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1.
Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a mammalian homologue of yeast vacuolar protein sorting (Vps) protein Vps27p; however, the role of Hrs in lysosomal trafficking is unclear. Here, we report that Hrs interacts with sorting nexin 1 (SNX1), a recently identified mammalian homologue of yeast Vps5p that recognizes the lysosomal targeting code of epidermal growth factor receptor (EGFR) and participates in lysosomal trafficking of the receptor. Biochemical analyses demonstrate that Hrs and SNX1 are ubiquitous proteins that exist in both cytosolic and membrane-associated pools, and that the association of Hrs and SNX occurs on cellular membranes but not in the cytosol. Furthermore, endogenous SNX1 and Hrs form a approximately 550-kDa complex that excludes EGFR. Immunofluorescence and subcellular fractionation studies show that Hrs and SNX1 colocalize on early endosomes. By using deletion analysis, we have mapped the binding domains of Hrs and SNX1 that mediate their association. Overexpression of Hrs or its SNX1-binding domain inhibits ligand-induced degradation of EGFR, but does not affect either constitutive or ligand-induced receptor-mediated endocytosis. These results suggest that Hrs may regulate lysosomal trafficking through its interaction with SNX1.  相似文献   

2.
Phosphorylation of Hrs downstream of the epidermal growth factor receptor.   总被引:2,自引:0,他引:2  
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.  相似文献   

3.
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.  相似文献   

4.
Li H  Ruano MJ  Villalobo A 《FEBS letters》2004,559(1-3):175-180
We have previously shown that exogenous calmodulin (CaM) binds to the epidermal growth factor receptor (EGFR) at its cytosolic juxtamembrane region inhibiting its tyrosine kinase activity. We demonstrate in this report that endogenous CaM binds to EGFR in intact cells as CaM co-immunoprecipitates with EGF-activated and non-activated receptors. We also show in living cells that cell-permeable CaM inhibitors prevent the full transphosphorylation of wild type EGFR but not the transphosphorylation of an insertional EGFR mutant in which the CaM-binding domain was divided into two parts. Overall these results suggest that CaM interacts with EGFR in vivo.  相似文献   

5.
Levels of the epidermal growth factor receptor (EGFR) at the cell surface are tightly regulated by a complex endocytic machinery. Following internalization, EGFR is either recycled back to the cell surface or transported to the late endosome/lysosome for degradation. Currently, the molecular machinery that regulates this sorting pathway is only partially defined. Eps15 (EGFR pathway substrate 15) is an endocytic adaptor protein that is well known to support clathrin-mediated internalization of EGFR at the plasma membrane. Using RT-PCR, we have identified a novel short form of Eps15 (Eps15S) from rat liver that lacks the 111 C-terminal amino acids present in the traditional Eps15 form. The goal of this study was to define the functional role of the novel Eps15S form in EGFR trafficking. Overexpression of a mutant form of Eps15S (Eps15S ΔEH2/EH3) did not block EGFR internalization but reduced its recycling to the cell surface. After knockdown of all Eps15 forms, re-expression of Eps15S significantly reduced EGFR degradation while promoting recycling back to the cell surface. In contrast, re-expression of Eps15 did not potentiate receptor recycling. Furthermore, overexpression of the mutant Eps15S substantially reduced cell proliferation, linking EGFR recycling to downstream mitogenic effects. Finally, we found that Eps15S is localized to the Rab11-positive recycling endosome that is disrupted in cells expressing the Eps15S mutant, leading to an accumulation of the EGFR in early endosomes. These findings suggest that distinct forms of Eps15 direct EGFR to either the late endosome/lysosome for degradation (Eps15) or to the recycling endosome for transit back to the cell surface (Eps15S).  相似文献   

