Met/Hepatocyte growth factor receptor ubiquitination suppresses transformation and is required for Hrs phosphorylation

The Met receptor tyrosine kinase (RTK) regulates epithelial remodeling, dispersal, and invasion and is deregulated in many human cancers. It is now accepted that impaired down-regulation, as well as sustained activation, of RTKs could contribute to their deregulation. Down-regulation of the Met receptor involves ligand-induced internalization, ubiquitination by Cbl ubiquitin ligases, and lysosomal degradation. Here we report that a ubiquitination-deficient Met receptor mutant (Y1003F) is tumorigenic in vivo. The Met Y1003F mutant is internalized, and undergoes endosomal trafficking with kinetics similar to the wild-type Met receptor, yet is inefficiently targeted for degradation. This results in sustained activation of Met Y1003F and downstream signals involving the Ras-mitogen-activated protein kinase pathway, cell transformation, and tumorigenesis. Although Met Y1003F undergoes endosomal trafficking and localizes with the cargo-sorting protein Hrs, it is unable to induce phosphorylation of Hrs. Fusion of monoubiquitin to Met Y1003F is sufficient to decrease Met receptor stability and prevent sustained MEK1/2 activation. In addition, this rescues Hrs tyrosine phosphorylation and decreases transformation in a focus-forming assay. These results demonstrate that Cbl-dependent ubiquitination is dispensable for Met internalization but is critical to target the Met receptor to components of the lysosomal sorting machinery and to suppress its inherent transforming activity.     

Epidermal growth factor receptor fate is controlled by Hrs tyrosine phosphorylation sites that regulate Hrs degradation

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is an endosomal protein essential for the efficient sorting of activated growth factor receptors into the lysosomal degradation pathway. Hrs undergoes ligand-induced tyrosine phosphorylation on residues Y329 and Y334 downstream of epidermal growth factor receptor (EGFR) activation. It has been difficult to investigate the functional roles of phosphoHrs, as only a small proportion of the cellular Hrs pool is detectably phosphorylated. Using an HEK 293 model system, we found that ectopic expression of the protein Cbl enhances Hrs ubiquitination and increases Hrs phosphorylation following cell stimulation with EGF. We exploited Cbl's expansion of the phosphoHrs pool to determine whether Hrs tyrosine phosphorylation controls EGFR fate. In structure-function studies of Cbl and EGFR mutants, the level of Hrs phosphorylation and rapidity of apparent Hrs dephosphorylation correlated directly with EGFR degradation. Differential expression of wild-type versus Y329,334F mutant Hrs in Hrs-depleted cells revealed that one or both tyrosines regulate ligand-dependent Hrs degradation, as well as EGFR degradation. By modulating Hrs ubiquitination, phosphorylation, and protein levels, Cbl may control the composition of the endosomal sorting machinery and its ability to target EGFR for lysosomal degradation.     

Hrs and SNX3 functions in sorting and membrane invagination within multivesicular bodies

After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs—an adaptor-like protein that binds membrane PtdIns3P via a FYVE motif—and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.     

The met receptor degradation pathway: requirement for Lys48-linked polyubiquitin independent of proteasome activity

Acute stimulation of the receptor for the hepatocyte growth factor/scatter factor Met leads to receptor monoubiquitination and down-regulation through the lysosomal degradation pathway. We have determined that the Met receptor undergoes multiple monoubiquitination as opposed to the appendage of polyubiquitin chains. Nevertheless, overexpression of ubiquitin in HEK293T cells enhances the rate of Met receptor degradation, in contrast to a point mutant of ubiquitin (K48R) that cannot form Lys(48)-linked polyubiquitin chains. Furthermore, an enhancement of Met degradation is also seen under conditions where the proteasome is inhibited by lactacystin. We propose that this reflects polyubiquitin-dependent sorting of Met, as the overexpression of ubiquitin but not K48R ubiquitin also restores hepatocyte growth factor-dependent phosphorylation of the endosomal coat protein Hrs from inhibition by lactacystin. Our data indicate a requirement for K48R-linked polyubiquitin for Met endosomal trafficking independent of its canonical function of targeting for proteasomal degradation.     

