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The UIM domain of Hrs couples receptor sorting to vesicle formation
Abstract
Hepatocyte growth factor regulated tyrosine kinase substrate (Hrs), a main component of the 'bilayered' clathrin coat on sorting endosomes, was originally identified as a substrate of activated tyrosine kinase receptors. We have analysed Hrs phosphorylation in response to epidermal growth factor (EGF) stimulation and show that the evolutionary conserved tyrosines Y329 and Y334 provide the principal phosphorylation sites. Hrs is proposed to concentrate ubiquitinated receptors within clathrin-coated regions via direct interaction with its UIM (ubiquitin interaction motif) domain. We show that the same UIM domain is necessary for EGF-stimulated tyrosine phosphorylation of Hrs. Over-expression of wild-type Hrs or a double mutant, Y329/334F, defective in EGF-dependent phosphorylation, both substantially retard EGF receptor (EGFR) degradation by inhibiting internal vesicle formation and thereby preventing EGFR incorporation into lumenal vesicles of the multivesicular bodies. In contrast, mutation or deletion of the Hrs-UIM domain strongly suppresses this effect. In addition the UIM-deletion and point mutants are also observed on internal membranes, indicating a failure to dissociate from the endosomal membrane prior to incorporation of the receptor complex into lumenal vesicles. Our data suggest a role for the UIM-domain of Hrs in actively retaining EGFR at the limiting membrane of endosomes as a prelude to lumenal vesicle formation.
... Ce domaine permet à HRS de ce lier au PI3P localisé sur les endosomes et de se lier spécifiquement à cette organelle (figure 22) (Raiborg et al., 2001b). D'autre part, HRS et STAM1/2 sont toutes les deux capables de lier l'ubiquitine grâce à leurs domaines UIM (Ubiquitin Interacting Motif) et VHS (Vps27, HRS, STAM), ce qui les localise spécifiquement au niveau des endosomes contenant des cargos ubiquitinés (Bilodeau et al., 2002;Mizuno et al., 2003;Urbé et al., 2003). HRS possède aussi un domaine CBD (Clathrin Binding Domain) permettant de lier la Clathrine et de recruter cette dernière au niveau des endosomes pour faciliter le regroupement des cargos et l'invagination de la membrane (Raiborg et al., , 2001aWenzel et al., 2018). ...
... De plus, les auteurs montrent une importante vacuolisation des tissus en absence de HRS avec une accumulation d'endosomes qui explique les défauts morphogéniques observé chez les embryons . interaction avec le inositide PI3P, qui sont distribués essentiellement aux niveaux des endosomes précoces Mao et al., 2000;Raiborg et al., 2001b) ; Un domaine VHS et un domaine UIM, lui permettant d'interagir avec les protéines/cargos ubiquitinés (Bache et al., 2003b;Lohi et al., 2002;Polo et al., 2002;Shekhtman and Cowburn, 2002;Urbé et al., 2003) ; Un domaine CBD lui permet de lier la ...
... La protéines HRS possède différents domaines qui lui permettent d'interagir avec les récepteurs membranaires ubiquitinés à dégrader et de se localiser sur les endosomes précoces afin de recruter les autres composants de la machinerie ESCRT (voir figure 24 dans ce manuscrit). Certains domaines participent aussi aux fonctions de HRS dans le recyclage de récepteurs membranaires Huang et al., 2009;Raiborg et al., 2006Urbé et al., 2003;Wenzel et al., 2018;Yan et al., 2005 (Henne et al., 2011a;Kojima et al., 2014;Takahashi et al., 2015). ...
La myogenèse est un mécanisme biologique à l’origine de la formation des muscles, et est induite également lors d’une blessure musculaire. Elle est orchestrée par les cellules satellites qui prolifèrent et deviennent des myoblastes capables de se différencier pour fusionner et donner naissance à des structures appelées myotubes, futurs composants des fibres musculaires néoformées lors de la réparation tissulaire. Ces étapes sont finement contrôlées par l’activation et/ou l’inhibition de facteurs de transcription myogéniques régulés par l’activation de récepteurs membranaires et des voies de signalisations associées en aval. Le processus d'endocytose régule de nombreuses voies de signalisation cellulaires en contrôlant le nombre de récepteurs fonctionnels disponibles à la surface cellulaire. Par ailleurs, de nombreuses études ont aussi révélé que des voies de signalisations pouvaient être activées sur les membranes des compartiments endosomaux. La machinerie ESCRT (endosomal Sorting Complex required for Transport) est composée de quatre sous-unités baptisées ESCRT-0 à ESCRT-III impliquées dans divers mécanismes cellulaires essentiels et notamment dans la biogenèse des compartiments endosomaux appelés corps multivésiculaires. De ce fait, la machinerie ESCRT est impliquée dans le transport, le recyclage ou la dégradation de récepteurs membranaires ubiquitinylés et par conséquent le contrôle de leur activité et des voies de signalisations qui leurs sont associées. L’implication de cette machinerie dans la régulation du processus de myogenèse reste néanmoins à ce jour non explorée. Mon travail de thèse s’est justement intéressé à caractériser le rôle de cette machinerie dans le processus de différentiation des myoblastes en myotubes. Au cours de ces quatre années, je me suis plus particulièrement intéressé à la protéine HRS/HGS (Hepatocyte growth factor Regulated tyrosine kinase Substrate) de la sous unité ESCRT-0 et à son implication lors du processus de myogenèse. Nos données expérimentales ont montré que l’expression et la distribution cellulaire de la protéine HRS/HGS étaient dynamiques au cours de la myogenèse et que son inhibition bloquait drastiquement la différenciation des myoblastes en myotubes. La recherche des mécanismes moléculaires et cellulaires associés nous a permis de mettre en évidence que l’inhibition de son expression dans les myoblastes empêchait la dégradation du récepteur à l’EGF (pour Epidermal Growth Factor) et conduisait alors à l’activation constitutive de la voie de signalisation Ras/Raf/MEK/ERK. Cette activation fut associée à une perturbation de la localisation et de l’expression de facteurs de transcription pro-myogéniques impliqués dans le processus de myogenèse. Nous avons également identifié que l’absence de HRS/HGS perturbait l’activité de la GSK3ß et participait à l’inhibition de ce processus. Enfin, nos résultats ont aussi montré que l’absence de cette protéine altérait négativement la voie autophagique indispensable au bon déroulement de la myogenèse. De façon collective, nos résultats montrent donc pour la première fois que la protéine HRS/HGS est un facteur central, multimodal et essentiel dans la régulation du processus de myogenèse.
... We selected six proteins to confirm binding to TRIAD3 in co-immunoprecipitation experiments: the ubiquitin E3 ligases WWP2 and RNF41, the Adaptor Protein complex subunit sigma (AP2s1), the Vacuolar Protein Sortingassociated Protein 35 (VPS35), Hepatocyte growth factorregulated tyrosine kinase substrate (HGS/HRS, in the following HRS) and the Ankyrin repeat domaincontaining protein 13D (Ankrd13D). We selected AP2s1, VPS35, HRS and Ankrd13D, because they were all connected to endocytic or intracellular trafficking pathways [22][23][24][25][26] , suggesting a functional connection of TRIAD3 with these processes. HA-tagged TRIAD3B was coexpressed with flag-tagged candidate proteins in HEK293T cells and anti-HA immunoprecipitations were performed. ...
... Two of these newly identified interactors were especially interesting in connection with the ubiquitin ligase TRIAD3: HRS carries one Ubiquitin Interacting Motif (UIM) and Ankrd13D carries even four of these ubiquitin binding domains, which have been well characterised for their ability to bind ubiquitin 27,28 . In particular, Ankrd13D and HRS bind to K63-linked ubiquitin chains and are both involved in endocytic processes 25,26,29 . We tested whether the interaction between TRIAD3 and HRS was mediated through ubiquitin chains, as for example TRIAD3 might be auto-ubiquitylated. ...
... A catalytically inactive mutant (C745A), which cannot auto-ubiquitylate, interacted with HRS similar to the wt TRIAD3 protein (Fig. 3f). Furthermore, we abrogated ubiquitin binding of HRS by either deleting the UIM of HRS (ΔUIM) or introduction of point mutations within the UIM (UIM*) 26 . Both proteins still interacted with TRIAD3 ( Fig. 3f), demonstrating that HRS and TRIAD3 bind to each other independent of ubiquitin chains. ...
TRIAD3/RNF216 is a ubiquitin ligase of the RING-in-between-RING family. Recent publications identified TRIAD3 mutations in patients with neurological diseases, including Gordon Holmes syndrome and Huntington-like disorder. To understand the functional relevance of these disease-associated mutations, we have tested the ubiquitin ligase activity of mutated TRIAD3 in vitro. Several of these point mutations completely abrogated TRIAD3’s catalytic activity. Using mass spectrometry, we identified new TRIAD3-interacting proteins/substrates from mouse brain lysate, which provide a new link between TRIAD3 and processes involving clathrin-mediated endocytosis. Strikingly, we found that TRIAD3 synthesises specifically lysine-63 (K63)-linked poly-ubiquitin chains in vitro, a chain type that usually plays a role in mediating signalling events rather than triggering proteasomal degradation. Therefore, this finding is of great importance to further understand TRIAD3’s cellular role and loss-of-function in disease.
... Hrs, and another ESCRT0 component, STAM1/2, are phosphorylated in response to growth factor stimulation [20] and intriguingly, both Hrs and STAM1/2 are also subject to dephosphorylation by PTP1B [5,21]. Although Hrs and STAM1/2 are soluble pro- teins and so would not necessarily require a contact site to interact with PTP1B, Hrs phosphorylation requires its recruitment to endosomes [22,23] and so would be well placed to be dephosphorylated by PTP1B at ER : endosome contact sites. The relationship between Hrs phosphorylation state and its ability to recruit clathrin would be an important indicator of the relationship between contact sites and the clathrin coat. ...
... The role of phosphorylation/dephosphorylation of Hrs and STAM1/2 in regulating activity of the ESCRT0 complex is also not clear. Several studies indicate that the major EGF-stimulated tyrosine phosphorylation sites within Hrs (Y329 and Y334) are important for efficient progression of EGFR along the endocytic pathway to the lysosome [23,25] and STAM2 phosphorylation mutants displayed altered endosomal localisation [21]. The ubiquitin-interacting motif of Hrs, which binds ubiquitinated cargo, is required for EGF-stimulated Hrs phos- phorylation [23] and can itself become ubiquitinated [26], preventing its interaction with ubiquitinated cargo. ...
... Several studies indicate that the major EGF-stimulated tyrosine phosphorylation sites within Hrs (Y329 and Y334) are important for efficient progression of EGFR along the endocytic pathway to the lysosome [23,25] and STAM2 phosphorylation mutants displayed altered endosomal localisation [21]. The ubiquitin-interacting motif of Hrs, which binds ubiquitinated cargo, is required for EGF-stimulated Hrs phos- phorylation [23] and can itself become ubiquitinated [26], preventing its interaction with ubiquitinated cargo. The relationship between Hrs ubiquitination and phosphorylation remains to be fully established, but when Hrs ubiquitination was enhanced by overexpression of the ubiquitin ligase, c-Cbl, Hrs became hyperphosphory- lated [25]. ...
Multivesicular endosomes/bodies (MVBs) sort membrane proteins between recycling and degradative pathways. Segregation of membrane proteins onto intraluminal vesicles (ILVs) of MVBs removes them from the recycling pathway and facilitates their degradation following fusion of MVBs with lysosomes. Sorting of many cargos onto ILVs depends on the ESCRT (Endosomal Sorting Complex Required for Transport) machinery, although ESCRT-independent mechanisms also exist. In mammalian cells, efficient sorting of ligand-stimulated epidermal growth factor receptors onto ILVs also depends on the tyrosine phosphatase, PTP1B, an ER-localised enzyme that interacts with endosomal targets at membrane contacts between MVBs and the ER. This review focuses on the potential roles played by ER : MVB membrane contact sites in regulating ESCRT-dependent ILV formation.
... Hrs, and another ESCRT0 component, STAM1/2, are phosphorylated in response to growth factor stimulation [20] and intriguingly, both Hrs and STAM1/2 are also subject to dephosphorylation by PTP1B [5,21]. Although Hrs and STAM1/2 are soluble proteins and so would not necessarily require a contact site to interact with PTP1B, Hrs phosphorylation requires its recruitment to endosomes [22,23] and so would be well placed to be dephosphorylated by PTP1B at ER: endosome contact sites. The relationship between Hrs phosphorylation state and its ability to recruit clathrin would be an important indicator of the relationship between contact sites and the clathrin coat. ...
... The role of phosphorylation/dephosphorylation of Hrs and STAM1/2 in regulating activity of the ESCRT0 complex is also not clear. Several studies indicate that the major EGF-stimulated tyrosine phosphorylation sites within Hrs (Y329 and Y334) are important for efficient progression of EGFR along the endocytic pathway to the lysosome [23,25] and STAM2 phosphorylation mutants displayed altered endosomal localisation [21]. The ubiquitin-interacting motif of Hrs, which binds ubiquitinated cargo, is required for EGF-stimulated Hrs phosphorylation [23] and can itself become ubiquitinated [26], preventing its interaction with ubiquitinated cargo. ...
... Several studies indicate that the major EGF-stimulated tyrosine phosphorylation sites within Hrs (Y329 and Y334) are important for efficient progression of EGFR along the endocytic pathway to the lysosome [23,25] and STAM2 phosphorylation mutants displayed altered endosomal localisation [21]. The ubiquitin-interacting motif of Hrs, which binds ubiquitinated cargo, is required for EGF-stimulated Hrs phosphorylation [23] and can itself become ubiquitinated [26], preventing its interaction with ubiquitinated cargo. The relationship between Hrs ubiquitination and phosphorylation remains to be fully established, but when Hrs ubiquitination was enhanced by overexpression of the ubiquitin ligase, c-Cbl, Hrs became hyperphosphorylated [25]. ...
