scholarly journals Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies

2002 ◽  
Vol 156 (5) ◽  
pp. 843-854 ◽  
Author(s):  
Karianne E. Longva ◽  
Frøydis D. Blystad ◽  
Espen Stang ◽  
Astrid M. Larsen ◽  
Lene E. Johannessen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ubiquitin Ligase ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Dominant Negative ◽  
Multivesicular Bodies ◽  
Proteasomal Activity ◽  
Lysosomal Sorting

EGF, but not TGFα, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGFα, whereas the ubiquitination was more sustained by incubation with EGF than with TGFα. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGFα, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGFα dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target.

scholarly journals Ubc4/5 and c-Cbl Continue to Ubiquitinate EGF Receptor after Internalization to Facilitate Polyubiquitination and Degradation

2008 ◽  
Vol 19 (8) ◽  
pp. 3454-3462 ◽  
Author(s):  
Kyohei Umebayashi ◽  
Harald Stenmark ◽  
Tamotsu Yoshimori
Keyword(s):  
Ubiquitin Ligase ◽  
Time Course ◽  
Kinase Activity ◽  
Egf Receptor ◽  
In Vitro Activity ◽  
Lysosomal Sorting ◽  
Time Course Experiment ◽  
Ubiquitin Conjugating Enzyme ◽  
Epidermal Growth

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.

scholarly journals Ear1p and Ssh4p Are New Adaptors of the Ubiquitin Ligase Rsp5p for Cargo Ubiquitylation and Sorting at Multivesicular Bodies

2008 ◽  
Vol 19 (6) ◽  
pp. 2379-2388 ◽  
Author(s):  
Sébastien Léon ◽  
Zoi Erpapazoglou ◽  
Rosine Haguenauer-Tsapis
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Ubiquitin Ligase ◽  
Multivesicular Bodies ◽  
Endosomal Sorting ◽  
Frame Fusion ◽  
Vacuolar Targeting

The ubiquitylation of membrane proteins destined for the vacuole/lysosome is essential for their recognition by the endosomal sorting machinery and their internalization into vesicles of multivesicular bodies (MVBs). In yeast, this process requires Rsp5p, an essential ubiquitin ligase of the Nedd4 family. We describe here two redundant proteins, Ear1p and Ssh4p, required for the vacuolar targeting of several cargoes originating from the Golgi or the plasma membrane. Ear1p is an endosomal protein that interacts with Rsp5p through its PPxY motifs, and it is required for the ubiquitylation of selected cargoes before their MVB sorting. In-frame fusion of cargo to ubiquitin overcomes the need for Ear1p/Ssh4p, confirming a role for these proteins in cargo ubiquitylation. Interestingly, Ear1p is itself ubiquitylated by Rsp5p and targeted to the vacuole. Finally, Ear1p overexpression leads to Rsp5p accumulation at endosomes, interfering with some of its functions in trafficking. Therefore, Ear1p/Ssh4p recruit Rsp5p and assist it in its function at MVBs by directing the ubiquitylation of specific cargoes.

scholarly journals Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane

2012 ◽  
Vol 303 (5) ◽  
pp. G657-G665 ◽  
Author(s):  
Se Won Park ◽  
Christopher M. Schonhoff ◽  
Cynthia R. L. Webster ◽  
M. Sawkat Anwer
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Membrane Localization ◽  
Plasma Membrane Localization ◽  
Phosphoinositide 3 Kinase ◽  
Interfering Rna ◽  
Protein Kinase Cδ

Cyclic AMP stimulates translocation of Na+/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.

scholarly journals Selective Stimulation of Caveolar Endocytosis by Glycosphingolipids and Cholesterol

2004 ◽  
Vol 15 (7) ◽  
pp. 3114-3122 ◽  
Author(s):  
Deepak K. Sharma ◽  
Jennifer C. Brown ◽  
Amit Choudhury ◽  
Timothy E. Peterson ◽  
Eileen Holicky ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Acute Treatment ◽  
Kinase Activity ◽  
Cultured Cells ◽  
Src Kinase ◽  
Growth Conditions ◽  
Dominant Negative ◽  
Caveolin 1 ◽  
Α Activity ◽  
Stimulation Of

Internalization of some plasma membrane constituents, bacterial toxins, and viruses occurs via caveolae; however, the factors that regulate caveolar internalization are still unclear. Here, we demonstrate that a brief treatment of cultured cells with natural or synthetic glycosphingolipids (GSLs) or elevation of cholesterol (either by acute treatment with mβ-cyclodextrin/cholesterol or by alteration of growth conditions) dramatically stimulates caveolar endocytosis with little or no effect on other endocytic mechanisms. These treatments also stimulated the movement of GFP-labeled vesicles in cells transfected with caveolin-1-GFP and reduced the number of surface-connected caveolae seen by electron microscopy. In contrast, overexpression of caveolin-1 decreased caveolar uptake, but treatment with GSLs reversed this effect and stimulated caveolar endocytosis. Stimulation of caveolar endocytosis did not occur using ceramide or phosphatidylcholine and was not due to GSL degradation because similar results were obtained using a nonhydrolyzable GSL analog. Stimulated caveolar endocytosis required src kinase and PKC-α activity as shown by i) use of pharmacological inhibitors, ii) expression of kinase inactive src or dominant negative PKCα, and iii) stimulation of src kinase activity upon addition of GSLs or cholesterol. These results suggest that caveolar endocytosis is regulated by a balance of caveolin-1, cholesterol, and GSLs at the plasma membrane.