Hrs recognizes a hydrophobic amino acid cluster in cytokine receptors during ubiquitin-independent endosomal sorting

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of the ESCRT-0 protein complex that captures ubiquitylated cargo proteins and sorts them to the lysosomal pathway. Although Hrs acts as a key transporter for ubiquitin-dependent endosomal sorting, we previously reported that Hrs is also involved in ubiquitin-independent endosomal sorting of interleukin-2 receptor β (IL-2Rβ). Here, we show direct interactions between bacterially expressed Hrs and interleukin-4 receptor α (IL-4Rα), indicating that their binding is not required for ubiquitylation of the receptors, similar to the case for IL-2Rβ. Examinations of the Hrs binding regions of the receptors reveal that a hydrophobic amino acid cluster in both IL-2Rβ and IL-4Rα is essential for the binding. Whereas the wild-type receptors are delivered to LAMP1-positive late endosomes, mutant receptors lacking the hydrophobic amino acid cluster are sorted to lysobisphosphatidic acid-positive late endosomes rather than LAMP1-positive late endosomes. We also show that the degradation of these mutant receptors is attenuated. Accordingly, Hrs functions during ubiquitin-independent endosomal sorting of the receptors by recognizing the hydrophobic amino acid cluster. These findings suggest the existence of a group of cargo proteins that have this hydrophobic amino acid cluster as a ubiquitin-independent sorting signal.     

Intrinsic tyrosine kinase activity is required for vascular endothelial growth factor receptor 2 ubiquitination, sorting and degradation in endothelial cells

The human endothelial vascular endothelial growth factor receptor 2 (VEGFR2/kinase domain region, KDR/fetal liver kinase-1, Flk-1) tyrosine kinase receptor is essential for VEGF-mediated physiological responses including endothelial cell proliferation, migration and survival. How VEGFR2 kinase activation and trafficking are co-coordinated in response to VEGF-A is not known. Here, we elucidate a mechanism for endothelial VEGFR2 response to VEGF-A dependent on constitutive endocytosis co-ordinated with ligand-activated ubiquitination and proteolysis. The selective VEGFR kinase inhibitor, SU5416, blocked the endosomal sorting required for VEGFR2 trafficking and degradation. Inhibition of VEGFR2 tyrosine kinase activity did not block plasma membrane internalization but led to endosomal accumulation. Lysosomal protease activity was required for ligand-stimulated VEGFR2 degradation. Activated VEGFR2 codistributed with the endosomal hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)/signal-transducing adaptor molecule (STAM) complex in a ligand and time-dependent manner, implying a role for this factor in sorting of ubiquitinated VEGFR2. Increased tyrosine phosphorylation of the Hrs subunit in response to VEGF-A links VEGFR2 activation and Hrs/STAM function. In contrast, VEGFR2 in quiescent cells was present on both the endothelial plasma membrane and early endosomes, suggesting constitutive recycling between these two compartments. This pathway was clathrin-linked and dependent on the AP2 adaptor complex as the A23 tyrphostin inhibited VEGFR2 trafficking. We propose a mechanism whereby the transition of endothelial VEGFR2 from a constitutive recycling itinerary to a degradative pathway explains ligand-activated receptor degradation in endothelial cells. This study outlines a mechanism to control the VEGF-A-mediated response within the vascular system.     

Ubc4/5 and c-Cbl continue to ubiquitinate EGF receptor after internalization to facilitate polyubiquitination and degradation

c-Cbl is the E3 ubiquitin ligase that ubiquitinates the epidermal growth factor (EGF) receptor (EGFR). On the basis of localization, knockdown, and in vitro activity analyses, we have identified the E2 ubiquitin-conjugating enzyme that cooperates with c-Cbl as Ubc4/5. Upon EGF stimulation, both Ubc4/5 and c-Cbl were relocated to the plasma membrane and then to Hrs-positive endosomes, strongly suggesting that EGFR continues to be ubiquitinated after internalization. Our time-course experiment showed that EGFR undergoes polyubiquitination, which seemed to be facilitated during the transport to Hrs-positive endosomes. Use of a conjugation-defective ubiquitin mutant suggested that receptor polyubiquitination is required for efficient interaction with Hrs and subsequent sorting to lysosomes. Abrupt inhibition of the EGFR kinase activity resulted in dissociation of c-Cbl from EGFR. Concomitantly, EGFR was rapidly deubiquitinated and its degradation was delayed. We propose that sustained tyrosine phosphorylation of EGFR facilitates its polyubiquitination in endosomes and counteracts rapid deubiquitination, thereby ensuring Hrs-dependent lysosomal sorting.     

Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes

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.     

Hrs and endocytic sorting of ubiquitinated membrane proteins

Endocytosed receptors are either recycled to the plasma membrane or trapped within intralumenal vesicles of multi-vesicular bodies for subsequent degradation in lysosomes. How the cell is able to sort receptors in endosomes has so far been largely unknown. The hepatocyte growth factor regulated tyrosine kinase substrate, Hrs, is an essential protein that has been implicated in cell signalling and intracellular membrane trafficking. Very recently, several reports have demonstrated a role for Hrs in endocytic sorting of ubiquitinated membrane proteins. Here, we review current knowledge about how Hrs recognises ubiquitinated cargo that is destined for lysosomal degradation, and how Hrs may act as a key regulator of the molecular machinery involved in receptor sorting and multivesicular body formation.     