The CRIB (Cdc42/Rac interactive binding) family of small G-protein effectors contain significant regions with intrinsic disorder. The G-protein-binding regions are contained within these intrinsically disordered regions. Most CRIB proteins also contain stretches of basic residues associated with their G-protein-binding regions. The basic region (BR) and G-protein-binding region together allow the CRIB effectors to bind to their cognate G-protein via a dock- and coalesce-binding mechanism. The BRs of these proteins take on multiple roles: steering G-protein binding, interacting with elements of the membrane and regulating intramolecular regulatory interactions. The ability of these regions of the CRIBs to undergo multivalent interactions and mediate charge neutralizations equips them with all the properties required to drive liquid-liquid phase separation and therefore to initiate and drive signalosome formation. It is only recently that the structural plasticity in these proteins is being appreciated as the driving force for these vital cellular processes.
... Hrs recognizes ubiquitinated receptors through the ubiquitin interacting motif (UIM), and together with signal-transduction adaptor-molecule (STAM) it acts as part of the sorting machinery for degradation via the ESCRT machinery 12,13 . Upon EGF-R activation, Hrs is tyrosine phosphorylated and monoubiquitinated 14,15 . ...
... The preceding experiments indicated a phosphorylation-mediated mechanism regulating the binding kinetics of Eps15-GFP and Hrs-YFP. To test whether phosphorylation is a prerequisite for the change in binding kinetics we point-mutated the known phosphorylation site Y850 in Eps15-GFP and in Hrs-YFP, the two adjacent phosphorylation sites Y329 and Y334 7,15 . ...
... Importantly, previous studies could show that the Ii induced enlargement did not change the membrane binding dynamics of the endosomal tethering-molecule EEA1 37 . We could furthermore confirm a similar EGF-R induced phosphorylation pattern for Eps15 and Hrs in M1 cells expressing Ii as in untransfected cells 7,9,15,38,39,44 . ...
Activation of EGF-R and PDGF-R triggers autophosphorylation and the recruitment of Eps15 and Hrs. These two endosomal proteins are important for specific receptor sorting. Hrs is recruiting ubiquitinated receptors to early endosomes to further facilitate degradation through the ESCRT complex. Upon receptor activation Hrs becomes phosphorylated and is relocated to the cytosol, important for receptor degradation. In this work we have studied the endosomal binding dynamics of Eps15 and Hrs upon EGF-R and PDGF-R stimulation. By analysing the fluorescence intensity on single endosomes after ligand stimulation we measured a time-specific decrease in the endosomal fluorescence level of Eps15-GFP and Hrs-YFP. Through FRAP experiments we could further register a specific change in the endosomal-membrane to cytosol binding properties of Eps15-GFP and Hrs-YFP. This specific change in membrane fractions proved to be a redistribution of the immobile fraction, which was not shown for the phosphorylation deficient mutants. We here describe a mechanism that can explain the previously observed relocation of Hrs from the endosomes to cytosol after EGF stimulation and show that Eps15 follows a similar mechanism. Moreover, this specific redistribution of the endosomal protein binding dynamics proved to be of major importance for receptor degradation.
... Hrs protein contains domains involved in sequence-directed recycling [27] and lysosomal-vacuolar sorting [28]. Outside of those domains, the protein comprises several potential phosphorylation sites on tyrosines (Y216, Y308, Y329 and Y334), where Y329 and Y334, specifically, have been recognized as being important for protein function [28,29,30]. ...
... Hrs protein contains domains involved in sequence-directed recycling [27] and lysosomal-vacuolar sorting [28]. Outside of those domains, the protein comprises several potential phosphorylation sites on tyrosines (Y216, Y308, Y329 and Y334), where Y329 and Y334, specifically, have been recognized as being important for protein function [28,29,30]. We transfected cells stably expressing TLR4-CFP/MD-2 with Hrs containing Y329F and Y334F (HrsY329/334F-EGFP) mutations. ...
... It is essential for sorting ubiquitinated proteins through the UIM (ubiquitin-interacting motif) domain into the lysosomal degradation pathway [20,39]. The UIM domain is necessary for the EGFstimulated (epidermal growth factor) tyrosine phosphorylation of Hrs [28]. Stern et al. proposed a mechanism where phosphorylation of Y329 and Y334 regulates Hrs degradation and showed that the level of Hrs phosphorylation and the speed of Hrs dephosphorylation correlated directly with EGFR degradation [29]. ...
There is a well-established association between exposure to air pollutants and pulmonary injuries. For example, metals found in ROFA (residual oil fly ash) increase susceptibility of mice as well as humans to microbial infections. In our research, we have found that vanadate substantially increased the response of several Toll-like receptors (TLRs) to stimulation with their ligands. Although vanadate caused generation of reactive oxygen species (ROS), the addition of ROS scavenger N-acetyl cysteine (NAC) had no effect on augmented lipopolysaccharide (LPS) stimulation. We further showed that vanadate inhibits endosome fusion. This effect was determined by measuring the size of endosomes, NF-κB activity and TLR4 degradation in Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) overexpressed cells. Moreover, we identified the role of Hrs phosphorylation in these processes. Based on our findings, we can conclude that vanadate potentiates TLR4 activity by increasing Hrs phosphorylation status, reducing the size of Hrs/TLR4-positive endosomes and impacting TLR4 degradation, thus contributing to the detrimental effects of air pollutants on human health.
... In humans the STAM family consists of STAM1, STAM2A and STAM2B-these three proteins share conserved domains important for their function and STAM2B is an inactive truncated form of STAM2A 22 . Hrs plays a central role in the regulation of receptor endocytosis by targeting ubiquitylated receptors to the degradative lysosomal pathway [23][24][25][26] . In this study we identify the interaction of STAM and Hrs with IFNAR as a key molecular switch that controls the spatiotemporal activation of JAK-STAT signalling between the plasma membrane and the early endosome. ...
... In contrast, we could not detect IFNAR1 in Hrs immunoprecipitates from cells stimulated by IFN-β (Fig. 1c). Hrs has been initially characterized as a tyrosine phosphorylated substrate of several growth factors, especially hepatocyte growth factor, EGF or PDGF [24][25][26]28,29 . We found that 10 min of stimulation by IFN-α notably increased Hrs tyrosine phosphorylation over basal levels. ...
Activation of the JAK–STAT pathway by type I interferons (IFNs) requires clathrin-dependent endocytosis of the IFN-α and -β receptor (IFNAR), indicating a role for endosomal sorting in this process. The molecular machinery that brings the selective activation of IFN-α/β-induced JAK–STAT signalling on endosomes remains unknown. Here we show that the constitutive association of STAM with IFNAR1 and TYK2 kinase at the plasma membrane prevents TYK2 activation by type I IFNs. IFN-α-stimulated IFNAR endocytosis delivers the STAM–IFNAR complex to early endosomes where it interacts with Hrs, thereby relieving TYK2 inhibition by STAM and triggering signalling of IFNAR at the endosome. In contrast, when stimulated by IFN-β, IFNAR signalling occurs independently of Hrs as IFNAR is sorted to a distinct endosomal subdomain. Our results identify the molecular machinery that controls the spatiotemporal activation of IFNAR by IFN-α and establish the central role of endosomal sorting in the differential regulation of JAK–STAT signalling by IFN-α and IFN-β.
... The downregulated phosphoproteins included SYK itself and, importantly, EGFR and ERBB2, providing evidence that the phosphorylation of these receptor tyrosine kinases was dependent on SYK activity. In addition, phosphorylation levels of several known EGFR downstream substrates decreased in tumors from animals treated with R406; these included the adapter molecule CRK [33], 1-phosphatidylinositol 4,5bisphosphate phosphodiesterase gamma-1 [34], hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) [35], GRB2 associated binding protein 1 (GAB1) [35], and signal transducing adapter molecule 2 (STAM2) [36] (Table S1). These findings provide compelling evidence that SYK inhibition affects EGFR phosphorylation signaling. ...
... The downregulated phosphoproteins included SYK itself and, importantly, EGFR and ERBB2, providing evidence that the phosphorylation of these receptor tyrosine kinases was dependent on SYK activity. In addition, phosphorylation levels of several known EGFR downstream substrates decreased in tumors from animals treated with R406; these included the adapter molecule CRK [33], 1-phosphatidylinositol 4,5bisphosphate phosphodiesterase gamma-1 [34], hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) [35], GRB2 associated binding protein 1 (GAB1) [35], and signal transducing adapter molecule 2 (STAM2) [36] (Table S1). These findings provide compelling evidence that SYK inhibition affects EGFR phosphorylation signaling. ...
Background:
Spleen tyrosine kinase (SYK) is frequently upregulated in recurrent ovarian carcinomas, for which effective therapy is urgently needed. SYK phosphorylates several substrates, but their translational implications remain unclear. Here, we show that SYK interacts with EGFR and ERBB2, and directly enhances their phosphorylation.
Methods:
We used immunohistochemistry and immunoblotting to assess SYK and EGFR phosphorylation in ovarian serous carcinomas. Association with survival was determined by Kaplan-Meier analysis and the log-rank test. To study its role in EGFR signaling, SYK activity was modulated using a small molecule inhibitor, a syngeneic knockout, and an active kinase inducible system. We applied RNA-seq and phosphoproteomic mass spectrometry to investigate the SYK-regulated EGF-induced transcriptome and downstream substrates.
Findings:
Induced expression of constitutively active SYK130E reduced cellular response to EGFR/ERBB2 inhibitor, lapatinib. Expression of EGFRWT, but not SYK non-phosphorylatable EGFR3F mutant, resulted in paclitaxel resistance, a phenotype characteristic to SYK active ovarian cancers. In tumor xenografts, SYK inhibitor reduces phosphorylation of EGFR substrates. Compared to SYKWT cells, SYKKO cells have an attenuated EGFR/ERBB2-transcriptional activity and responsiveness to EGF-induced transcription. In ovarian cancer tissues, pSYK (Y525/526) levels showed a positive correlation with pEGFR (Y1187). Intense immunoreactivity of pSYK (Y525/526) correlated with poor overall survival in ovarian cancer patients.
Interpretation:
These findings indicate that SYK activity positively modulates the EGFR pathway, providing a biological foundation for co-targeting SYK and EGFR. FUND: Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, NIH/NCI, Ovarian Cancer Research Foundation Alliance, HERA Women's Cancer Foundation and Roseman Foundation. Funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript and eventually in the decision to submit the manuscript.
... We found that the majority of the endocytosed IFNAR1 subunits were present in early endosomes, as shown by colocalization with the bona fide endosomal marker EEA1 (Fig. 1A). These endosomes were also positive for hepatocyte growth factorregulated tyrosine kinase substrate (Hrs), a member of the endosomal sorting complex required for transport (ESCRT)-0 that binds to ubiquitylated cargo proteins and sorts them to the degradative lysosomal pathway (8)(9)(10). Whereas IFNAR1 and Hrs immunostainings were perfectly superimposed upon cell stimulation with IFN-α, we found that for IFN-β, IFNAR1 staining was juxtaposed to Hrs, suggesting that IFNAR was localized in a different endosomal subdomain. ...
... Hrs was initially characterized as a tyrosine phosphorylated substrate of several growth factors, especially hepatocyte growth factor (8)(9)(10)(11)(12)(13). We found that a 10 min stimulation by IFN-α significantly increased Hrs tyrosine phosphorylation over basal levels found in unstimulated cells. ...
Activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway by type I interferons (IFN) requires clathrin-dependent endocytosis of the IFN-α/β receptor (IFNAR). The molecular machinery that brings about the selective activation of IFN-α/β-induced JAK/STAT signaling on endosomes remains unknown. Here we show that the constitutive association of STAM with IFNAR1 and the TYK2 Janus kinase at the plasma membrane prevents the activation of TYK2 by type I IFNs. IFN-α stimulated endocytosis leads to the interaction of IFNAR1 with Hrs on early endosomes, which then relieves TYK2 inhibition by STAM and thereby allows for TYK2 and IFNAR signaling. In contrast, IFN-β stimulation results in sorting of IFNAR to a distinct endosomal subdomain where the receptor is activated independently from Hrs. Our results identify the molecular machinery that controls the spatiotemporal activation of TYK2 and establish the central role of endosomal sorting in the differential regulation of JAK/STAT signaling by IFN-α and IFN-β.
Summary
The spatiotemporal activation of JAK/STAT signaling by IFN-α is controlled by STAM association with Hrs at the early endosome.
... ErbB3 was then found to co-precipitate along with Hrs both in the presence and absence of heregulin ( Supplementary Fig. 9). Moreover, it has been demonstrated that EGF induces tyrosine phosphorylation of Hrs [39], and that Hrs phosphorylation controls the EGFR fate [40,41]. To study if heregulin induces Hrs phosphorylation, Hrs was precipitated under denaturing conditions from MCF-7 cells incubated with or without heregulin or with EGF. ...
... Our current findings that overexpression of Hrs or DN Vps 4 (both of which impair EGFR trafficking and degradation [35][36][37]55,56]) causes endosomal accumulation of ErbB3, indicate a role for the ESCRT complexes in degradative sorting of ErbB3. EGFinduced phosphorylation of Hrs is reported to be important for degradative sorting of EGFR [40,41,57]. Our results showing that ErbB3 constitutively colocalizes with and interacts with Hrs, and that heregulin induces phosphorylation of Hrs, strongly support the idea that the ESCRT machinery regulates endosomal sorting of ErbB3. ...
... Another target of PTP1B upon EGF stimulation is the early endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) (39). TRPV1-mediated Ca 2+ influx and subsequent calpain activation should therefore also affect Hrs Y334 phosphorylation, the major phosphorylated tyrosine residue in response to EGF (40). Indeed, we found that overexpression of TRPV1 resulted in suppression of Hrs Y334 phosphorylation upon EGF stimulation (Supplemental Figure 6F). ...
... ACC-030). Anti-phospho-Hrs (Y334) antibody was provided by S. Urbé (University of Liverpool, Liverpool, United Kingdom; ref. 40). ...