6.
Rin1, the prototype of a new family of multidomain Rab5 exchange factors, has been shown to play an important role in the endocytosis of the epidermal growth factor receptor (EGFR). Herein, we examined the role of Rin1 in the down-regulation of EGFR following EGF stimulation. We observed that overexpression of Rin1 accelerates EGFR degradation in EGF-stimulated cells. In concordance, depletion of endogenous Rin1 by RNA interference resulted in a substantial reduction of EGFR degradation. We showed that Rin1 interacts with signal-transducing adaptor molecule 2 (STAM2), a protein that associates with hepatocyte growth factor-regulated substrate and plays a key role in the endosomal sorting machinery. Green fluorescent protein (GFP)-Rin1 co-localizes with hemagglutinin (HA)-STAM2 and with endogenous hepatocyte growth factor-regulated substrate. Furthermore, wild type STAM2, but not a deletion mutant lacking the SH3 domain, co-immunoprecipitates with endogenous Rin1. This interaction is dependent on the proline-rich domain (PRD) of Rin1 as Rin1DeltaPRD, a mutant lacking the PRD, does not interact with STAM2. Moreover, EGFR degradation was not accelerated by expression of the Rin1DeltaPRD mutant. Together these results suggest that Rin1 regulates EGFR degradation in cooperation with STAM, defining a novel role for Rin1 in regulating endosomal trafficking.  相似文献   

7.
Ligand-stimulated growth factor receptors are rapidly internalized and transported to early endosomes. Unstimulated receptors are also internalized constitutively, although at a slower rate, and delivered to the same organelle. At early endosomes, stimulated receptors are sorted for the lysosomal degradation pathway, whereas unstimulated receptors are mostly recycled back to the cell surface. To investigate the role of Hrs, an early endosomal protein, in this sorting process, we overexpressed Hrs in HeLa cells and examined the intracellular trafficking of epidermal growth factor receptor (EGFR) in EGF-stimulated and unstimulated cells. Overexpression of Hrs inhibited the trafficking of EGFR from early endosomes, resulting in an accumulation of EGFR on early endosomes in both ligand-stimulated and unstimulated cells. On the other hand, overexpression of Hrs mutants with a deletion or a point mutation within the FYVE domain did not inhibit the trafficking. These results suggest that Hrs regulates the sorting of ligand-stimulated and unstimulated growth factor receptors on early endosomes, and that the FYVE domain, which is required for Hrs to reside in a microdomain of early endosomes, plays an essential role in the function of Hrs.  相似文献   

8.
4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly inhibited the binding of low concentrations (less than 10(-9 m) of 125I-epidermal growth factor (EGF) to A431 human epidermoid carcinoma cells. However, very little change in the binding of 125-I-EGF at high concentrations (greater than 10(-8) M) was observed in response to PMA. Affinity labeling of the 170,000-dalton EGF receptor with 125I-EGF and disuccinimidyl suberate was also decreased by the tumor promoter at low, but not high, concentrations of 125I-EGF. In order to examine this action of PMA on the EGF receptor, the receptor phosphorylation state was evaluated in A431 cells that had been incubated with [32P]phosphate for 3 h prior to the addition of PMA. The 32P content of the EGF receptor purified with EGF-Sepharose was increased by 38% compared with the same amount of receptor isolated from control cells. The increase in EGF receptor phosphorylation was dose-dependent with a half-maximal effect between 0.1 and 1 nM PMA and was specific for tumor promoting analogues of phorbol diesters. Phosphoamino acid analysis indicated that the increase in the 32P content of the EGF receptor was mainly due to phosphoserine. These results demonstrate that the EGF receptor is a target for PMA action and suggest that the mechanism of PMA action on the response of cells to epidermal growth factor may be mediated in part by phosphorylation of the EGF receptor.  相似文献   

9.
Recently, we demonstrated that hydrogen peroxide (H2O2) inhibits the internalization of the epidermal growth factor (EGF) receptor and the EGF-induced mono-ubiquitination of EGF receptor pathway substrate clone #15 (Eps15) in fibroblasts. In addition, it was suggested that EGF receptor internalization might be inhibited by H2O2 by inhibition of ubiquitination of proteins involved in endocytosis. Here, we show that H2O2 also inhibits the poly-ubiquitination of the EGF receptor in fibroblasts. Furthermore, recovery of the cells resulted in re-establishment of ubiquitination of both the EGF receptor and Eps15 and coincided with restoration of internalization of those receptors that had bound EGF in the presence of H2O2. In addition, EGF receptor internalization was inhibited by the sulphydryl reagent N-ethylmaleimide (NEM), indicating that intact SH groups might be required for receptor-mediated endocytosis. Furthermore, H2O2 rapidly induced an increase in the cellular ratio of GSSG:GSH (oxidized glutathione:reduced glutathione) and removal of H2O2 resulted in a fast restoration of the ratio of GSSG:GSH. Therefore, these results suggest a relation between the inhibition of internalization ubiquitination and an increase in GSSG:GSH ratio, which strengthens the hypothesis that H2O2 inhibits EGF receptor internalization by an inhibition of ubiquitination of proteins involved in EGF receptor-mediated endocytosis.  相似文献   