Dominant Negative Effect of Palmitoylation-Deficient NRAS In Suppression of BCR/ABL Leukemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3157-3157
Author(s):  
Benjamin Cuiffo ◽  
Ruibao Ren
Keyword(s):  
Plasma Membrane ◽  
Hematological Malignancies ◽  
Kinase Activity ◽  
Effective Therapy ◽  
Dominant Negative ◽  
Myelogenous Leukemia ◽  
Dominant Negative Effect ◽  
Membrane Microdomains ◽  
Ras Signaling ◽  
Negative Effect

Abstract Abstract 3157 RAS mutations are found in ∼30% of all human cancers, with NRAS being the RAS isoform found most frequently mutated in hematological malignancies including acute myelogenous leukemia (AML) and chronic myelomonocytic leukemia (CMML). We have previously shown that expression of oncogenic NRAS efficiently induces a CMML- or AML-like disease in mice. Like all RAS proteins, NRAS must undergo a series of post-translational modifications for differential targeting to distinct cellular membrane microdomains. We have previously found that palmitoylation, in addition to prenylation, is an essential process for NRAS leukemogenesis. The results suggest that targeting RAS palmitoylation may be an effective therapy for hematological malignancies as well as other NRAS related cancers. In addition to the direct activation by mutations, RAS can also be functionally activated by other oncogenic mutations, including activated protein tyrosine kinases such as BCR/ABL. While significant advances have been made in the treatment of chronic myelogenous leukemia (CML) by targeting the kinase activity of BCR/ABL, these treatments do not constitute a cure, as BCR/ABL-positive cells persist and eventually develop mutations that cause resistance to the kinase inhibitors. In addition, although advances have also been achieved in the treatment of BCR/ABL+ B-acute lymphoblastic lymphoma (B-ALL) through the use of combination chemotherapy along with the ABL kinase inhibitor imatinib, BCR/ABL+ B-ALL continues to pose a huge challenge; they are highly refractory to imatinib, and are still the least treatable subtype of ALL. The identification of molecular targets that impede the progression of BCR/ABL+ leukemias not relying on targeting the kinase activity of BCR/ABL is likely beneficial. Previous studies have shown that RAS is a critical downstream transducer of the oncogenic signaling by BCR/ABL. We have shown that palmitoylation-deficient oncogenic NRAS is mislocalized away from the plasma membrane yet still capable of binding GTP in cells. This finding suggests that palmitoylation-deficient, activated RAS may have a dominant negative effect in RAS signaling. As a proof-of-concept study for treating cancers driven by RAS regulators through blocking RAS palmitoylation, we evaluated the effect of blocking RAS palmitoylation in BCR/ABL leukemogenesis. We found that expression of palmitoylation-deficient oncogenic NRAS or palmitoylation-deficient wild type NRAS significantly impeded progression of BCR/ABL induced B-ALL- and CML-like diseases in mice. The palmitoylation-deficient NRAS suppresses the activation of downstream effectors of RAS, possibly by sequestrating RAS effectors away from the plasma membrane. The results suggest that targeting RAS palmitoylation may also constitute an effective therapy in cancers driven by oncogenes upstream of RAS. Disclosures: No relevant conflicts of interest to declare.

Role of Exosomes in the Exchange of Spermatozoa after Leaving the Seminiferous Tubule: A Review

2020 ◽  
Vol 21 (5) ◽  
pp. 330-338
Author(s):  
Luming Wu ◽  
Yuan Ding ◽  
Shiqiang Han ◽  
Yiqing Wang
Keyword(s):  
Drug Delivery ◽  
Plasma Membrane ◽  
Seminiferous Tubule ◽  
Metabolic Diseases ◽  
Inflammatory Diseases ◽  
Multivesicular Bodies ◽  
Extensive Search ◽  
Neurological Research ◽  
The One

Background: Exosomes are extracellular vesicles (EVs) released from cells upon fusion of an intermediate endocytic compartment with the plasma membrane. They refer to the intraluminal vesicles released from the fusion of multivesicular bodies with the plasma membrane. The contents and number of exosomes are related to diseases such as metabolic diseases, cancer and inflammatory diseases. Exosomes have been used in neurological research as a drug delivery tool and also as biomarkers for diseases. Recently, exosomes were observed in the seminal plasma of the one who is asthenozoospermia, which can affect sperm motility and capacitation. Objective: The main objective of this review is to deeply discuss the role of exosomes in spermatozoa after leaving the seminiferous tubule. Methods: We conducted an extensive search of the literature available on relationships between exosomes and exosomes in spermatozoa on the bibliographic database. Conclusion: : This review thoroughly discussed the role that exosomes play in the exchange of spermatozoa after leaving the seminiferous tubule and its potential as a drug delivery tool and biomarkers for diseases as well.