The intestinal epithelium has a high rate of turnover, and dysregulation of pathways that regulate regeneration can lead to tumor development; however, the negative regulators of oncogenic events in the intestinal epithelium are not fully understood. Here we identified a feedback loop between the epidermal growth factor receptor (EGFR), a known mediator of proliferation, and the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), in intestinal epithelial cells (IECs). We found that TRPV1 was expressed by IECs and was intrinsically activated upon EGFR stimulation. Subsequently, TRPV1 activation inhibited EGFR-induced epithelial cell proliferation via activation of Ca2+/calpain and resulting activation of protein tyrosine phosphatase 1B (PTP1B). In a murine model of multiple intestinal neoplasia (ApcMin/+ mice), TRPV1 deficiency increased adenoma formation, and treatment of these animals with an EGFR kinase inhibitor reversed protumorigenic phenotypes, supporting a functional association between TRPV1 and EGFR signaling in IECs. Administration of a TRPV1 agonist suppressed intestinal tumorigenesis in ApcMin/+ mice, similar to - as well as in conjunction with - a cyclooxygenase-2 (COX-2) inhibitor, which suggests that targeting both TRPV1 and COX-2 has potential as a therapeutic approach for tumor prevention. Our findings implicate TRPV1 as a regulator of growth factor signaling in the intestinal epithelium through activation of PTP1B and subsequent suppression of intestinal tumorigenesis.
... Endocytosis is important for the function of many plasma membrane receptors 20 , and conjugation of ubiquitin to these membrane proteins is the major component of the regulatory mechanism for their internalization and lysosomal degradation [21][22][23][24] . Deubiquitination, the opposite process, is also critically involved in regulating the degradation of several RTKs by removing monoubiqutin and polyubiquitin chains from ubiquitin-conjugated proteins, resulting in inhibition of protein degradation [25][26][27] . ...
... Hepatocyte growth factor-regulated Tyr-kinase substrate (Hrs) functions by binding to ubiquitin via ubiquitin-interacting motif (UIM), which is essential for efficient sorting of ubiquitinated membrane proteins 22,23 . Thus, Hrs is proposed to be a sorting receptor that recognizes the ubiquitin moieties of membrane proteins and introduces them into multivesicular bodies in mammalian cells 24 . ...
The Met receptor tyrosine kinase is an attractive target for cancer therapy as it promotes invasive tumor growth. SAIT301 is a novel anti-Met antibody, which induces LRIG1-mediated Met degradation and inhibits tumor growth. However, detailed downstream mechanism by which LRIG1 mediates target protein down-regulation is unknown. In the present study, we discovered that SAIT301 induces ubiquitination of LRIG1, which in turn promotes recruitment of Met and LRIG1 complex to the lysosome through its interaction with Hrs, resulting in concomitant degradation of both LRIG1 and Met. We also identified USP8 as a LRIG1-specific deubiquitinating enzyme, reporting the interaction between USP8 and LRIG1 for the first time. SAIT301 triggers degradation of LRIG1 by inhibiting the interaction of LRIG1 and USP8, which regulates ubiquitin modification and stability of LRIG1. In summary, SAIT301 employs ubiquitination of LRIG1 for its highly effective Met degradation. This unique feature of SAIT301 enables it to function as a fully antagonistic antibody without Met activation. We found that USP8 is involved in deubiquitination of LRIG1, influencing the efficiency of Met degradation. The relation of Met, LRIG1 and USP8 strongly supports the potential clinical benefit of a combination treatment of a USP8 inhibitor and a Met inhibitor, such as SAIT301.
... Exosomes are generated by the process of cellular membrane internalization, resulting in the formation of endosomes. The invagination of the endosomal membrane leads to the formation of many intraluminal vesicles, which are commonly referred to as multivesicular bodies (Urbé et al. 2003). There is a growing body of evidence indicating that these vesicles can serve as intercellular transmitters, facilitating the transfer of their contents, specifically miRNA, across cells (Rechavi et al. 2009). ...
Pheochromocytoma (PCC) is a neuroendocrine tumor that produces and secretes catecholamine from either the adrenal medulla or extra-adrenal locations. MicroRNAs (miRNAs, miR) can be used as biomarkers to detect cancer or the return of a previously treated disease. Blood-borne miRNAs might be envisioned as noninvasive markers of malignancy or prognosis, and new studies demonstrate that microRNAs are released in body fluids as well as tissues. MiRNAs have the potential to be therapeutic targets, which would greatly increase the restricted therapy options for adrenal tumors. This article aims to consolidate and synthesize the most recent studies on miRNAs in PCC, discussing their potential clinical utility as diagnostic and prognostic biomarkers while also addressing their limitations.
... Some proteins are only found in exosomes produced by the ESCRT-dependent pathway while others require the ESCRT-independent route for their secretion via exosomes. For example loading of ILVs with the epidermal growth factor (EGFR) requires Hrs and STAM1 of ESCRT complexes (Bache et al., 2003;Raiborg et al., 2002;Urbe et al., 2003). This was further confirmed by Stuffers et al who showed that depletion of ESCRT components inhibit EGFR sorting into ILVs (Stuffers et al., 2009). ...
With a diverse set of neuronal and glial cell populations, Central Nervous System (CNS) has one of the most complex structures in the body. Intercellular communication is therefore highly important to coordinate cell-to-cell interactions. Besides electrical and chemical messengers, CNS cells also benefit from another communication route, what is known as extracellular vesicles, to harmonize their interactions. Extracellular Vesicles (EVs) and their subtype exosomes are membranous particles secreted by cells and contain information packaged in the form of biomolecules such as small fragments of DNA, lipids, miRNAs, mRNAs, and proteins. They are able to efficiently drive changes upon their arrival to recipient cells. EVs actively participate in all stages of CNS development by stimulating neural cell proliferation, differentiation, synaptic formation, and mediating reciprocal interactions between neurons and oligodendrocyte for myelination process. The aim of the present review is to enlighten the presence and contribution of EVs at each CNS developmental milestone.
... Receptor) et le GHR (pour Growth Hormone Receptor) sont enrichis dans ces structures, mais pas le récepteur à la transferrine (TfR) (Sachse, Urbé, et al., 2002). Pourtant, le résultat de la fusion du TfR et de l'ubiquitine localise dans ces sous-domaines de façon dépendante de Hrs (Raiborg et al., 2002;Urbe et al., 2003). Le manteau de clathrine étant cytosolique et la vésicule s'invaginant vers l'intérieur de l'endosome, la topologie est différente des autres cas de formation de vésicules médiées par la clathrine. ...
Plus d’une soixantaine de protéines différentes sont insérées dans la membrane du lysosome et participent aux différentes fonctions de cet organite. Mais les voies de transport intracellulaire qui mènent les protéines membranaires lysosomales (LMP) jusqu’à cet organite ne sont pas bien comprises. De façon intéressante, certaines LMP présentent des localisations intracellulaires anormales dans certains cancers. C’est notamment le cas de la glycoprotéine LAMP1 qui s’avère surexposée à la surface cellulaire dans plusieurs cancers, où elle y joue plusieurs rôles dans l’agressivité tumorale. Grâce au système RUSH (pour Retention Using Selective Hooks), permettant la synchronisation du transport le long de la voie de sécrétion, nous avons montré que LAMP1, après néosynthèse, passe par la membrane plasmique avant d’accéder aux endosomes. La comparaison des voies empruntées par différentes LMP a aussi permis de révéler que LAMP1 et LIMP2 sont triées au niveau de l’appareil de Golgi, LIMP2 étant concentrée dans des structures vésiculaires caractéristiques et dépourvues de clathrine. Nous avons aussi montré que, de façon surprenante, ce tri au niveau de l’appareil de Golgi est indépendant des signaux de recrutement d’adaptateurs à la clathrine portés dans les queues C-terminales de ces deux LMP. Afin d’étudier quels mécanismes pourraient être impliqués dans la surexposition de LAMP1 à la surface cellulaire de certains cancers, nous avons aussi réalisé un criblage d’inactivation de gènes basé sur la technologie CRISPR-Cas9. Nous avons sélectionné les gènes dont l’inactivation affectait les niveaux de LAMP1 en surface et nous avons obtenu de nombreux gènes candidats qui sont à l’étude.
... The ubiquitination and ubiquitin-like modifiers represent one of regulatory systems in protein location, stability and function [88]. Recent evidences point that ESCRT family involved in sorting ubiquitinated proteins into endosome compartments prior to exosome budding and scission (Fig. 2) [89]. ESCRT complex performs three functions: it recognizes ubiquitylated cargoes and prevents their degradation; then, it deforms the membrane and sorting cargo in or out; final, it forms ILVs which contain the sorted cargo [90]. ...
Cellular communication can be mediated by the exchange of biological information, mainly in the form of proteins and RNAs. This can occur when extracellular vesicles, such as exosomes, secreted by a donor cell are internalized by an acceptor cell. Exosomes bear specific repertoires of proteins and RNAs, indicating the existence of mechanisms that control the sorting of molecules into them. Knowledge about loadings and processes and mechanisms of cargo sorting of exosomes is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis. In this review, we will discuss the molecular mechanisms associated with exosome secretion and their specific cargo sorting, with special attention to the sorting of RNAs and proteins, and thus the outcome and the emerging therapeutic opportunities of the communication between the exosome-producer and recipient cells.
... The response of established endosomal recycling factors has not been investigated in great detail. HRS is an interesting candidate as it has been shown to be tyrosine phosphorylated downstream of RTKs, notably the MET receptor and EGFR [80][81][82]. Activation of either receptor interestingly creates different phosphorylation profiles of HRS [82]. The phosphorylation of HRS controls the cytosolic to endosomal ratio of HRS, with the phosphorylated protein being dissociated from the endosome. ...
Endocytosis is an essential process where proteins and lipids are internalised from the plasma membrane in membrane-bound carriers, such as clathrin-coated vesicles. Once internalised into the cell these vesicles fuse with the endocytic network where their contents are sorted towards degradation in the lysosome or recycling to their origin. Initially, it was thought that cargo recycling is a passive process, but in recent years the identification and characterisation of specialised recycling complexes has established a hitherto unthought-of level of complexity that actively opposes degradation. This review will summarise recent developments regarding the composition and regulation of the recycling machineries and their relationship with the degradative pathways of the endosome.
... Lippincott-Schwartz (Cole et al., 1996) (Thuenauer et al., 2014). GFP-Hrs was a gift from Dr. S. Urbé (Urbé et al., 2003). Plasmid coding superfolder GFP was a gift from Dr. E.L. Snapp (Aronson et al., 2011). ...
Peripherin 2 (PRPH2) is a tetraspanin protein concentrated in the light-sensing cilium (called the outer segment) of the vertebrate photoreceptor. The mechanism underlying the ciliary targeting of PRPH2 and the etiology of cone dystrophy caused by PRPH2 mutations remain elusive. Here we show that the late endosome (LE) is the main waystation that critically sorts newly synthesized PRPH2 to the cilium. PRPH2 is expressed in the luminal membrane of the LE. We delineate multiple C-terminal motifs of PRPH2 that distinctively regulate its LE and ciliary targeting through ubiquitination and binding to ESCRT (Endosomal Sorting Complexes Required for Transport) component Hrs. Using the newly developed TetOn-inducible system in transfected male and female mouse cones in vivo, we show that the entry of nascent PRPH2 into the cone outer segment can be blocked by either cone dystrophy-causing C-terminal mutations of PRPH2, or by short-term perturbation of the LE or recycling endosomal traffic. These findings open new avenues of research to explore the biological role of the LE in the biosynthetic pathway and the etiology of cone dystrophy caused by PRPH2 mutations and/or malfunctions of the LE.SIGNIFICANCE STATEMENT Peripherin 2 (PRPH2) is a tetraspanin protein abundantly expressed in the light-sensing cilium, the outer segment, of the vertebrate photoreceptor. The mechanism underlying the ciliary transport of PRPH2 is unclear. The present study reveals a novel ciliary targeting pathway, in which the newly synthesized PRPH2 is first targeted to the lumen of the late endosome (LE) en route to the cilia. We deciphered the protein motifs and the machinery that regulates the LE trafficking of PRPH2. Using a novel TetOn-inducible system in transfected mouse cones, we showed that the LE pathway of PRPH2 is critical for its outer segment expression. A cone dystrophy-causing mutation impairs the LE and ciliary targeting of PRPH2, implicating the relevance of LE to cone/macular degenerative diseases.
... To address this hypothesis experimentally, we co-transfected PLD3 together with GFP-tagged Hrs, an essential ubiquitin-binding subunit of the ESCRT-0 complex. Overexpression of Hrs traps ESCRT cargo within enlarged endosomes and prevents efficient formation of ILVs and cargo sorting into ILVs of MVBs by increased recruitment of clathrin to endosomes (Raiborg et al., 2001;Urbé et al., 2003). PLD3 extensively co-localized with GFP-tagged Hrs in the limiting membrane of enlarged endosomes ( Figure 5A). ...
Variants in the phospholipase D3 (PLD3) gene have genetically been linked to late-onset Alzheimer's disease. We present a detailed biochemical analysis of PLD3 and reveal its endogenous localization in endosomes and lysosomes. PLD3 reaches lysosomes as a type II transmembrane protein via a (for mammalian cells) uncommon intracellular biosynthetic route that depends on the ESCRT (endosomal sorting complex required for transport) machinery. PLD3 is sorted into intraluminal vesicles of multivesicular endosomes, and ESCRT-dependent sorting correlates with ubiquitination. In multivesicular endosomes, PLD3 is subjected to proteolytic cleavage, yielding a stable glycosylated luminal polypeptide and a rapidly degraded N-terminal membrane-bound fragment. This pathway closely resembles the delivery route of carboxypeptidase S to the yeast vacuole. Our experiments reveal a biosynthetic route of PLD3 involving proteolytic processing and ESCRT-dependent sorting for its delivery to lysosomes in mammalian cells.
... concentrates ubiquitinated receptors by direct interaction with its ubiquitin-interaction motif (UIM) 42 . Therefore we investigated if the UIM of HRS was essential for its regulation of CLR degradation. ...
Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 2 (RAMP2) comprise a receptor for adrenomedullin (AM). Although it is known that AM induces internalization of CLR•RAMP2, little is known about the molecular mechanisms that regulate the trafficking of CLR•RAMP2. Using HEK and HMEC-1 cells, we observed that AM-induced activation of CLR•RAMP2 promoted ubiquitination of CLR. A mutant (CLRΔ9KR), lacking all intracellular lysine residues was functional and trafficked similar to the wild-type receptor, but was not ubiquitinated. Degradation of CLR•RAMP2 and CLRΔ9KR•RAMP2 was not dependent on the duration of AM stimulation or ubiquitination and occurred via a mechanism that was partially prevented by peptidase inhibitors. Degradation of CLR•RAMP2 was sensitive to overexpression of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), but not to HRS knockdown, whereas CLRΔ9KR•RAMP2 degradation was unaffected. Overexpression, but not knockdown of HRS, promoted hyperubiquitination of CLR under basal conditions. Thus, we propose a role for ubiquitin and HRS in the regulation of AM-induced degradation of CLR•RAMP2.