10.
We have demonstrated previously that c-Cbl requires the presence of a functional ubiquitin interacting motif (UIM) in Eps15 to mediate epidermal growth factor receptor (EGFR) endocytosis. Both the ubiquitin ligase activity of c-Cbl and the UIM of Eps15 were necessary for plasma membrane recruitment of Eps15 and entry of ligand-bound EGFR into coated pits and vesicles containing Eps15. This is consistent with a scenario in which ubiquitin moieties appended to activated EGFR complexes act as docking sites for Eps15 and thereby recruit receptors into clathrin coated pits. Here, we have investigated which additional structural features of c-Cbl are required for this process. We find that c-Cbl can guide ligand-bound EGFR into the Eps15 internalization route by two distinct mechanisms. These are either dependent on the phosphotyrosine binding domain of c-Cbl that directly binds to the EGFR or on the region C-terminal of the Ring finger, which allows for indirect binding to an alternative site on the receptor. No strict requirement exists for either ubiquitin modified EGFR or the Cbl binding ubiquitination substrate CIN85 as docking site for the UIM of Eps15. Only in the phosphotyrosine binding-dependent pathway, the EGFR is ubiquitinated and may serve as a site of recruitment for Eps15. Only in this pathway, Eps15 is tyrosine-phosphorylated, but this appears unrelated to its capacity to participate in EGFR internalization.  相似文献   

11.
To reach the lysosomes, down-regulated receptors such as the epidermal growth factor receptor must first be sorted into internal vesicles of late endosomes (multivesicular bodies), a ubiquitin-dependent event that requires the coordinated function of the endosome sorting complex required for transport (ESCRT) proteins. Here we report that CHMP3, an ESCRT-III complex component, and associated molecule of SH3 domain of STAM (AMSH), a deubiquitinating enzyme, interact with each other in cells. A dominant-negative version of CHMP3, which specifically prevents targeting of AMSH to endosomes, inhibits degradation but not internalization of EGFR, suggesting that endosomal AMSH is a functional component of the multivesicular body pathway.  相似文献   

12.
Heterotrimeric G proteins have been implicated in the regulation of membrane trafficking, but the mechanisms involved are not well understood. Here, we report that overexpression of the stimulatory G protein subunit (Galphas) promotes ligand-dependent degradation of epidermal growth factor (EGF) receptors and Texas Red EGF, and knock-down of Galphas expression by RNA interference (RNAi) delays receptor degradation. We also show that Galphas and its GTPase activating protein (GAP), RGS-PX1, interact with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a critical component of the endosomal sorting machinery. Galphas coimmunoprecipitates with Hrs and binds Hrs in pull-down assays. By immunofluorescence, exogenously expressed Galphas colocalizes with myc-Hrs and GFP-RGS-PX1 on early endosomes, and expression of either Hrs or RGS-PX1 increases the localization of Galphas on endosomes. Furthermore, knock-down of both Hrs and Galphas by double RNAi causes greater inhibition of EGF receptor degradation than knock-down of either protein alone, suggesting that Galphas and Hrs have cooperative effects on regulating EGF receptor degradation. These observations define a novel regulatory role for Galphas in EGF receptor degradation and provide mechanistic insights into the function of Galphas in endocytic sorting.  相似文献   

13.
Cbl proteins function as ubiquitin protein ligases for the activated epidermal growth factor receptor and, thus, negatively regulate its activity. Here we show that Cbl-b is ubiquitinated and degraded upon activation of the receptor. Epidermal growth factor (EGF)-induced Cbl-b degradation requires intact RING finger and tyrosine kinase binding domains and requires binding of the Cbl-b protein to the activated EGF receptor (EGFR). Degradation of both the EGFR and the Cbl-b protein is blocked by lysosomal and proteasomal inhibitors. Other components of the EGFR-signaling complex (i.e. Grb2 and Shc) are also degraded in an EGF-induced Cbl-b-dependent fashion. Our results suggest that the ubiquitin protein ligase function of Cbl-b is regulated by coordinated degradation of the Cbl-b protein along with its substrate. Furthermore, the data demonstrate that Cbl-b mediates degradation of multiple proteins in the EGFR-signaling complex.  相似文献   