The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.     

UBE4B Protein Couples Ubiquitination and Sorting Machineries to Enable Epidermal Growth Factor Receptor (EGFR) Degradation

The signaling of plasma membrane proteins is tuned by internalization and sorting in the endocytic pathway prior to recycling or degradation in lysosomes. Ubiquitin modification allows recognition and association of cargo with endosomally associated protein complexes, enabling sorting of proteins to be degraded from those to be recycled. The mechanism that provides coordination between the cellular machineries that mediate ubiquitination and endosomal sorting is unknown. We report that the ubiquitin ligase UBE4B is recruited to endosomes in response to epidermal growth factor receptor (EGFR) activation by binding to Hrs, a key component of endosomal sorting complex required for transport (ESCRT) 0. We identify the EGFR as a substrate for UBE4B, establish UBE4B as a regulator of EGFR degradation, and describe a mechanism by which UBE4B regulates endosomal sorting, affecting cellular levels of the EGFR and its downstream signaling. We propose a model in which the coordinated action of UBE4B, ESCRT-0, and the deubiquitinating enzyme USP8 enable the endosomal sorting and lysosomal degradation of the EGFR.     

Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity

Immune responses are initiated when molecules of microbial origin are sensed by the Toll-like receptors (TLRs). We now report the identification of essential molecular components for the trafficking of the lipopolysaccharide (LPS) receptor complex. LPS was endocytosed by a receptor-mediated mechanism dependent on dynamin and clathrin and colocalized with TLR4 on early/sorting endosomes. TLR4 was ubiquitinated and associated with the ubiquitin-binding endosomal sorting protein hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs. Inhibition of endocytosis and endosomal sorting increased LPS signaling. Finally, the LPS receptor complex was sorted to late endosomes/lysosomes for degradation and loading of associated antigens onto HLA class II molecules for presentation to CD4+ T cells. Our results show that endosomal trafficking of the LPS receptor complex is essential for signal termination and LPS-associated antigen presentation, thus controlling both innate and adaptive immunity through TLR4.     

Rapid transport of internalized P-selectin to late endosomes and the TGN: roles in regulating cell surface expression and recycling to secretory granules

Prior studies on receptor recycling through late endosomes and the TGN have suggested that such traffic may be largely limited to specialized proteins that reside in these organelles. We present evidence that efficient recycling along this pathway is functionally important for nonresident proteins. P-selectin, a transmembrane cell adhesion protein involved in inflammation, is sorted from recycling cell surface receptors (e.g., low density lipoprotein [LDL] receptor) in endosomes, and is transported from the cell surface to the TGN with a half-time of 20-25 min, six to seven times faster than LDL receptor. Native P-selectin colocalizes with LDL, which is efficiently transported to lysosomes, for 20 min after internalization, but a deletion mutant deficient in endosomal sorting activity rapidly separates from the LDL pathway. Thus, P-selectin is sorted from LDL receptor in early endosomes, driving P-selectin rapidly into late endosomes. P-selectin then recycles to the TGN as efficiently as other receptors. Thus, the primary effect of early endosomal sorting of P-selectin is its rapid delivery to the TGN, with rapid turnover in lysosomes a secondary effect of frequent passage through late endosomes. This endosomal sorting event provides a mechanism for efficiently recycling secretory granule membrane proteins and, more generally, for downregulating cell surface receptors.     

SNX12 role in endosome membrane transport

In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.     

Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes

Hrs and the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are involved in the endosomal sorting of membrane proteins into multivesicular bodies and lysosomes or vacuoles. The ESCRT complexes are also required for formation of intraluminal endosomal vesicles and for budding of certain enveloped RNA viruses such as HIV. Here, we show that Hrs binds to the ESCRT-I subunit Tsg101 via a PSAP motif that is conserved in Tsg101-binding viral proteins. Depletion of Hrs causes a reduction in membrane-associated ESCRT-I subunits, a decreased number of multivesicular bodies and an increased size of late endosomes. Even though Hrs mainly localizes to early endosomes and Tsg101 to late endosomes, the two proteins colocalize on a subpopulation of endosomes that contain lyso-bisphosphatidic acid. Overexpression of Hrs causes accumulation of Tsg101 on early endosomes and prevents its localization to late endosomes. We conclude that Hrs mediates the initial recruitment of ESCRT-I to endosomes and, thereby, indirectly regulates multivesicular body formation.     