... 7 It has been shown that overexpression of Hrs impairs endosomal sorting of ubiquitinated cargo by inhibition of intralumenal vesicle formation. 22,26 Thus, in Hrs-overexpressing cells, the transferrin receptor-ubiquitin fusion is bound by Hrs and accumulates in endosomes, which can be followed using fluorescently labelled transferrin. 7 Strong endosomal accumulation of transferrin was observed in control cells expressing WT Hrs (Figure 6a, expression of transfected proteins in Supplementary Figures S4a and b). ...
Ubiquitin-dependent sorting of membrane proteins in endosomes directs them to lysosomal degradation. In the case of receptors such as the epidermal growth factor receptor (EGFR), lysosomal degradation is important for the regulation of downstream signalling. Ubiquitinated proteins are recognised in endosomes by the endosomal sorting complexes required for transport (ESCRT) complexes, which sequentially interact with the ubiquitinated cargo. Although the role of each ESCRT complex in sorting is well established, it is not clear how the cargo is passed on from one ESCRT to the next. We here show that flotillin-1 is required for EGFR degradation, and that it interacts with the subunits of ESCRT-0 and -I complexes (hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and Tsg101). Flotillin-1 is required for cargo recognition and sorting by ESCRT-0/Hrs and for its interaction with Tsg101. In addition, flotillin-1 is also required for the sorting of human immunodeficiency virus 1 Gag polyprotein, which mimics ESCRT-0 complex during viral assembly. We propose that flotillin-1 functions in cargo transfer between ESCRT-0 and -I complexes.
... STAMBP is known to interact with ESCRT proteins to modulate the endolysosomal sorting and degradation of membrane receptors such as the epidermal growth factor receptor (EGFR) and the chemokine receptor CXCR4. Stimulation by EGF results in ubiquitination and trafficking of EGFR to the endosomal pathway through interactions with the ESCRT-0 complex 17,28,36,37 . Degradation of EGFR is enhanced by STAMBP knockdown following stimulation with EGF 19 . ...
Inflammasomes regulate innate immune responses by facilitating maturation of inflammatory cytokines, interleukin (IL)-1b and IL-18. NACHT, LRR and PYD domains-containing protein 7 (NALP7) is one inflammasome constituent, but little is known about its cellular handling. Here we show a mechanism for NALP7 protein stabilization and activation of the inflammasome by Toll-like receptor (TLR) agonism with bacterial lipopolysaccharide (LPS) and the synthetic acylated lipopeptide Pam3CSK4. NALP7 is constitutively ubiquitinated and recruited to the endolysosome for degradation. With TLR ligation, the deubiquitinase enzyme, STAM-binding protein (STAMBP) impedes NALP7 trafficking to lysosomes to increase NALP7 abundance. STAMBP deubiquitinates NALP7 and STAMBP knockdown abrogates LPS or Pam3CSK4-induced increases in NALP7 protein. A small-molecule inhibitor of STAMBP deubiquitinase activity, BC-1471, decreases NALP7 protein levels and suppresses IL-1b release after TLR agonism. These findings describe a unique pathway of inflammasome regulation with the identification of STAMBP as a potential therapeutic target to reduce pro-inflammatory stress.
... Exosomes are homologous small vesicles (50-90 nm) and have an endosomal origin (Camussi et al. 2010). Exosomes are formed by invagination of the membrane of endosomes to produce intraluminal vesicles, thus rendering these organelles multivesicular bodies (Urbe et al. 2003). In response to cell stimulation, budding of endosomes occurs, a process dependent on calcium influx, calpain and cytoskeleton reorganization (Johnstone 2006). ...
MicroRNAs are a class of endogenously initiated, small non coding RNAs typically regulates the expression of mRNAs in post transcriptional level either via translational repression or mRNA degradation. Recently, it has been revealed that miRNAs are not only present in cellular environment but also in extracellular milieu specially in different bio-fluids including blood plasma, follicular fluid and even in cell culture media. Such circulating miRNAs are remarkably stable in the extracellular harsh environment with the presence of high RNAse activity. Although the precise cellular release mechanisms of miRNAs remain largely unknown, the first studies revealed that these extracellular miRNAs may be delivered to recipient cells, where they can regulate translation of target genes. In this review, we will discuss the nature of the stability of extracellular miRNAs that present in the bloodstream and other bio fluids and their release mechanisms. Furthermore, based on available evidences we will shed lights on the possible role of these circulating miRNAs in distant cell-to-cell communication.
... Exosomes are formed by internalization of the cell membrane to produce endosomes. Invagination of the membrane of endosomes results several intraluminal vesicles, these organelles are known as multivesicular bodies (Urbé et al., 2003). Budding of endosomes occurs in response to different cellular stimulations. ...
MicroRNAs (miRNAs) are endogenously initiated, small non-coding RNAs and typically regulate the expression of mRNAs in post transcriptional level either via translational repression or mRNA degradation. Aberrant expression of miRNAs is observed in diverse disease and altered physiological states. Recently, it has been revealed that miRNAs are not only present in cells but also in extracellular milieu especially in different bio-fluids including blood plasma, follicular fluid and even in cell culture media. Such extracellular miRNAs (ECmiRNAs) are remarkably stable in the extracellular harsh environment with the presence of high RNAse activity. Although the precise mechanisms of release of cellular miRNAs to extracellular environment remain largely unknown, recent studies suggest that the expression of these ECmiRNAs can be associated with patho-physiological condition of an organism. Moreover, these ECmiRNAs may deliver to the recipient cells via certain pathways where they can regulate translational activity of target genes. This review will discuss the nature and stability of ECmiRNAs along with their release mechanisms. Furthermore, based on recent evidences, it also summarizes the possible function of these ECmiRNAs in distant cell-to-cell communication and the difficulties we may face during ECmiRNA research.
... The following antibodies were used: phospho-CREB (CST, 9198, 1:200), EEA1 (CST, 3288, 1:200), Flag (Sigma, M2, 1:500), PKAC (BD, 610981, 1:200), GFP (Abcam, ab13970, 1:1,000), ZO-1 (Santa Cruz, sc33725, 1:1,000) ARHGAP36 (Atlas, HPA002064, 1:100), RIIa (Santa Cruz, sc-909, 1:200) and RIIb (BD, 610626, 1;200). Rabbit polyclonal anti-HRS (1:500) and rabbit polyclonal anti-AQP2 (H27, 1:1,000) were custom made 61,63 . ...
Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease.
... Hrs modifications disrupt the interaction of its ubiquitin identification motif (UIM) domain with ubiquitinated (Ub-) cargoes and relocate Hrs to the cytosol to facilitate transfer of Ub-cargoes to downstream Ub-binding ESCRT proteins. Also, relocation of ubiquitinated Hrs from endosomal membranes permits replacement by nonubiquitinated Hrs to sustain endosomal sorting [200][201][202][203][204]. At the level of ESCRT-I, mahogunin-1 monoubiquitinates TSG101 to favour endolysosomal cargo degradation [205][206][207], while TAL (Tsg101 associated ligase) specifically polyubiquitinates and aims Tsg101 towards degradation, thus inhibiting endosomal trafficking [208,209]. ...
Originally discovered as regulators of cargo sorting during endosomal trafficking, ESCRT (endosomal sorting complexes required for transport) proteins are emerging as flexible machines that shape the behaviour of membranes throughout the cell. Deregulation of ESCRT activity is observed in neuro-degenerative diseases, virus infection and cancer. However, the mechanisms of pathogenesis involving ESCRTs have not yet fully come into focus. Here, we review the current knowledge of ESCRT function in health and disease and provide educated guesses for future research and focused therapeutic intervention.
... Both ESCRT-I and ESCRT-II are heterotetrameric protein complexes [86]. Subunits from ESCRT-0, -I, and -II all recognize ubiquitinated cargo [87][88][89] and function as adaptors to recruit ESCRT-III, which then facilitates vesicle formation via recruitment of curvature-bending proteins [77,79,90,91]. Mutations in several genes causative of HSPs have been linked to failure of the ESCRT machinery, revealing a role of altered endo/ lysosomal trafficking in these motor neuron pathologies. ...
Neurodegenerative diseases are amongst the most devastating of human disorders. New technologies have led to a rapid increase in the identification of disease-related genes with an enhanced appreciation of the key roles played by genetics in the etiology of these disorders. Importantly, pinpointing the normal function of disease gene proteins leads to new understanding of the cellular machineries and pathways that are altered in the disease process. One such emerging pathway is membrane trafficking in the endosomal system. This key cellular process controls the localization and levels of a myriad of proteins and is thus critical for normal cell function. In this review we will focus on three neurodegenerative diseases; Parkinson disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegias, for which a large number of newly discovered disease genes encode proteins that function in endosomal membrane trafficking. We will describe how alterations in these proteins affect endosomal function and speculate on the contributions of these disruptions to disease pathophysiology.
... While HGS is better known for its ability to target the ubiquitin-modified cargoes [67,68], it has also been shown to recognize a motif of clustering hydrophobic amino acids in cytokine receptors IL-2Rb and IL-4Ra, in an ubiquitin-independent manner [69,70]. Similarly, our result from the co-IP experiment suggested that the HGS protein could be associated with the assembly domain of HBc in a manner independent of the potential ubiquitination sites in both HGS (UIM domain) and HBc (K7, K96) (Fig 7). ...
The Endosomal Sorting Complex Required for Transport (ESCRT) is an important cellular machinery for the sorting and trafficking of ubiquitinated cargos. It is also known that ESCRT is required for the egress of a number of viruses. To investigate the relationship between ESCRT and hepatitis B virus (HBV), we conducted an siRNA screening of ESCRT components for their potential effect on HBV replication and virion release. We identified a number of ESCRT factors required for HBV replication, and focused our study here on HGS (HRS, hepatocyte growth factor-regulated tyrosine kinase substrate) in the ESCRT-0 complex. Aberrant levels of HGS suppressed HBV transcription, replication and virion secretion. Hydrodynamic delivery of HGS in a mouse model significantly suppressed viral replication in the liver and virion secretion in the serum. Surprisingly, overexpression of HGS stimulated the release of HBV naked capsids, irrespective of their viral RNA, DNA, or empty contents. Mutant core protein (HBc 1-147) containing no arginine-rich domain (ARD) failed to secrete empty virions with or without HGS. In contrast, empty naked capsids of HBc 1-147 could still be promoted for secretion by HGS. HGS exerted a strong positive effect on the secretion of naked capsids, at the expense of a reduced level of virions. The association between HGS and HBc appears to be ubiquitin-independent. Furthermore, HBc is preferentially co-localized with HGS near the cell periphery, instead of near the punctate endosomes in the cytoplasm. In summary, our work demonstrated the importance of an optimum level of HGS in HBV propagation. In addition to an effect on HBV transcription, HGS can diminish the pool size of intracellular nucleocapsids with ongoing genome maturation, probably in part by promoting the secretion of naked capsids. The secretion routes of HBV virions and naked capsids can be clearly distinguished based on the pleiotropic effect of HGS involved in the ESCRT-0 complex.
... Les protéines de surface destinées à la dégradation sont marquées par leur ubiquitinylation (Hicke, 2001). Excepté ESCRT-III, les complexes ESCRT possèdent tous des domaines de liaison à l'ubiquitine qui sont impliqués dans la reconnaissance de ce signal Bilodeau et al., 2002;Shih et al., 2002;Mizuno et al., 2003;Urbe et al., 2003;Alam et al., 2004). Les critères exacts que doit remplir un cargo pour être pris en charge par ESCRT sont encore inconnus. ...
This work focused on CHMP2B, an ESCRT-III complex subunit. This protein complex is involved in several membrane fission processes with an inversed topology (virus budding, multivesicular body genesis, cytokisesis, autophagy). Mutations in the gene coding for CHMP2B have been linked to neurodegenerative diseases such as fronto temporal demencia (FTD) and Spinal Lateral Amyotrophy (SLA). This work consisted in understanding molecular mechanisms of CHMP2B's action in order to better understand it' s links with these pathologies. The project was organized around three research axis: - The potential involvement of CHMP2B in the mitochondrial dynamics. - The role of CHMP2B and the impact of the FTD linked mutations on the morphology and the physiology of dendritic spines. - The study of helical CHMP2B containing complexes that deform the plasma membrane into tubular structures. Our experiments led us to the following observations: Extinction of CHMP2B inhibits mitochondrial fission by an unknown mechanism. The FTD linked CHMP2B mutants disrupt the normal pattern of spine development. This alteration could participate to the progressive appearance of FTD. CHMP2B is thought to be involved in the remodeling of cytoplasmic organelles such as late endosomes, autophagosomes, centrosome... Our work demonstrate that the protein is manly recruited at the plasma membrane where it assembles in rigid tubular structure that can deform the lipid bilayer.
... The association with endosomal membranes is mediated by the N-terminal FYVE domain of HRS, that is a double zinc-finger domain (Mao, Nickitenko et al. 2000) able to specifically recognize a highly abundant phosphoinositide in these membranes -phosphatidylinositol-3phosphate (PtdIns3P) (Raiborg, Bremnes et al. 2001). HRS also binds ubiquitin through its ubiquitin-interacting motif (UIM) and this is critical for initiating the sorting of ubiquitylated membrane proteins (Urbe, Sachse et al. 2003). Both HRS and STAM harbour VHS domains that are common N-terminal elements found in several proteins involved in intracellular trafficking and are thought to participate in cargo binding. ...