14.
Activated Cdc42-associated kinase-2 (ACK2) is a non-receptor tyrosine kinase that serves as a specific effector for Cdc42, a Rho family small G-protein. Recently, we have found that ACK2 directly interacts with clathrin heavy chain through a clathrin-binding motif that is conserved in all endocytic adaptor proteins and regulates clathrin assembly, suggesting that ACK2 plays a role in clathrin-coated vesicle endocytosis (Yang, W., Lo, C. G., Dispenza, T., and Cerione, R. A. (2001) J. Biol. Chem. 276, 17468-17473). Here we report the identification of another binding partner for ACK2 that has previously been implicated in endocytosis, namely the sorting nexin protein SH3PX1 (sorting nexin 9). The interaction occurs between a proline-rich domain of ACK2 and the Src homology 3 domain (SH3) of SH3PX1. Co-immunoprecipitation studies indicate that ACK2, clathrin, and SH3PX1 form a complex in cells. Epidermal growth factor (EGF) stimulated the tyrosine phosphorylation of SH3PX1, whereas co-transfection of ACK2 with SH3PX1 resulted in the constitutive phosphorylation of SH3PX1. However, co-transfection of the kinase-dead mutant ACK2(K158R) with SH3PX1 blocked EGF-induced tyrosine phosphorylation of SH3PX1, indicating that the EGF-stimulated phosphorylation of SH3PX1 is mediated by ACK2. EGF receptor levels were significantly decreased following EGF stimulation of cells co-expressing ACK2 and SH3PX1, thus highlighting a novel role for ACK2, working together with SH3PX1 to promote the degradation of the EGF receptor.  相似文献   

15.
Whereas poly-ubiquitination targets protein substrates for proteasomal degradation, mono-ubiquitination is known to regulate protein trafficking in the endosomal system and to target cargo proteins for lysosomal degradation. The role of the de-ubiquitinating enzymes AMSH and UBPY in endosomal trafficking of cargo proteins such as the epidermal growth factor receptor (EGFR) has only very recently been the subject of study and is already a matter of debate. Although one report (Mizuno, E., Iura, T., Mukai, A., Yoshimori, T., Kitamura, N., and Komada, M. (2005) Mol. Biol. Cell 16, 5163-5174) concludes that UBPY negatively regulates EGFR degradation by de-ubiquitinating the EGFR on endosomes, another report (Row, P. E., Prior, I. A., McCullough, J., Clague, M. J., and Urbe, S. (2006) J. Biol. Chem. 281, 12618-12624) concludes that UBPY-mediated EGFR de-ubiquitination is essential for EGFR degradation. Here, we demonstrate that Usp8/UBPY, the mammalian ortholog of budding yeast Ubp4/Doa4, constitutively co-precipitates in a bivalent manner with the EGFR. Moreover, UBPY is a substrate for Src-family tyrosine kinases that are activated after ligand-induced EGFR activation. Using overexpression of three different recombinant dominant negative UBPY mutants (UBPY C748A mutant, UBPY 1-505, and UBPY 640-1080) in NIH3T3 and HEK293 cells, we demonstrate that UBPY affects both constitutive and ligand-induced (i) EGFR ubiquitination, (ii) EGFR expression levels, and (iii) the appearance of intermediate EGFR degradation products as well as (iv) downstream mitogen-activated protein kinase signal transduction. Our findings provide further evidence in favor of the model that UBPY-mediated EGFR de-ubiquitination promotes EGFR degradation.  相似文献   

16.
A method which allows direct cloning of intracellular substrates for receptor tyrosine kinases (RTKs) was developed. By applying this technique to the study of the epidermal growth factor receptor (EGFR) signaling pathway, we have isolated a cDNA, designated eps8, which predicts a approximately 92 kDa protein containing an SH3 domain. Eps8 also contains a putative nuclear targeting sequence. Antibodies specific to the eps8 gene product recognize a protein of M(r) 97 kDa and a minor 68 kDa component, which are closely related, as demonstrated by V8 proteolytic mapping. The product of the eps8 gene is tyrosine-phosphorylated in vivo following EGF stimulation of intact cells and associates with the EGFR, despite the lack of a functional SH2 domain. Several other RTKs are also able to phosphorylate p97eps8. Thus, the eps8 gene product represents a novel substrate for RTKs. Adoptive expression of the eps8 cDNA in fibroblastic or hematopoietic target cells expressing the EGFR resulted in increased mitogenic response to EGF, implicating the eps8 gene product in the control of mitogenic signals.  相似文献   

