Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

Stang, Espen; Blystad, Frøydis D.; Kazazic, Maja; Bertelsen, Vibeke; Brodahl, Tonje; Raiborg, Camilla; Stenmark, Harald; Madshus, Inger Helene

doi:10.1091/mbc.e04-01-0041

Cited by 145 publications

(181 citation statements)

References 55 publications

(81 reference statements)

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“…These latter findings also indirectly stress the importance of Cbl-executed ubiquitination of other downstream endocytic proteins for proper clathrin endocytosis. A recent report by Stang et al (25), which shows that Cbl-dependent ubiquitination is required for progression of the EGFR into coated pits, further reinforces this notion. Taken together with these results, our findings suggest a mechanism through which ubiquitination of endocytic machinery, but not cargo ubiquitination, is required for internalization into coated pits.…”

Section: Discussionmentioning

confidence: 59%

Clathrin-independent endocytosis of ubiquitinated cargos

Sigismund

Woelk

Puri

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

736

852

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Plasma membrane receptors can be endocytosed through clathrindependent and clathrin-independent pathways. Here, we show that the epidermal growth factor (EGF) receptor (EGFR), when stimulated with low doses of EGF, is internalized almost exclusively through the clathrin pathway, and it is not ubiquitinated. At higher concentrations of ligand, however, a substantial fraction of the receptor is endocytosed through a clathrin-independent, lipid raft-dependent route, as the receptor becomes ubiquitinated. An ubiquitination-impaired EGFR mutant was internalized through the clathrin pathway, whereas an EGFR͞ubiquitin chimera, that can signal solely through its ubiquitin (Ub) moiety, was internalized exclusively by the non-clathrin pathway. Non-clathrin internalization of ubiquitinated EGFR depends on its interaction with proteins harboring the Ub-interacting motif, as shown through the ablation of three Ub-interacting motif-containing proteins, eps15, eps15R, and epsin. Thus, eps15s and epsin perform an important function in coupling ubiquitinated cargo to clathrin-independent internalization.internalization ͉ rafts ͉ caveolae ͉ ubiquitination ͉ ubiquitin-interacting motif U biquitination is a posttranslational modification whereby substrate proteins are conjugated to a short highly conserved peptide, ubiquitin (Ub), through the action of Ub ligases (E3 enzymes). Polyubiquitination, in which a chain of Ub is appended, targets proteins to proteasomal degradation (1). However, when a single Ub moiety is appended (monoubiquitination), the modification functions as a signaling device through interactions with intracellular proteins harboring Ub-binding domains, such as the Ub-interacting motif (UIM) (2). In yeast, monoubiquitination has been known to act as an internalization signal for quite some time (3). In mammals, however, this connection has remained more elusive.We are interested in the mechanisms of internalization of receptor tyrosine kinases and, in particular, the epidermal growth factor receptor (EGFR). The EGFR is monoubiquitinated at multiple sites (4) through the action of the E3 enzyme Cbl. Although there is consensus on the function of Cbl and receptor ubiquitination in intracellular sorting of the EGFR, their role in the internalization step of endocytosis is less clear (5, 6). To gain insight into this issue, we generated a chimera in which the extracellular and transmembrane domains of the EGFR are fused to a mutant Ub (Ubmut), unable to form polyUb chains (EGFR͞Ubmut). With this chimera, we showed that ubiquitination is sufficient for internalization (4). The present studies were undertaken to elucidate the molecular mechanisms through which receptor ubiquitination directs internalization. Materials and MethodsTransfection and Biochemical Studies. Transfections were performed by using Lipofectamine or Oligofectamine (Invitrogen). For biochemical experiments, cells were serum-starved and then stimulated with EGF (100 ng͞ml, unless otherwise indicated) at 37°C. Lysis, immunoprecipitation, and immunoblot...