Alix is an adaptor protein involved in several cellular processes including apoptosis, endocytic membrane trafficking, budding of retroviruses (e.g. HIV-1, EIAV) and cytokinesis. Alix is organized in three major domains: an N-terminal BRO domain, a V-shaped domain in the middle (Alix-V) and a C-terminal proline rich domain (PRD). We have shown that a C-terminal truncated form lacking the PRD (Alix-∃PRD) forms monomers and dimers in solution and that the V-shaped domain is sufficient to mediate dimerization. Small angle X-ray scattering analyses revealed that Alix-∃PRD folds into an elongated curved structure that resembles membrane bending BAR domains. Although we determined that Alix interacts efficiently with membranes in vitro its potential deformation capability has yet to be confirmed. We further determined by isothermal titration calorimetry measurements that both monomeric and dimeric Alix-V interact with a peptide derived from EIAV Gag p9 with micromolar affinities. We obtained crystals of dimeric Alix-V which, however, diffracted X-rays no better than 10Å. We further crystallized a mutant of Alix-V (Mut1) that no longer dimerizes and folds into an open elongated monomeric structure as determined by small angle X-ray scattering. The crystals diffracted X-rays to 3Å resolution and structure determination is underway. Moreover, we showed that the deficient release of virus-like particles (VLP) upon overexpression of a human Alix-∃Bro form (residues 358-868), was rescued by generating a Mut1 version of this form, thus suggesting a role for dimerization in viral release. Dimeric Alix-V was also used to produce an Alix antiserum, which showed that endogenous Alix co-localizes with recycling endosomes. Finally, we showed that CHMP4B forms polymeric ring-like structures that are able to bind Alix. Together our data give insight into the conformational flexibility of Alix and its potential implications in concert with CHMP4 ring-like polymers in membrane budding processes. Our work also provides the framework for further functional analyses on the physiological relevance of dimeric Alix namely in HIV-1 infected cells.
... Among the different ESCRT complexes, ESCRT-0, and in particular Hrs, is considered as the best candidate for endosomal Ub-sorting receptor. Indeed, studies in mammalian cells demonstrate that loss of the Ub-binding domain of Hrs (a UIM variant that has two Ub-binding surfaces) disrupts Hrs ability to bind to ubiquitinated proteins and to retain Ub receptors on endosomes (55, 77, 78). Notably, we have observed that Hrs depletion sequesters ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation (70), demonstrating a key role for Hrs in regulating the fate of internalized FcεRI complexes. ...
Most immune cells, particularly phagocytes, express various receptors for the Fc portion of the different immunoglobulin isotypes (Fc receptors, FcRs). By binding to the antibody, they provide a link between the adaptive immune system and the powerful effector functions triggered by innate immune cells such as mast cells, neutrophils, macrophages, and NK cells. Upon ligation of the immune complexes, the downstream signaling pathways initiated by the different receptors are quite similar for different FcR classes leading to the secretion of preformed and de novo synthesized pro-inflammatory mediators. FcR engagement also promotes negative signals through the combined action of several molecules that limit the extent and duration of positive signaling. To this regard, ligand-induced ubiquitination of FcRs for IgE (FcεR) and IgG (FcγR) has become recognized as a key modification that generates signals for the internalization and/or delivery of engaged receptor complexes to lysosomes or cytoplasmic proteasomes for degradation, providing negative-feedback regulation of Fc receptor activity. In this review, we discuss recent advances in our understanding of the molecular mechanisms that ensure the clearance of engaged Fcε and Fcγ receptor complexes from the cell surface with an emphasis given to the cooperation between the ubiquitin pathway and endosomal adaptors including the endosomal sorting complex required for transport (ESCRT) in controlling receptor internalization and sorting along the endocytic compartments.
... Similarly, Cherry-GASP-1 was mainly distributed throughout the cytoplasm but also colocalized with Ga s -GFP and endogenous HRS on sorting endosomes. Interestingly, the overexpression of Ga s together with either dysbindin or GASP-1 altered the morphology of the early endosomes, as previously reported for overexpressed HRS [38][39][40] . Taken together, these data suggested that the components of this sorting complex localize together on early endosomes and could facilitate the sorting of GPCRs into the degradative pathway. ...
The role of Gαs in G protein-coupled receptor (GPCR) signalling at the cell surface is well established. Recent evidence has revealed the presence of Gαs on endosomes and its capacity to elicit GPCR-promoted signalling from this intracellular compartment. Here, we report an unconventional role for Gαs in the endocytic sorting of GPCRs to lysosomes. Cellular depletion of Gαs specifically delays the lysosomal degradation of GPCRs by disrupting the transfer of GPCRs into the intraluminal vesicles (ILVs) of multivesicular bodies. We show that Gαs interacts with GPCR-associated binding protein-1 (GASP1) and dysbindin, two key proteins that serve as linkers between GPCRs and the endosomal-sorting complex required for transport (ESCRT) machinery involved in receptor sorting into ILVs. Our findings reveal that Gαs plays a role in both GPCR signalling and trafficking pathways, providing another piece in the intertwining molecular network between these processes.
... However, other deubiquitinating enzymes such as AMSH and USP8 have previously been implicated in the regulation of EGFR endosomal trafficking after its internalisation [22]. In particular, EGFR has been shown to be ubiquitinated upon activation [23], prior to being internalised and trafficked to early endosomes from where it can either be recycled or sent to the lysosome for degradation [24]. AMSH can deubiquitinate EGFR and prompt recycling to the plasma membrane [25], whilst USP8 is required for the degradation of EGFR in the lysosome [26,27,28,29]. ...
Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of 'CaaX' motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis.
... By IEM it was shown that cargo molecules destined for lysosomal degradation, such as EGFR, growth hormone receptor, and the melanosomal proteins MART-1 and OA1, are concentrated in the coat, but recycling proteins like TfR are not (De Maziere et al. 2002;Sachse et al. 2002b;Giordano et al. 2011). Ubiquitination of TfR redirects this protein from the recycling to the degradative pathway and its localization from uncoated to coated areas of the SE, dependent on the Hrs ubiquitin interacting domain (Raiborg et al. 2002;Urbe et al. 2003). Thus, ubiquitinated cargo is concentrated in the SE clathrin coats before sorting into ILVs. ...
Live-cell imaging reveals the endolysosomal system as a complex and highly dynamic network of interacting compartments. Distinct types of endosomes are discerned by kinetic, molecular, and morphological criteria. Although none of these criteria, or combinations thereof, can capture the full complexity of the endolysosomal system, they are extremely useful for experimental purposes. Some membrane domain specializations and specific morphological characteristics can only be seen by ultrastructural analysis after preparation for electron microscopy (EM). Immuno-EM allows a further discrimination of seemingly identical compartments by their molecular makeup. In this review we provide an overview of the ultrastructural characteristics and membrane organization of endosomal compartments, along with their organizing machineries.
... Rather, the role of Grb2 in the recruitment of c-Cbl as an adaptor added an additional level of complexity to the mechanism of EGFR sorting under oxidative stress. Importantly, the degree of intricacy was further expanded by other studies that demonstrated that several proteins with ubiquitin interaction domains are in fact required for EGFR transfer from the early endosomes to other vesicular bodies for degradation (11,213). For example, the proteins Hrs and Tsg 101 were shown to be involved in a large sorting complex that is ''somehow'' responsible for coupling EGFR transfer between early and late endosomes. ...
Chronic obstructive pulmonary disease (COPD) and lung cancer are frequently caused by tobacco smoking. However, these diseases present opposite phenotypes involving redox signaling at the cellular level. While COPD is characterized by excessive airway epithelial cell death and lung injury, lung cancer is caused by uncontrolled epithelial cell proliferation. Notably, epidemiological studies have demonstrated that lung cancer incidence is significantly higher in patients who have pre-existing emphysema/ lung injury. However, the molecular link and common cell signaling events underlying lung injury diseases and lung cancer are poorly understood (3). This review focuses on studies of molecular mechanism(s) underlying smoking-related lung injury (COPD) and lung cancer. Specifically, the role of the ceramide generating machinery during cigarette smoke-induced oxidative stress leading to both apoptosis and proliferation of lung epithelial cells is emphasized. Over recent years, it has been established that ceramide is a sphingolipid playing a major role in lung epithelia structure/ function leading to lung injury in chronic pulmonary diseases. However, new and unexpected findings draw attention to its potential role in lung development, cell proliferation and tumorigenesis. To address this dichotomy in detail, evidence is presented regarding several protein targets, including Src, p38 mitogen activated protein kinase (MAPK) and neutral sphingomyelinase 2 (nSMase2), the major sphingomyelinase that controls ceramide generation during oxidative stress. Furthermore, their roles are presented not only in apoptosis and lung injury but also in enhancing cell proliferation, lung cancer development and resistance to EGF receptor (EGFR)-targeted therapy for treating lung cancer.
... Only the knockdown of ESCRT subunits 0 (Hrs) and I (Tsg101 and Vps37) caused continuous EGFR activation and MAPK signaling (Raiborg et al., 2008). Consistently, over-expression of mutant Hrs that can no longer bind to ubiquitinylated membrane proteins resulted in delay of EGFR degradation (Urbe et al., 2003). However, continued MAPK signaling was not seen for EAP30 (ESCRT-II) or Chmp3 (ESCRT-III) depletion. ...
Abstract The endosomal sorting complexes required for transport (ESCRT) are needed for three distinct cellular functions in higher eukaryotes: (i) Multivesicular body formation for the degradation of transmembrane proteins in lysosomes, (ii) midbody abscission during cytokinesis and (iii) retroviral budding. Not surprisingly, loss of ESCRT function has severe consequences, which include the failure to down-regulate growth factor receptors leading to deregulated mitogenic signaling. While it is clear that the function of the ESCRT machinery is important for embryonic development, its role in cancer is more controversial. Various experimental approaches in different model organisms arrive at partially divergent conclusions regarding the contribution of ESCRTs to tumorigenesis. Therefore the aim of this review is to provide an overview on different model systems used to study the role of the ESCRT machinery in cancer development, to highlight common grounds and present certain controversies in the field.
... Ubiquitination Assay-To assess the amount of ubiquitinated TAP in airway epithelial cells, a protocol was adapted from Urbe et al. (21) and recently published by our laboratory (18). Briefly, polarized airway epithelial cells were treated with P. aeruginosa OMVs as noted in figure legends in the presence of 37.5 M MG132 to prevent degradation of ubiquitinated proteins. ...
Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters
(ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to
determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1
and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen
clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human
airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased
polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation
into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation
at the plasma membrane of airway epithelial cells and recognition by CD8+ T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.
From the discovery of ubiquitin and its function as signal for proteasomal degradation over 20 years ago to this days, it became evident that ubiquitin is a universal signal in eukaryotic cells. Ubiquitin in its different forms is involved in many versatile cellular processes. Knowing that the ubiquitin signal is differently translated, depending on its occurrences as mono-ubiquitin or poly-ubiquitin, raises the question: how do cells distinguish between the different occurrences of ubiquitin and translate it into the proper response? Proteins interacting with ubiquitin contain so called ubiquitin binding domains (UBDs), whereas the affinities to ubiquitin vary from a few _M to mM. So far only three (K63, K48 and linear chains) out of the eight possible chain-linkages can be produced in sufficient amounts to characterize their interaction with UBDs. K48- and K63- linked ubiquitin chains regulate different cellular events and need to be recognized by different proteins. Thus, it is of prime importance to characterize the binding of different UBDs to these two kinds of ubiquitin chains, as it can give important clues related to the general mechanism of chain discrimination by ubiquitin adapter proteins. Some isolated UBDs exhibit a preference for one chain linkage type over the other, whereas others do not discriminate between mono-ubiquitin or K63- and K48-linked chains. Interestingly, many ubiquitin adapter proteins harbor more than one UBD. STAM2 is a ubiquitin adapter protein, that is involved in endosomal receptor sorting and supposed to preferentially bind mono-ubiquitin and K63- over K48-linked ubiquitin. STAM2 contains two UBDs (a VHS and UIM domain) that were shown to bind to ubiquitin . The current manuscript shows that STAM2’s SH3 domain binds ubiquitin as well. To understand the function of the sequential arrangement of three UBDs in one protein, first binding of the individual VHS and UIM domains to monoubiquitin as well as K48- and K63-linked di-ubiquitin was investigated. This work shows, that the VHS domain displays a different mode of binding for K63- and K48-linked diubiquitin. In spite of the fact, that the apparent Kd for both chains is the same, only one VHS domain can bind to K48-linked di-ubiquitin chains (with a preference for the distal domain), whereas K63-linked di-ubiquitin can accommodate two VHS domains at a time. Since no conclusion can be drawn with respect to the apparent Kds, the different binding modes might gain more impact in consideration of the ensemble of three UBDs. Results presented in this manuscript, based on a construct containing the VHS and UIM domain, show that binding to K63- but not K48-linked di-ubiquitin is cooperative
Calcitonin gene-related peptide (CGRP) is a promiscuous peptide, similar to many other members of the calcitonin family of peptides. The potential of CGRP to act on many different receptors with differing affinities and efficacies makes deciphering the signalling from the CGRP receptor a challenging task for researchers.Although it is not a typical G protein-coupled receptor (GPCR), in that it is composed not just of a GPCR, the CGRP receptor activates many of the same signalling pathways common for other GPCRs. This includes the family of G proteins and a variety of protein kinases and transcription factors. It is now also clear that in addition to the initiation of cell-surface signalling, GPCRs, including the CGRP receptor, also activate distinct signalling pathways as the receptor is trafficking along the endocytic conduit.Given CGRP's characteristic of activating multiple GPCRs, we will first consider the complex of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) as the CGRP receptor. We will discuss the discovery of the CGRP receptor components, the molecular mechanisms controlling its internalization and post-endocytic trafficking (recycling and degradation) and the diverse signalling cascades that are elicited by this receptor in model cell lines. We will then discuss CGRP-mediated signalling pathways in primary cells pertinent to migraine including neurons, glial cells and vascular smooth muscle cells.Investigation of all the CGRP- and CGRP receptor-mediated signalling cascades is vital if we are to fully understand CGRP's role in migraine and will no doubt unearth new targets for the treatment of migraine and other CGRP-driven diseases.