17.
Rapid engagement of the extracellular signal-regulated kinase (ERK) cascade via the Gq/11-coupled GnRH receptor (GnRHR) is mediated by transactivation of the epidermal growth factor receptor (EGFR). Here we show that the cross-talk between GnRHR and EGFR in gonadotropic cells is accomplished via gelatinases A and B (matrix metalloproteinases (MMPs) 2 and 9), identifying gelatinases as the first distinct members of the MMP family mediating EGFR transactivation by G protein-coupled receptors. Using a specific MMP2 and MMP9 inhibitor, Ro28-2653, GnRH-dependent EGFR transactivation was abrogated. Proving the specificity of the effect, transient transfection of alphaT3-1 cells with ribozymes directed against MMP2 or MMP9 specifically blocked EGFR tyrosine phosphorylation in response to GnRH stimulation. GnRH challenge of alphaT3-1 cells furthered the release of active MMP2 and MMP9 and increased their gelatinolytic activities within 5 min. Rapid release of activated MMP2 or MMP9 was inhibited by ribozyme-targeted down-regulation of MT1-MMP or MMP2, respectively. We found that GnRH-induced Src, Ras, and ERK activation were also gelatinase-dependent. Thus, gelatinase-induced EGFR transactivation was required to engage the extracellular-signal regulated kinase cascade. Activation of c-Jun N-terminal kinase and p38 MAPK by GnRH was unaffected by EGFR or gelatinase inhibition that, however, suppressed GnRH induction of c-Jun and c-Fos. Our findings suggest a novel role for gelatinases in the endocrine regulation of pituitary gonadotropes.  相似文献   

18.
The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family. Ligand (epidermal growth factor or EGF) binding to the EGFR results in the coordinated activation and integration of biochemical signaling events to mediate cell growth, migration, and differentiation. One mechanism the cell utilizes to orchestrate these events is ligand-mediated endocytosis through the canonical clathrin-mediated endocytic pathway. Identification of proteins that regulate the intracellular movement of the EGF.EGFR complex is an important first step in dissecting how specificity of EGFR signaling is conferred. We examined the role of the small molecular weight guanine nucleotide-binding protein (G-protein) rab7 as a regulator of the distal stages of the endocytic pathway. Through the transient expression of activating and inactivating mutants of rab7 in HeLa cells, we have determined that rab7 activity directly correlates with the rate of radiolabeled EGF and EGFR degradation. Furthermore, when inhibitory mutants of rab7 are expressed, the internalized EGF.EGFR complex accumulates in high-density endosomes that are characteristic of the late endocytic pathway. Thus, we conclude that rab7 regulates the endocytic trafficking of the EGF.EGFR complex by regulating its lysosomal degradation.  相似文献   

19.
We describe a novel syntaxin family member, syntaxin 9 (Syn 9), which does not possess a typical C-terminal hydrophobic tail anchor. Syn 9 has, however, a Q-SNARE domain and an overall homology to syntaxins (with the highest overall homology with mammalian syntaxin 11). Syn 9 is enriched in some epithelial cells, particularly that of the stomach lining and the skin. At the skin, it is found in the epidermal layers as well as structures associated with hair follicles. A biochemical interaction screen revealed that Syn 9 interacts specifically with the epidermal growth factor (EGF) receptor. Overexpression of Syn 9 perturbed EGF receptor endocytosis but does not appear to affect the internalization of the transferrin receptor. Syn 9 may therefore have a role in EGF receptor transport and signaling in certain epithelial cell types.  相似文献   

20.
The mechanisms that mediate implantation of the human embryo remain poorly understood and represent a fundamental problem in reproductive biology. Candidate molecules that mediate and facilitate implantation have been identified in animal studies, and include heparin binding epidermal growth factor. Here we demonstrate a potential function for the transmembrane form of heparin-binding epidermal growth factor in mediating blastocyst attachment to the endometrium, in two different novel in vitro models for human implantation. Furthermore, we demonstrate specific localisation of the heparin-binding epidermal growth factor receptor ErbB4, on the surface of the trophectoderm in peri-implantation human blastocysts. Our data lead the way for further dissection of the molecular mechanisms of implantation of the human embryo, and have implications for infertility, in vitro fertilization and contraception.  相似文献   

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