The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4

Ubiquitination of the chemokine receptor CXCR4 serves as a targeting signal for lysosomal degradation, but the mechanisms mediating ubiquitination and lysosomal sorting remain poorly understood. Here we report that the Nedd4-like E3 ubiquitin ligase AIP4 mediates ubiquitination of CXCR4 at the plasma membrane, and of the ubiquitin binding protein Hrs on endosomes. CXCR4 activation promotes CXCR4 colocalization with AIP4 and Hrs within the same region of endosomes. Endosomal sorting of CXCR4 is dependent on Hrs as well as the AAA ATPase Vps4, the latter involved in regulating the ubiquitination status of both CXCR4 and Hrs. We propose a model whereby AIP4, Hrs, and Vps4 coordinate a cascade of ubiquitination and deubiquitination events that sort CXCR4 to the degradative pathway.     

PI3P signaling regulates receptor sorting but not transport in the endosomal pathway

While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.     

Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway.

The Met tyrosine kinase receptor is a widely expressed molecule which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). In this communication we demonstrate that significant Met degradation is induced by HGF/SF and that this degradation can be blocked by lactacystin, an inhibitor of proteasome activity. We also show that Met is rapidly polyubiquitinated in response to ligand and that polyubiquitinated Met molecules, which are normally unstable, are stabilized by lactacystin. Both HGF/SF-induced degradation and polyubiquitination of Met were shown to be dependent on the receptor possessing intact tyrosine kinase activity. Finally, we found that a normally highly labile 55-kDa fragment of the Met receptor is stabilized by lactacystin and demonstrate that it represents a cell-associated remnant that is generated following the ligand-independent proteolytic cleavage of the Met receptor in its extracellular domain. This truncated Met molecule encompasses the kinase domain of the receptor and is itself tyrosine phosphorylated. We conclude that the ubiquitin-proteasome pathway plays a significant role in the degradation of the Met tyrosine kinase receptor as directed by ligand-dependent and -independent signals. We propose that this proteolytic pathway may be important for averting cellular transformation by desensitizing Met signaling following ligand stimulation and by eliminating potentially oncogenic fragments generated via extracellular cleavage of the Met receptor.     

Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking

A ubiquitin-binding endosomal protein machinery is responsible for sorting endocytosed membrane proteins into intraluminal vesicles of multivesicular endosomes (MVEs) for subsequent degradation in lysosomes. The Hrs-STAM complex and endosomal sorting complex required for transport (ESCRT)-I, -II and -III are central components of this machinery. Here, we have performed a systematic analysis of their importance in four trafficking pathways through endosomes. Neither Hrs, Tsg101 (ESCRT-I), Vps22/EAP30 (ESCRT-II), nor Vps24/CHMP3 (ESCRT-III) was required for ligand-mediated internalization of epidermal growth factor (EGF) receptors (EGFRs) or for recycling of cation-independent mannose 6-phosphate receptors (CI-M6PRs) from endosomes to the trans-Golgi network (TGN). In contrast, both Hrs and ESCRT subunits were equally required for degradation of both endocytosed EGF and EGFR. Whereas depletion of Hrs or Tsg101 caused enhanced recycling of endocytosed EGFRs, this was not the case with depletion of Vps22 or Vps24. Depletion of Vps24 instead caused a strong increase in the levels of CI-M6PRs and a dramatic redistribution of the Golgi and the TGN. These results indicate that, although Hrs-STAM and ESCRT-I, -II and -III have a common function in degradative protein sorting, they play differential roles in other trafficking pathways, probably reflecting their functions at distinct stages of the endocytic pathway.     

hSef potentiates EGF-mediated MAPK signaling through affecting EGFR trafficking and degradation

Sef (similar expression to fgf genes) was identified as an effective antagonist of fibroblast growth factor (FGF) in vertebrates. Previous reports have demonstrated that Sef interacts with FGF receptors (FGFRs) and inhibits FGF signaling, however, its role in regulating epidermal growth factor receptor (EGFR) signaling remains unclear. In this report, we found that hSef localizes to the plasma membrane (PM) and is subjected to rapid internalization and well localizes in early/recycling endosomes while poorly in late endosomes/lysosomes. We observed that hSef interacts and functionally colocalizes with EGFR in early endosomes in response to EGF stimulation. Importantly, we demonstrated that overexpression of hSef attenuates EGFR degradation and potentiates EGF-mediated mitogen-activated protein kinase (MAPK) signaling by interfering EGFR trafficking. Finally, our data showed that, with overexpression of hSef, elevated levels of Erk phosphorylation and differentiation of rat pheochromocytoma (PC12) cells occur in response to EGF stimulation. Taken together, these data suggest that hSef plays a positive role in the EGFR-mediated MAPK signaling pathway. This report, for the first time, reveals opposite roles for Sef in EGF and FGF signalings.