FcγRI cross-linking on monocytes may trigger clathrin-mediated endocytosis, likely through interaction of multiple intracellular molecules that are controlled by phosphorylation and dephosphorylation events. However, the identity of phospho-proteins and their regulation are unknown. We proposed the leukocyte immunoglobulin-like receptor B4 (LILRB4) that inhibits FcγRI-mediated cytokine production via Tyr dephosphorylation of multiple kinases, may also regulate endocytosis/phagocytosis through similar mechanisms. FcγRI and/or LILRB4 were antibody-ligated on THP-1 cells, lysates immunoprecipitated using anti-pTyr antibody and peptides sequenced by mass spectrometry. Mascot Search identified 25 Tyr phosphorylated peptides with high confidence. Ingenuity Pathway Analysis revealed that the most significantly affected pathways were clathrin-mediated endocytosis and Fc-receptor dependent phagocytosis. Tyr phosphorylation of key candidate proteins in these pathways included common γ-chain of the Fc receptors, Syk, clathrin, E3 ubiquitin protein ligase Cbl, hepatocyte growth factor-regulated tyrosine kinase substrate, tripartite motif-containing 21 and heat shock protein 70. Importantly, co-ligation of LILRB4 with FcγRI caused significant dephosphorylation of these proteins and was associated with suppression of Fc receptor-dependent uptake of antibody-opsonised bacterial particles, indicating that LILRB4. These results suggest that Tyr phosphorylation may be critical in FcγRI-dependent endocytosis/phagocytosis that may be regulated by LILRB4 by triggering dephosphorylation of key signalling proteins.
Secretory Carrier Membrane Proteins (SCAMPs) are transmembrane proteins that function in the plasma membrane, endosomes, and trans-Golgi network. Here we show that SCAMP 3 is a novel regulator of endosomal morphology and composition. Under certain nutrient-starved conditions, SCAMP 3 concentrates in enlarged early endosomes. The enlarged contain ubiquitylated and non-ubiquitylated SCAMP 3 as well as other SCAMPs, EEA1, and the ESCRT-0 protein Hrs. We demonstrate that SCAMP 3 is sufficient to recruit Hrs to the enlarged endosomes. Taken together, our results suggest a novel role for SCAMP 3 in modifying endosome structure through interactions that involve its ubiquitylation and ESCRT proteins.
Vascular endothelial growth factors (VEGFs) bind receptor tyrosine kinases (VEGFRs) to regulate vascular and lymphatic development and homeostasis. Such interactions are also implicated in pathological conditions ranging from cancer to heart disease. Increasingly, it is evident that ubiquitination plays a central role in regulating VEGFR function and the cellular response to VEGFs. E1, E2, and E3 ubiquitination enzymes deliver ubiquitin-specific modifications to protein substrates but there is much debate on the exact enzymes involved. The deubiquitinase (DUB) enzymes remove such modifications and are attracting increasing interest as potential therapeutic targets in a host of different disease states. Understanding how these enzyme families regulate VEGFR function in different cells and tissues is a major challenge. An understanding of the fundamental mechanisms underlying such biochemical regulation is needed for providing new therapeutics that target diseases such as cancer and heart disease.
IntroductionIdentification of Ubiquitin as an Endosomal Sorting SignalUbiquitin-mediated Sorting at the Endosome: The MVB Sorting Machinery Endosome-associated Ubiquitin Interacting Domains: Structure and FunctionThe Hrs – STAM Complex and the Endosomal Clathrin CoatGGA and Tom1: Alternative Sorting Adapters?The ESCRT MachineryVps4 – SKD1Ubiquitin Ligases and Endosomal Sorting Nedd4 Familyc-CblEndosomal DUBs Ubp1 and Ubp2Doa4UBPYAMSHPolyubiquitin Linkages and Endocytosis Proteasome Involvement in Endocytic SortingK63-linked UbiquitinFuture DirectionsAcknowledgementsReferences Endosome-associated Ubiquitin Interacting Domains: Structure and FunctionThe Hrs – STAM Complex and the Endosomal Clathrin CoatGGA and Tom1: Alternative Sorting Adapters?The ESCRT MachineryVps4 – SKD1 Nedd4 Familyc-Cbl Ubp1 and Ubp2Doa4UBPYAMSH Proteasome Involvement in Endocytic SortingK63-linked Ubiquitin
Following activation by its cognate ligand(s), the epidermal growth factor receptor (EGFR) is rapidly routed to the lysosome for degradation in a ubiquitination-dependent fashion. This pathway represents the major mechanism of long-term attenuation of EGFR signaling, and its deregulation is a significant feature in different types of cancers. Here we demonstrate, through a systematic RNAi-based approach, that several deubiquitinating (DUB) enzymes extend or decrease EGFR half-life upon EGF stimulation. We focus on USP9X, whose depletion severely affects EGFR turnover, interfering with its internalization and trafficking. We identify the endocytic protein Eps15 as one of the critical substrates of USP9X, and we map the Eps15 ubiquitination sites. We found that Eps15 monoubiquitination occurs already at minimal dose of EGF stimulation and is essential for EGFR internalization. Overall, our findings identify USP9X as a novel regulator of EGFR endocytosis and suggest a model whereby cycles of ubiquitination and deubiquitination events on endocytic accessory proteins may regulate the internalization and trafficking of the EGFR toward the lysosomes.
Epidermal growth factor receptor (EGFR) dysregulation drives several human pathological conditions, such as glioblastoma and non-small cell lung cancer, and as a result, EGFR activation, internalization, and downregulation are extensively studied for their contributions to tumorigenesis. However, while the mechanisms for ligand-dependent EGFR activation and internalization are quite well understood, current knowledge of ligand-independent mechanisms for EGFR activation and trafficking is obscured by somewhat conflicting data. Thus, unraveling the “unorthodox” machinery driving stress-induced EGFR activation and trafficking remains a very important task.
We present in this chapter the progression of the EGFR field, acknowledging mechanism(s) of stress-dependent activation, internalization, and trafficking of the EGFR. Emphasis is given within the context of cellular oxidative stress, which appears to be a common outcome of several types of stressors and pathological conditions, as observed in cancers and chemotherapy. In addition, c-Cbl, p38 MAPK, c-Src, and caveolin-1 are provided as examples of proteins contributing to the aberrant EGFR internalization and trafficking phenotype observed during cellular stress. Lastly, the role of the membrane itself in stress-induced EGFR internalization is discussed within the context of ceramide, a membrane sphingolipid generated during oxidative stress, and ceramide-enriched lipid rafts.
The MET gene encodes a tyrosine kinase receptor and its ligand is hepatocyte growth factor/scatter factor (HGF/SF). MET is expressed in many tissues, organs, and types of cells, but is generally expressed in cells of epithelial origin. HGF/SF, on the other hand, is produced in stromal cells of mesenchymal origin. The MET receptor is synthesized as a 185 kDa single-chain precursor, which is proteolytically cleaved to yield a highly glycosylated extracellular α-subunit and a transmembrane β-subunit. The two subunits are linked by disulfide bonds to form the mature receptor. Paracrine activation of the MET receptor by HGF/SF activates multiple signaling pathways, including RAS/MAPK, PI3K/AKT, SRC, and STAT3, leading to pleiotropic cellular responses such as mitogenesis, motogenesis, and morphogenesis. The HGF/SF–MET pathway is essential for embryonic development, organ regeneration, and wound healing during adulthood. Disruption of HGF/SF–MET signaling has been implicated in several human diseases including autism, diabetes, and most types of human cancer. In tumor cells, aberrant activation of MET signaling occurs through amplification, mutation, and/or overexpression. Ligand-dependent activation promotes tumor progression by facilitating cell proliferation, migration, invasion, survival, and angiogenesis. The prevalence of the HGF/SF–MET pathway in human cancers has led to great interest in the pathway as a cancer drug target. Small-molecule inhibitors or biologics such as monoclonal antibodies directed against MET or HGF/SF impede tumor growth in cells expressing activated MET. Promising results have been reported from clinical trials with compounds targeting HGF/SF–MET signaling. Treatment using HGF/SF–MET antagonists on select patient subpopulations that have aberrant MET activity may improve therapeutic efficacy.
The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing
adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal
accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study,
we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part
of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, thus suggesting that
exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes. A fluorescently labeled Hrs, introduced into the
cells by membrane permeabilization, exhibited endosomal localization in the absence of STAM1 and gradually dissociated from
the endosomes upon the sequential addition of recombinant STAM1. Furthermore, when microinjected into cells, the fluorescently
labeled Hrs also showed endosomal accumulation; however, ESCRT-0 complexes formed prior to the microinjection did not. Analysis
of the state of the complex in HeLa cells using blue-native PAGE revealed that the membrane-associated Hrs exists partly as
a monomer and not only in the STAM1-bound form. Thus, our data suggest that the membrane binding and dissociation cycle of
the ESCRT-0 proteins on the endosomal membrane is a critical step during the cargo sorting process.
The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study, we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes, and a recovery of the impaired cargo protein degradation process, thus suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes. A fluorescently labeled Hrs, introduced into the cells by membrane-permeabilization, exhibited endosomal localization in the absence of STAM1 and gradually dissociated from the endosomes upon the sequential addition of recombinant STAM1. Furthermore, when microinjected into cells, the fluorescently labeled Hrs also showed endosomal accumulation; however, ESCRT-0 complexes formed prior to the microinjection did not. Analysis of the state of the complex in HeLa cells using blue-native PAGE revealed that the membrane-associated Hrs exists partly as a monomer and not only in the STAM1-bound form. Thus, our data suggest that the membrane binding and dissociation cycle of the ESCRT-0 proteins on the endosomal membrane is a critical step during the cargo sorting process.
Unlabelled:
Coxsackievirus A9 (CVA9) is a member of the human enterovirus B species in the Enterovirus genus of the family Picornaviridae. According to earlier studies, CVA9 binds to αVβ3 and αVβ6 integrins on the cell surface and utilizes β2-microglobulin, dynamin, and Arf6 for internalization. However, the structures utilized by the virus for internalization and uncoating are less well understood. We show here, based on electron microscopy, that CVA9 is found in multivesicular structures 2 h postinfection (p.i.). A neutral red labeling assay revealed that uncoating occurs mainly around 2 h p.i., while double-stranded RNA is found in the cytoplasm after 3 h p.i. The biogenesis of multivesicular bodies (MVBs) is crucial for promoting infection, as judged by the strong inhibitory effect of the wild-type form of Hrs and dominant negative form of VPS4 in CVA9 infection. CVA9 infection is dependent on phospholipase C at the start of infection, whereas Rac1 is especially important between 1 and 3 h p.i., when the virus is in endosomes. Several lines of evidence implicate that low pH does not play a role in CVA9 infection. The infection is not affected by Bafilomycin A1. In addition, CVA9 is not targeted to acidic late endosomes or lysosomes, and the MVBs accumulating CVA9 have a neutral pH. Thus, CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger neutral MVBs, which are important for virus infection.
Importance:
We demonstrate here that the enterovirus coxsackievirus A9 (CVA9) uses a nonclathrin and nonacidic pathway to infect cells. CVA9 does not accumulate in conventional late endosomes or lysosomes. We found that inhibitors of phospholipase C (PLC), Rac1, and the Na(+)/H(+) exchanger decreased CVA9 infection. The PLC inhibitor acts on early entry, the Rac1 inhibitor acts between 1 and 3 h, when the virus is in endosomes, and the Na(+)/H(+) exchange inhibitor acts during various steps during the virus life cycle. The infection depends on the formation of novel neutral multivesicular bodies (MVBs), which accumulate CVA9 during the first hours of entry. Thus, CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger formation of neutral MVBs. The data show that these enteroviruses favor nonacidic conditions and complex MVBs to promote virus infection.
We previously reported a new type of signal-transducing adaptor molecule, STAM, which was shown to be involved in cytokine-mediated
intracellular signal transduction. In this study, we molecularly cloned a 110-kDa phosphotyrosine protein inducible by stimulation
with interleukin 2 (IL-2). The 110-kDa molecule was found to be a human counterpart of mouse Hrs (hepatocyte growth factor-regulated
tyrosine kinase substrate) and to be associated with STAM. Tyrosine phosphorylation of Hrs is induced rapidly after stimulation
with IL-2 and granulocyte-macrophage colony-stimulating factor as well as hepatocyte growth factor. The mutual association
sites of Hrs and STAM include highly conserved coiled-coil sequences, suggesting that their association is mediated by the
coiled-coil structures. Exogenous introduction of the wild-type Hrs significantly suppressed DNA synthesis upon stimulation
with IL-2 and granulocyte-macrophage colony-stimulating factor, while the Hrs mutant deleted of the STAM-binding site lost
such suppressive ability. These results suggest that Hrs counteracts the STAM function which is critical for cell growth signaling
mediated by the cytokines.
We have constructed a PRC1-SUC2 gene fusion that directs the synthesis in Saccharomyces cerevisiae of a hybrid polypeptide consisting of a 433-residue amino-terminal domain derived from the yeast vacuolar protease carboxypeptidase Y (CPY; EC 3.4.16.1) and a 511-residue carboxyl-terminal domain derived from the secreted yeast enzyme invertase (EC 3.2.1.26). Fractionation data indicated that this amount of CPY primary sequence is sufficient to quantitatively divert invertase to the yeast vacuole. The phenotypic consequence of localizing active invertase to the vacuole has enabled us to select for mutants that "mislocalize" the hybrid protein to the cell surface. The corresponding mutations that lead to this effect are all trans-acting and recessive, and they define at least eight complementation groups. These vacuolar protein targeting (vpt) mutants also exhibit hybrid protein independent defects in wild-type CPY delivery to the yeast vacuole. Precursor forms of CPY accumulate in the mutants and are secreted into the yeast periplasm and extracellular medium. The vpt mutants should provide useful information pertaining to the mechanisms by which yeast cells regulate vacuolar protein traffic.
