However, the functions of PTEN in regulating chondrocytic adaptation to stresses remain largely unknown. In this study, we have created chondrocyte-specific Pten knockout mice (Pten(co/co);Col2a1-Cre) using the Cre-loxP system. Following AKT activation, Pten mutant mice exhibited dyschondroplasia resembling human enchondroma. Cartilaginous nodules originated from Pten mutant resting chondrocytes that suffered from impaired proliferation and differentiation, and this was coupled with enhanced endoplasmic reticulum (ER) stress. We further found that ER stress in Pten mutant chondrocytes only occurred under hypoxic stress, characterized by an upregulation of unfolded protein response-related genes as well as an engorged and fragmented ER in which collagens were trapped. An upregulation of hypoxia-inducible factor 1alpha (HIF1alpha) and downstream targets followed by ER stress induction was also observed in Pten mutant growth plates and in cultured chondrocytes, suggesting that PI3K/AKT signaling modulates chondrocytic adaptation to hypoxic stress via regulation of the HIF1alpha pathway. These data demonstrate that PTEN function in chondrocytes is essential for their adaptation to stresses and for the inhibition of dyschondroplasia.     

Essential role for nuclear PTEN in maintaining chromosomal integrity

A broad spectrum of mutations in PTEN, encoding a lipid phosphatase that inactivates the P13-K/AKT pathway, is found associated with primary tumors. Some of these mutations occur outside the phosphatase domain, suggesting that additional activities of PTEN function in tumor suppression. We report a nuclear function for PTEN in controlling chromosomal integrity. Disruption of Pten leads to extensive centromere breakage and chromosomal translocations. PTEN was found localized at centromeres and physically associated with CENP-C, an integral component of the kinetochore. C-terminal PTEN mutants disrupt the association of PTEN with centromeres and cause centromeric instability. Furthermore, Pten null cells exhibit spontaneous DNA double-strand breaks (DSBs). We show that PTEN acts on chromatin and regulates expression of Rad51, which reduces the incidence of spontaneous DSBs. Our results demonstrate that PTEN plays a fundamental role in the maintenance of chromosomal stability through the physical interaction with centromeres and control of DNA repair. We propose that PTEN acts as a guardian of genome integrity.     

The E3 ubiquitin ligase adaptor Ndfip1 regulates Th17 differentiation by limiting the production of proinflammatory cytokines

Ndfip1 is an adaptor for the E3 ubiquitin ligase Itch. Both Ndfip1- and Itch-deficient T cells are biased toward Th2 cytokine production. In this study, we demonstrate that lungs from Ndfip1(-/-) mice showed increased numbers of neutrophils and Th17 cells. This was not because Ndfip1(-/-) T cells are biased toward Th17 differentiation. In fact, fewer Ndfip1(-/-) T cells differentiated into Th17 cells in vitro due to high IL-4 production. Rather, Th17 differentiation was increased in Ndfip1(-/-) mice due to increased numbers of IL-6-producing eosinophils. IL-6 levels in mice that lacked both Ndfip1 and IL-4 were similar to wild-type controls, and these mice had fewer Th17 cells in their lungs. These results indicate that Th2 inflammation, such as that observed in Ndfip1(-/-) mice, can increase Th17 differentiation by recruiting IL-6-producing eosinophils into secondary lymphoid organs and tissues. This may explain why Th17 cells develop within an ongoing Th2 inflammatory response.     

Essential role of AKT-1/protein kinase B alpha in PTEN-controlled tumorigenesis

PTEN is mutated at high frequency in many primary human cancers and several familial cancer predisposition disorders. Activation of AKT is a common event in tumors in which the PTEN gene has been inactivated. We previously showed that deletion of the murine Pten gene in embryonic stem (ES) cells led to increased phosphatidylinositol triphosphate (PIP(3)) accumulation, enhanced entry into S phase, and better cell survival. Since PIP(3) controls multiple signaling molecules, it was not clear to what degree the observed phenotypes were due to deregulated AKT activity. In this study, we mutated Akt-1 in Pten(-/-) ES cells to directly assess the role of AKT-1 in PTEN-controlled cellular processes, such as cell proliferation, cell survival, and tumorigenesis in nude mice. We showed that AKT-1 is one of the major downstream effectors of PTEN in ES cells and that activation of AKT-1 is required for both the cell survival and cell proliferation phenotypes observed in Pten(-/-) ES cells. Deletion of Akt-1 partially reverses the aggressive growth of Pten(-/-) ES cells in vivo, suggesting that AKT-1 plays an essential role in PTEN-controlled tumorigenesis.     

Allosteric auto‐inhibition and activation of the Nedd4 family E3 ligase Itch

The Nedd4 family E3 ligases are key regulators of cell growth and proliferation and are often misregulated in human cancers and other diseases. The ligase activities of Nedd4 E3s are tightly controlled via auto‐inhibition. However, the molecular mechanism underlying Nedd4 E3 auto‐inhibition and activation is poorly understood. Here, we show that the WW domains proceeding the catalytic HECT domain play an inhibitory role by binding directly to HECT in the Nedd4 E3 family member Itch. Our structural and biochemical analyses of Itch reveal that the WW2 domain and a following linker allosterically lock HECT in an inactive state inhibiting E2‐E3 transthiolation. Binding of the Ndfip1 adaptor or JNK1‐mediated phosphorylation relieves the auto‐inhibition of Itch in a WW2‐dependent manner. Aberrant activation of Itch leads to migration defects of cortical neurons during development. Our study provides a new mechanism governing the regulation of Itch.