scholarly journals E3-13.7 Integral Membrane Proteins Encoded by Human Adenoviruses Alter Epidermal Growth Factor Receptor Trafficking by Interacting Directly with Receptors in Early Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3559-3572 ◽  
Author(s):  
Denise Crooks ◽  
Song Jae Kil ◽  
J. Michael McCaffery ◽  
Cathleen Carlin
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Viral Protein ◽  
Egf Receptor ◽  
Receptor Trafficking ◽  
Model Systems ◽  
Egf Receptors ◽  
Down Regulation ◽  
Human Adenoviruses ◽  
Early Endosomes

Animal cell viruses provide valuable model systems for studying many normal cellular processes, including membrane protein sorting. The focus of this study is an integral membrane protein encoded by the E3 transcription region of human adenoviruses called E3-13.7, which diverts recycling EGF receptors to lysosomes without increasing the rate of receptor internalization or intrinsic receptor tyrosine kinase activity. Although E3-13.7 can be found on the plasma membrane when it is overexpressed, its effect on EGF receptor trafficking suggests that the plasma membrane is not its primary site of action. Using cell fractionation and immunocytochemical experimental approaches, we now report that the viral protein is located predominantly in early endosomes and limiting membranes of endosome-to-lysosome transport intermediates called multivesicular endosomes. We also demonstrate that E3-13.7 physically associates with EGF receptors undergoing E3-13.7–mediated down-regulation in early endosomes. Receptor–viral protein complexes then dissociate, and EGF receptors proceed to lysosomes, where they are degraded, while E3-13.7 is retained in endosomes. We conclude that E3-13.7 is a resident early endocytic protein independent of EGF receptor expression, because it has identical intracellular localization in mouse cells lacking endogenous receptors and cells expressing a human cytomegalovirus-driven receptor cDNA. Finally, we demonstrate that EGF receptor residues 675–697 are required for E3-13.7–mediated down-regulation. Interestingly, this sequence includes a known EGF receptor leucine-based lysosomal sorting signal used during ligand-induced trafficking, which is also conserved in the viral protein. E3-13.7, therefore, provides a novel model system for determining the molecular basis of selective membrane protein transport in the endocytic pathway. Our studies also suggest new paradigms for understanding EGF receptor sorting in endosomes and adenovirus pathogenesis.

scholarly journals NF-κB-Inducing Kinase (NIK) Mediates Skeletal Muscle Insulin Resistance: Blockade by Adiponectin

Endocrinology ◽  
2011 ◽  
Vol 152 (10) ◽  
pp. 3622-3627 ◽  
Author(s):  
Sanjeev Choudhary ◽  
Sandeep Sinha ◽  
Yanhua Zhao ◽  
Srijita Banerjee ◽  
Padma Sathyanarayana ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Muscle Insulin Resistance ◽  
Akt Phosphorylation ◽  
Skeletal Muscle Insulin Resistance ◽  
Obese Subjects

Enhanced levels of nuclear factor (NF)-κB-inducing kinase (NIK), an upstream kinase in the NF-κB pathway, have been implicated in the pathogenesis of chronic inflammation in diabetes. We investigated whether increased levels of NIK could induce skeletal muscle insulin resistance. Six obese subjects with metabolic syndrome underwent skeletal muscle biopsies before and six months after gastric bypass surgery to quantitate NIK protein levels. L6 skeletal myotubes, transfected with NIK wild-type or NIK kinase-dead dominant negative plasmids, were treated with insulin alone or with adiponectin and insulin. Effects of NIK overexpression on insulin-stimulated glucose uptake were estimated using tritiated 2-deoxyglucose uptake. NF-κB activation (EMSA), phosphatidylinositol 3 (PI3) kinase activity, and phosphorylation of inhibitor κB kinase β and serine-threonine kinase (Akt) were measured. After weight loss, skeletal muscle NIK protein was significantly reduced in association with increased plasma adiponectin and enhanced AMP kinase phosphorylation and insulin sensitivity in obese subjects. Enhanced NIK expression in cultured L6 myotubes induced a dose-dependent decrease in insulin-stimulated glucose uptake. The decrease in insulin-stimulated glucose uptake was associated with a significant decrease in PI3 kinase activity and protein kinase B/Akt phosphorylation. Overexpression of NIK kinase-dead dominant negative did not affect insulin-stimulated glucose uptake. Adiponectin treatment inhibited NIK-induced NF-κB activation and restored insulin sensitivity by restoring PI3 kinase activation and subsequent Akt phosphorylation. These results indicate that NIK induces insulin resistance and further indicate that adiponectin exerts its insulin-sensitizing effect by suppressing NIK-induced skeletal muscle inflammation. These observations suggest that NIK could be an important therapeutic target for the treatment of insulin resistance associated with inflammation in obesity and type 2 diabetes.

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