The amiloride-sensitive epithelial sodium channel (ENaC) plays a major role in sodium transport in kidney and other epithelia, and in regulating blood pressure. The channel is composed of three subunits (alphabetagamma) each containing two proline-rich sequences (P1 and P2) at its C-terminus. The P2 regions in human beta and gammaENaC, identical to the rat betagammarENaC, were recently shown to be deleted in patients with Liddle's syndrome (a hereditary form of hypertension), leading to hyperactivation of the channel. Using a yeast two-hybrid screen, we have now identified the rat homologue of Nedd4 (rNedd4) as the binding partner for the P2 regions of beta and gammarENaC. rNedd4 contains a Ca2+ lipid binding (CaLB or C2) domain, three WW domains and a ubiquitin ligase (Hect) domain. Our yeast two-hybrid and in vitro binding studies revealed that the rNedd4-WW domains mediate this association by binding to the P2 regions, which include the PY motifs (XPPXY) of either betarENaC (PPPNY) or gammarENaC (PPPRY). SH3 domains were unable to bind these sequences. Moreover, mutations to Ala of Pro616 or Tyr618 within the betarENaC P2 sequence (to PPANY or PPPNA, respectively), recently described in Liddle's patients, led to abrogation of rNedd4-WW binding. Nedd4-WW domains also bound to the proline-rich C-terminus (containing the sequence PPPAY) of alpharENaC, and endogenous Nedd4 co-immunoprecipitated with alpharENaC expressed in MDCK cells. These results demonstrate that the WW domains of rNedd4 bind to the PY motifs deleted from beta or gammaENaC in Liddle's syndrome patients, and suggest that Nedd4 may be a regulator (suppressor) of the epithelial Na+ channel.
Epidermal growth factor (EGF) receptor pathway substrate clone 15 (Eps15) has been described as a 142-kDa EGF receptor substrate.
It has been shown to bind to the EGF receptor, adaptor protein-2, and clathrin and is present at clathrin-coated pits and
vesicles. Upon stimulation of cells with EGF or transforming growth factor α, Eps15 becomes rapidly and transiently phosphorylated
on tyrosine residues. This phosphorylation coincides with an increase of 8 kDa in molecular mass. Here we show that this increase
in molecular mass is not due to tyrosine phosphorylation. Instead, we found both by Western blotting and protein sequencing
that this EGF-induced increase in molecular mass is the result of monoubiquitination. Eps15 ubiquitination but not tyrosine
phosphorylation was inhibited under conditions that blocked EGF-induced internalization of the EGF receptor. Our results establish
ubiquitination as a second form of EGF-stimulated covalent modification of Eps15.
Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase
receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which
specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required
both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1
and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with
wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P
required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A)
to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide
a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor
(or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.
The hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, has been implicated in intracellular trafficking and signal transduction. Hrs contains a phosphatidylinositol 3-phosphate-binding FYVE domain that contributes to its endosomal targeting. Here we show that Hrs and EEA1, a FYVE domain protein involved in endocytic membrane fusion, are localized to different regions of early endosomes. We demonstrate that Hrs co-localizes with clathrin, and that the C-terminus of Hrs contains a functional clathrin box motif that interacts directly with the terminal beta-propeller domain of clathrin heavy chain. A massive recruitment of clathrin to early endosomes was observed in cells transfected with Hrs, but not with Hrs lacking the C-terminus. Furthermore, the phosphatidylinositol 3-kinase inhibitor wortmannin caused the dissociation of both Hrs and clathrin from endosomes. While overexpression of Hrs did not affect endocytosis and recycling of transferrin, endocytosed epidermal growth factor and dextran were retained in early endosomes. These results provide a molecular mechanism for the recruitment of clathrin onto early endosomes and suggest a function for Hrs in trafficking from early to late endosomes.
Yeast endosomes, like those in animal cells, invaginate their membranes to form internal vesicles. The resulting multivesicular bodies fuse with the vacuole, the lysosome equivalent, delivering the internal vesicles for degradation. We have partially purified internal vesicles and analysed their content. Besides the known component carboxypeptidase S (Cps1p), we identified a polyphosphatase (Phm5p), a presumptive haem oxygenase (Ylr205p/Hmx1p) and a protein of unknown function (Yjl151p/Sna3p). All are membrane proteins, and appear to be cargo molecules rather than part of the vesicle-forming machinery. We show that both Phm5p and Cps1p are ubiquitylated, and that in a doa4 mutant, which has reduced levels of free ubiquitin, Cps1p, Phm5p and Hmx1p are mis-sorted to the vacuolar membrane. Mutation of Lys 6 in the cytoplasmic tail of Phm5p disrupts its sorting, but sorting is restored, even in doa4 cells, by the biosynthetic addition of a single ubiquitin chain. In contrast, Sna3p enters internal vesicles in a ubiquitin-independent manner. Thus, ubiquitin acts as a signal for the partitioning of some, but not all, membrane proteins into invaginating endosomal vesicles.
Phosphorylation is one of the most common forms of protein modification. The most frequent targets for protein phosphorylation in eukaryotes are serine and threonine residues, although tyrosine residues also undergo phosphorylation. Many of the currently applied methods for the detection and localization of protein phosphorylation sites are mass spectrometry-based and are biased against the analysis of tyrosine-phosphorylated residues because of the stability and low reactivity of phosphotyrosines. To overcome this lack of sensitive methods for the detection of phosphotyrosine-containing peptides, we have recently developed a method that is not affected by the more predominant threonine or serine phosphorylation within cells. It is based on the specific detection of immonium ion of phosphotyrosine at 216.043 Da and does not require prior knowledge of the protein sequence. In this report, we describe the first application of this new method in a proteomic strategy. Using anti-phosphotyrosine antibodies for immunoprecipitation and one-dimensional gel electrophoresis, we have identified 10 proteins in the epidermal growth factor receptor signaling pathway, of which 8 have been shown previously to be involved in epidermal growth factor signaling. Most importantly, in addition to several known tyrosine phosphorylation sites, we have identified five novel sites on SHIP-2, Hrs, Cbl, STAM, and STAM2, most of which were not predicted to be phosphorylated. Because of its sensitivity and selectivity, this approach will be useful in proteomic approaches to study tyrosine phosphorylation in a number of signal transduction pathways.
The Golgi apparatus is a highly complex organelle comprised of a stack of cisternal membranes on the secretory pathway from the ER to the cell surface. This structure is maintained by an exoskeleton or Golgi matrix constructed from a family of coiled-coil proteins, the golgins, and other peripheral membrane components such as GRASP55 and GRASP65. Here we find that TMP21, p24a, and gp25L, members of the p24 cargo receptor family, are present in complexes with GRASP55 and GRASP65 in vivo. GRASPs interact directly with the cytoplasmic domains of specific p24 cargo receptors depending on their oligomeric state, and mutation of the GRASP binding site in the cytoplasmic tail of one of these, p24a, results in it being transported to the cell surface. These results suggest that one function of the Golgi matrix is to aid efficient retention or sequestration of p24 cargo receptors and other membrane proteins in the Golgi apparatus.
There is increasing evidence that ubiquitination of receptors provides an important endosomal sorting signal. Here we report that mammalian class E vacuolar protein-sorting (vps) proteins recognize ubiquitin. Both tumor susceptibility gene 101 (TSG101)/human VPS (hVPS)28 and hepatocyte growth factor receptor substrate (Hrs) cytosolic complexes bind ubiquitin-agarose. TSG101 and hVPS28 are localized to endosomes that contain internalized EGF receptor and label strongly for ubiquitinated proteins. Microinjection of anti-hVPS28 specifically retards EGF degradation and leads to endosomal accumulation of ubiquitin-protein conjugates. Likewise, depletion of TSG101 impairs EGF trafficking and causes dramatic relocalization of ubiquitin to endocytic compartments. Similar defects are found in cells overexpressing Hrs, further emphasizing the links between class E protein function, receptor trafficking, and endosomal ubiquitination.
Ubiquitination is a post-translation modification in which ubiquitin chains or single ubiquitin molecules are appended to target proteins, giving rise to poly- or monoubiquitination, respectively. Polyubiquitination targets proteins for destruction by the proteasome. The role of monoubiquitination is less understood, although a function in membrane trafficking is emerging, at least in yeast. Here we report that a short amino-acid stretch at the carboxy-termini of the monoubiquitinated endocytic proteins Eps15 and eps15R is indispensable for their monoubiquitination. A similar sequence, also required for this modification, is found in other cytosolic endocytic proteins, such as epsins and Hrs. These sequences comprise a protein motif, UIM (ref. 6), which has been proposed to bind to ubiquitin. We confirm this for the UIMs of eps15, eps15R, epsins and Hrs. Thus, the same motif in several endocytic proteins is responsible for ubiquitin recognition and monoubiquitination. Our results predict the existence of a UIM:ubiquitin-based intracellular network. Eps15/eps15R, epsins and Hrs may function as adaptors between ubiquitinated membrane cargo and either the clathrin coat or other endocytic scaffolds. In addition, through their own ubiquitination, they may further contribute to the amplification of this network in the endocytic pathway.
In many cells endosomal vacuoles show clathrin coats of which the function is unknown. Herein, we show that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. By immunoelectron microscopy we show that the coat contains clathrin heavy as well as light chain, but lacks the adaptor complexes AP1, AP2, and AP3, by which it differs from clathrin coats on endocytic vesicles and recycling endosomes. The coat is insensitive to short incubations with brefeldin A, but disappears in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin. No association of endosomal coated areas with tracks of tubulin or actin was found. By quantitative immunoelectron microscopy, we found that the lysosomal-targeted receptors for growth hormone (GHR) and epidermal growth factor are concentrated in the coated membrane areas, whereas the recycling transferrin receptor is not. In addition, we found that the proteasomal inhibitor MG 132 induces a redistribution of a truncated GHR (GHR-369) toward recycling vesicles, which coincided with a redistribution of endosomal vacuole-associated GHR-369 to the noncoated areas of the limiting membrane. Together, these data suggest a role for the bilayered clathrin coat on vacuolar endosomes in targeting of proteins to lysosomes.
After endocytosis, some membrane proteins recycle from early endosomes to the plasma membrane whereas others are transported to late endosomes and lysosomes for degradation. Conjugation with the small polypeptide ubiquitin is a signal for lysosomal sorting. Here we show that the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, is involved in the endosomal sorting of ubiquitinated membrane proteins. Hrs contains a clathrin-binding domain, and by electron microscopy we show that Hrs localizes to flat clathrin lattices on early endosomes. We demonstrate that Hrs binds directly to ubiquitin by way of a ubiquitin-interacting motif (UIM), and that ubiquitinated proteins localize specifically to Hrs- and clathrin-containing microdomains. Whereas endocytosed transferrin receptors fail to colocalize with Hrs and rapidly recycle to the cell surface, transferrin receptors that are fused to ubiquitin interact with Hrs, localize to Hrs- and clathrin-containing microdomains and are sorted to the degradative pathway. Overexpression of Hrs strongly and specifically inhibits recycling of ubiquitinated transferrin receptors by a mechanism that requires a functional UIM. We conclude that Hrs sorts ubiquitinated membrane proteins into clathrin-coated microdomains of early endosomes, thereby preventing their recycling to the cell surface.
An important negative control mechanism in the signaling of epidermal growth factor (EGF) is the endocytosis and subsequent degradation of activated EGF receptors. Eps15 and its related partner Eps15R play a key role in clathrin-mediated endocytosis of transmembrane receptors. Upon EGF stimulation of the cell, Eps15 becomes both phosphorylated on tyrosine residues and monoubiquitinated. Although tyrosine phosphorylation of Eps15 has been implicated in EGF receptor internalization, the function of Eps15 ubiquitination is not known. Using a mutational approach, we have found that the second ubiquitin-interacting motif (UIM) of Eps15 and Eps15R is essential for their ubiquitination. This UIM partially overlaps with the recently characterized nuclear export signal in Eps15. We show that these two overlapping motifs have different structural requirements with respect to nuclear export signal versus ubiquitination signal activity. Our data demonstrate that the UIM does not contain the ubiquitin acceptor site but functions as a recruitment site for the ubiquitination machinery leading to the monoubiquitination of both Eps15 and Eps15R.
Ligand-dependent endocytosis of the epidermal growth factor receptor (EGFR) involves recruitment of a ubiquitin ligase, and sorting of ubiquitylated receptors to lysosomal degradation. By studying Hgs, a mammalian homolog of a yeast vacuolar-sorting adaptor, we provide information on the less understood, ligand-independent pathway of receptor endocytosis and degradation. Constitutive endocytosis involves receptor ubiquitylation and translocation to Hgs-containing endosomes. Whereas the lipid-binding motif of Hgs is necessary for receptor endocytosis, the ubiquitin-interacting motif negatively regulates receptor degradation. We demonstrate that the ubiquitin-interacting motif is endowed with two functions: it binds ubiquitylated proteins and it targets self-ubiquitylation by recruiting Nedd4, an ubiquitin ligase previously implicated in endocytosis. Based upon the dual function of the ubiquitin-interacting motif and its wide occurrence in endocytic adaptors, we propose a ubiquitin-interacting motif network that relays ubiquitylated membrane receptors to lysosomal degradation through successive budding events.
Ubiquitination of key cellular proteins involved in signal transduction, gene transcription and cell-cycle regulation usually condemns those proteins to proteasomal or lysosomal degradation. Additionally, cycles of reversible ubiquitination regulate the function of certain proteins in a manner analogous to phosphorylation. In this short review we describe the current methodology for measuring ubiquitin and ubiquitination, provide examples which illustrate how various techniques have been used to study protein ubiquination, alert the readers of pitfalls to avoid, and offer guidelines to investigators newly interested in this novel post-translational protein modification.
The mammalian tumor susceptibility gene tsg101 encodes the homologue of Vps23p, a class E Vps protein essential for normal membrane trafficking in the late endosome/multivesicular body of yeast. Both proteins assemble into large (∼350 kDa) cytosolic protein complexes and we show that the yeast complex contains another class E Vps protein, Vps28p. tsg101 mutant cells exhibit defects in sorting and proteolytic maturation of the lysosomal hydrolase cathepsin D, as well as in the steady-state distribution of the mannose-6-phosphate receptor. Additionally, endocytosed EGF receptors that are normally sorted to the lysosome are instead rapidly recycled back to the cell surface in tsg101 mutant cells. We propose that tsg101 mutant cells are defective in the delivery of cargo proteins to late endosomal compartments. One consequence of this endosomal trafficking defect is the delayed down-regulation/degradation of activated cell surface receptors, resulting in prolonged signaling. This may contribute to the tumorigenic phenotype exhibited by the tsg101 mutant fibroblasts.
Several algorithms have been described in the literature for protein identification by searching a sequence database using mass spectrometry data. In some approaches, the experimental data are peptide molecular weights from the digestion of a protein by an enzyme. Other approaches use tandem mass spectrometry (MS/MS) data from one or more peptides. Still others combine mass data with amino acid sequence data. We present results from a new computer program, Mascot, which integrates all three types of search. The scoring algorithm is probability based, which has a number of advantages: (i) A simple rule can be used to judge whether a result is significant or not. This is particularly useful in guarding against false positives. (ii) Scores can be compared with those from other types of search, such as sequence homology. (iii) Search parameters can be readily optimised by iteration. The strengths and limitations of probability-based scoring are discussed, particularly in the context of high throughput, fully automated protein identification.
The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.
The activation of growth factor receptor tyrosine kinases leads to tyrosine phosphorylation of many intracellular proteins which are thought to play crucial roles in growth factor signaling pathways. We previously showed that tyrosine phosphorylation of a 115-kDa protein is rapidly induced in cells treated with hepatocyte growth factor. To clarify the structure and possible function of the 115-kDa protein (designated Hrs for hepatocyte growth factor-regulated tyrosine kinase substrate), we purified this protein from B16-F1 mouse melanoma cells by anti-phosphotyrosine immunoaffinity chromatography and determined its partial amino acid sequences. On the basis of the amino acid sequences, we molecularly cloned the cDNA for mouse Hrs. The nucleotide sequence of the cDNA revealed that Hrs is a novel 775-amino-acid protein with a putative zinc finger domain that is structurally conserved in several other proteins. This protein also contained a proline-rich region and a proline- and glutamine-rich region. The expression of Hrs mRNA was detected in all adult mouse tissues tested and also in embryos. To analyze the Hrs cDNA product, we prepared a polyclonal antibody against bacterially expressed Hrs. Using this antibody, we showed by subcellular fractionation that Hrs is localized to the cytoplasm; we also showed that that tyrosine phosphorylation of Hrs is induced in cells treated with epidermal growth factor or platelet-derived growth factor. These results suggest that Hrs plays a unique and important role in the signaling pathway of growth factors.
Addition of wortmannin to normal rat kidney cells caused a redistribution of the lysosomal type I integral membrane proteins Igp110 and Igp120 to a swollen vacuolar compartment. This compartment did not contain the cation independent mannose 6-phosphate receptor and was depleted in acid hydrolases. It was distinct from another swollen vacuolar compartment containing the cation independent mannose 6-phosphate receptor. The swollen Igp110-positive compartment was accessible to a monoclonal antibody against Igp120 added extracellularly, showing that it had the characteristics of an endosomal compartment. Wortmannin had no gross morphological effect on the trans-Golgi network or lysosomes nor any effect on the delivery to the trans-Golgi network of endocytosed antibodies against the type I membrane protein TGN38. We propose that the observed effects of wortmannin were due to inhibition of membrane traffic between cation independent mannose 6-phosphate receptor-positive late endosomes and the trans-Golgi network and to inhibition of membrane traffic between a novel Igp120-positive, cation independent mannose 6-phosphate receptor-negative late endosomal compartment and lysosomes. The effects of wortmannin suggest a function for a phosphatidylinositol 3-kinase(s) in regulating membrane traffic in the late endocytic pathway.
Proteins from silver-stained gels can be digested enzymatically and the resulting peptide analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nano-electrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
Ubiquitination of key cellular proteins involved in signal transduction, gene transcription and cell-cycle regulation usually condemns those proteins to proteasomal or lysosomal degradation. Additionally, cycles of reversible ubiquitination regulate the function of certain proteins in a manner analogous to phosphorylation. In this short review we describe the current methodology for measuring ubiquitin and ubiquitination, provide examples which illustrate how various techniques have been used to study protein ubiquination, alert the readers of pitfalls to avoid, and offer guidelines to investigators newly interested in this novel post-translational protein modification.
G-protein-coupled receptors and transporters in Saccharomyces cerevisiae are modified with ubiquitin in response to ligand biding. In most cases, the proteasome does not recognize these ubiquitinated proteins. Instead, ubiquitination serves to trigger internalization and degradation of plasma membrane proteins in the lysosome-like vacuole. A number of mammalian receptors and at least one ion channel undergo ubiquitination at the plasma membrane, and this modification is required for their downregulation. Some of these cell-surface proteins appear to be degraded by both the proteasome and lysosomal proteases. Recent evidence indicates that other proteins required for receptor internalization might also be regulated by ubiquitination, suggesting that ubiquitin plays diverse roles in regulating plasma membrane protein activity.
Hrs is an early endosomal protein homologous to Vps27p, a yeast protein required for vesicular trafficking. Hrs has a FYVE double zinc finger domain, which specifically binds phosphatidylinositol(3)-phosphate and is conserved in several proteins involved in vesicular traffic. To understand the physiological role of Hrs, we generated mice carrying a null mutation of the gene. Hrs homozygous mutant embryos developed with their ventral region outside of the yolk sac, had two independent bilateral heart tubes (cardia bifida), lacked a foregut, and died around embryonic day 11 (E11). These phenotypes arise from a defect in ventral folding morphogenesis that occurs normally around E8.0. Significant apoptosis was detected in the ventral region of mutant embryos within the definitive endoderm, suggesting an important role of this germ layer in ventral folding morphogenesis. Abnormally enlarged early endosomes were detected in the mutants in several tissues including definitive endoderm, suggesting that a deficiency in vesicular transport via early endosomes underlies the mutant phenotype. The vesicular localization of Hrs was disrupted in cells treated with wortmannin, implicating Hrs in the phosphatidylinositol 3-kinase pathway of membrane trafficking.
Hrs (hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate) is an early endosomal protein that is rapidly tyrosine-phosphorylated in cells stimulated with growth factors. Hrs is thought to play a regulatory role in the endocytosis of growth factor/receptor complexes through early endosomes. In this study, we searched for Hrs-interacting molecules which may regulate the function of Hrs, using a yeast two-hybrid system.
We isolated a cDNA clone encoding a novel Src homology 3 (SH3)-containing protein, and named it 'Hrs binding protein' (Hbp). Hbp was co-immunoprecipitated with Hrs, and its intracellular localization was similar to that of Hrs. The association between Hbp and Hrs was mediated through the coiled coil motifs in Hbp and Hrs. Deletion mutants of Hbp lacking either the SH3 domain or the Hrs binding domain showed dominantly negative effects on the intracellular degradation of a growth factor and its receptor, but not on the internalization of growth factor/receptor complexes.
Hbp is thought to be closely associated with Hrs on early endosomes. Hbp, together with Hrs may play a regulatory role in the vesicular transport of growth factor/receptor complexes through early endosomes, for their degradation.
Responses to extracellular stimuli are often transduced from cell-surface
receptors to protein tyrosine kinases which, when activated, initiate the
formation of protein complexes that transmit signals throughout the cell.
A prominent component of these complexes is the product of the proto-oncogene
c-Cbl, which specifically targets activated protein tyrosine kinases
and regulates their signalling. How, then, does this multidomain protein shape
the responses generated by these signalling complexes?
Ubiquitination generally serves as a signal for targeting cytoplasmic and nuclear proteins to the proteasome for subsequent degradation. Recently, evidence has accumulated indicating that ubiquitination also plays an important role in targeting integral membrane proteins for degradation by the lytic vacuole or the lysosome. This article describes a conserved protein motif, based on a sequence of the proteasomal component Rpn10/S5a, that is known to recognize ubiquitin. The presence of this motif in Eps15, Epsin and HRS, proteins involved in ligand-activated receptor endocytosis and degradation, suggest a more general role in ubiquitin recognition.
The multivesicular body (MVB) pathway is responsible for both the biosynthetic delivery of lysosomal hydrolases and the downregulation of numerous activated cell surface receptors which are degraded in the lysosome. We demonstrate that ubiquitination serves as a signal for sorting into the MVB pathway. In addition, we characterize a 350 kDa complex, ESCRT-I (composed of Vps23, Vps28, and Vps37), that recognizes ubiquitinated MVB cargo and whose function is required for sorting into MVB vesicles. This recognition event depends on a conserved UBC-like domain in Vps23. We propose that ESCRT-I represents a conserved component of the endosomal sorting machinery that functions in both yeast and mammalian cells to couple ubiquitin modification to protein sorting and receptor downregulation in the MVB pathway.
Many studies have demonstrated a role for ubiquitin (Ub) in the down-regulation of cell surface proteins. In yeast, down-regulation is marked by the internalization of proteins, followed by their delivery to the lumen of the vacuole where both the cytosolic and lumenal domains are degraded. It is generally believed that the regulatory step of this process is internalization from the plasma membrane and that protein delivery to the lysosome or vacuole is by default. By separating the process of internalization from degradation, we demonstrate that incorporation of proteins into intralumenal vesicles represents a distinct sorting step along the endocytic pathway that is controlled by recognition of ubiquitin. We show that attachment of a single ubiquitin can serve as a specific sorting signal for the degradative pathway by redirecting recycling Golgi proteins and resident vacuolar proteins into intralumenal vesicles of the yeast vacuole. This pathway is independent of PtdIns(3,5) P2 and does not rely on the specific composition of transmembrane domain segments. These data provide a physiological basis for how ubiquitination of cell surface proteins guides their degradation and removal from the recycling pathway.
Signaling through tyrosine kinase receptors (TKRs) is thought to be modulated by receptor-mediated endocytosis and degradation of the receptor in the lysosome. However, factors that regulate endosomal sorting of TKRs are largely unknown. Here, we demonstrate that Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate) is one such factor. Electron microscopy studies of hrs mutant larvae reveal an impairment in endosome membrane invagination and formation of multivesicular bodies (MVBs). hrs mutant animals fail to degrade active epidermal growth factor (EGF) and Torso TKRs, leading to enhanced signaling and altered embryonic patterning. These data suggest that Hrs and MVB formation function to downregulate TKR signaling.
Ubiquitin functions as a signal for sorting cargo at multiple steps of the endocytic pathway and controls the activity of trans-acting components of the endocytic machinery (reviewed in refs 1, and 2). By contrast to proteasome degradation, which generally requires a polyubiquitin chain that is at least four subunits long, internalization and sorting of endocytic cargo at the late endosome are mediated by mono-ubiquitination. Here, we demonstrate that ubiquitin-interacting motifs (UIMs) found in epsins and Vps27p (ref. 9) from Saccharomyces cerevisiae are required for ubiquitin binding and protein transport. Epsin UIMs are important for the internalization of receptors into vesicles at the plasma membrane. Vps27p UIMs are necessary to sort biosynthetic and endocytic cargo into vesicles that bud into the lumen of a late endosomal compartment, the multivesicular body. We propose that mono-ubiquitin regulates internalization and endosomal sorting by interacting with modular ubiquitin-binding domains in core components of the protein transport machinery. UIM domains are found in a broad spectrum of proteins, consistent with the idea that mono-ubiquitin can function as a regulatory signal to control diverse biological activities.
Membrane proteins that are degraded in the vacuole of Saccharomyces cerevisiae are sorted into discrete intralumenal vesicles, analogous to the internal membranes of multi-vesiculated bodies (MVBs). Recently, it has shown that the attachment of ubiquitin (Ub) mediates sorting into lumenal membranes. We describe a complex of Vps27p and Hse1p that localizes to endosomal compartments and is required for the recycling of Golgi proteins, formation of lumenal membranes and sorting of ubiquitinated proteins into those membranes. The Vps27p-Hse1p complex binds to Ub and requires multiple Ub Interaction Motifs (UIMs). Mutation of these motifs results in specific defects in the sorting of ubiquitinated proteins into the vacuolar lumen. However, the recycling of Golgi proteins and the generation of lumenal membranes proceeds normally in Delta UIM mutants. These data support a model in which the Vps27p-Hse1p complex has multiple functions at the endosome, one of which is as a sorting receptor for ubiquitinated membrane proteins destined for degradation.
Lysosomally directed receptors are concentrated at a 'bilayered' clathrin coat on the face of sorting endosomes. This coat is highly enriched in Hrs protein, which can potentially serve as an adaptor between ubiquitinated receptors and clathrin.
The hepatocyte growth factor-regulated tyrosine kinase substrate Hrs is an early endosomal protein that is thought to play a regulatory role in the trafficking of growth factor/receptor complexes through early endosomes. Stimulation of cells with epidermal growth factor (EGF) rapidly leads to phosphorylation of Hrs, raising the question whether the receptor tyrosine kinase phosphorylates Hrs directly. Here, we present evidence that a downstream kinase, rather than the active receptor kinase is responsible. We show that the nonreceptor tyrosine kinase Src is able to phosphorylate Hrs in vitro and in vivo, but that Hrs is nevertheless phosphorylated in Src-, Yes- and Fyn-negative cells. Moreover, we show that only 10-20% of Hrs is phosphorylated following EGF stimulation, and that phosphorylation occurs at multiple tyrosines located in different parts of Hrs. These results suggest that Hrs is a substrate for several kinases downstream of the EGF receptor.
Sorting of ubiquitinated endosomal membrane proteins into the MVB pathway is executed by the class E Vps protein complexes ESCRT-I, -II, and -III, and the AAA-type ATPase Vps4. This study characterizes ESCRT-II, a soluble approximately 155 kDa protein complex formed by the class E Vps proteins Vps22, Vps25, and Vps36. This protein complex transiently associates with the endosomal membrane and thereby initiates the formation of ESCRT-III, a membrane-associated protein complex that functions immediately downstream of ESCRT-II during sorting of MVB cargo. ESCRT-II in turn functions downstream of ESCRT-I, a protein complex that binds to ubiquitinated endosomal cargo. We propose that the ESCRT complexes perform a coordinated cascade of events to select and sort MVB cargoes for delivery to the lumen of the vacuole/lysosome.
The sorting of transmembrane proteins (e.g., cell surface receptors) into the multivesicular body (MVB) pathway to the lysosomal/vacuolar lumen requires the function of the ESCRT protein complexes. The soluble coiled-coil-containing proteins Vps2, Vps20, Vps24, and Snf7 are recruited from the cytoplasm to endosomal membranes where they oligomerize into a protein complex, ESCRT-III. ESCRT-III contains two functionally distinct subcomplexes. The Vps20-Snf7 subcomplex binds to the endosomal membrane, in part via the myristoyl group of Vps20. The Vps2-Vps24 subcomplex binds to the Vps20-Snf7 complex and thereby serves to recruit additional cofactors to this site of protein sorting. We provide evidence for a role for ESCRT-III in sorting and/or concentration of MVB cargoes.