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Section: Discussionmentioning

confidence: 59%

Clathrin-independent endocytosis of ubiquitinated cargos

Sigismund

Woelk

Puri

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

736

852

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“…We have found that ubiquitination of the EGFR is necessary for its recruitment into coated pits (Stang et al, 2004). It has been found that ubiquitination of the EGFR by Cbl can occur both via the direct interaction between the EGFR and Cbl and via indirect interactions through Grb2 (Waterman et al, 2002).…”

Section: Ubiquitination and Endocytosismentioning

confidence: 92%

“…Epsin is localized to clathrin-coated pits (Stang et al, 2004;Hawryluk et al, 2006) and has been found to be involved in clathrin-mediated endocytosis Wendland et al, 1999). Whereas some authors have proposed that epsin functions as an adaptor in clathrin-mediated endocytosis (reviewed in Wendland, 2002), others have shown that epsin is involved in the formation of membrane curvature within clathrincoated pits.…”

Section: Additional Adaptor Proteins In Clathrin-mediated Endocytosismentioning

confidence: 99%

“…Inhibition of EGFR ubiquitination by overexpression of the Cbl-binding protein Sprouty, known to inhibit Cbl's interactions with E2 enzymes (Fong et al, 2003), was found to block the progression of the EGFR into clathrin-coated pits and to block endocytosis of the EGFR (Stang et al, 2004). However, others have found that Cbl-mediated ubiquitination is required for EGFR degradation, but not for internalization (Duan et al, 2003).…”

Section: Ubiquitination As Internalization Signal For Egfrmentioning

confidence: 99%

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Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

Grøvdal

Stang

Sorkin

et al. 2004

Experimental Cell Research

Self Cite

158

142

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“…de Melker et al 2004;Stang et al 2004;Sigismund et al 2005 membrane -cytosol interface (Jahnke et al 2012;Kajikawa et al 2012). Another major class of E3 ligases is the neural precursor-cell-expressed, developmentally down-regulated 4 (Nedd4) family, which contains a catalytic HECT (homologous E6-AP carboxy-terminal) domain (Rotin and Kumar 2009).…”

Section: Eps15mentioning

confidence: 99%

Ubiquitin-Dependent Sorting in Endocytosis

Piper

Đikić

Lukacs

2014

Cold Spring Harbor Perspectives in Biology

181

170

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When ubiquitin (Ub) is attached to membrane proteins on the plasma membrane, it directs them through a series of sorting steps that culminate in their delivery to the lumen of the lysosome where they undergo complete proteolysis. Ubiquitin is recognized by a series of complexes that operate at a number of vesicle transport steps. Ubiquitin serves as a sorting signal for internalization at the plasma membrane and is the major signal for incorporation into intraluminal vesicles of multivesicular late endosomes. The sorting machineries that catalyze these steps can bind Ub via a variety of Ub-binding domains. At the same time, many of these complexes are themselves ubiquitinated, thus providing a plethora of potential mechanisms to regulate their activity. Here we provide an overview of how membrane proteins are selected for ubiquitination and deubiquitination within the endocytic pathway and how that ubiquitin signal is interpreted by endocytic sorting machineries. HOW PROTEINS ARE SELECTED FOR UBIQUITINATIONU biquitin (Ub) is covalently attached to substrate proteins by the concerted action of E2-conjugating enzymes and E3 ligases (Varshavsky 2012). Most of the specificity and regulation of ubiquitination is at the level of the E3 ligases, which vastly outnumber the E2-conjugating enzymes. Ubiquitination results from the transfer of Ub, held either by the E2 or in some cases by the E3 as a high-energy thioester bond, onto a substrate protein, typically on the terminal amide of a substrate acceptor lysine side chain. Owing to the intense interest in the ubiquitination system, many of the simple rules and distinctions concerning different types of ligases, their specificity for forming particular polyUb chains, and even the kinds of residues in substrate proteins that can be ubiquitin modified, have been blurred with the discovery of mixed poly-Ub chains, new enzymatic mechanisms for Ub transfer, and ubiquitination of nonlysine residues (Kulathu and Komander 2012;Wenzel and Klevit 2012;McDowell and Philpott 2013). Nonetheless, in basic terms, Ub ligases function as platforms that coordinate substrate recognition with Ub transfer as well as configuring poly-Ub chain topology by the E2-conjugating enzyme. Substrates can be bound directly by the E3 ligase or by adaptor proteins that in turn bind to ligases, and selecEditors:

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Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

Cited by 145 publications

References 55 publications

Clathrin-independent endocytosis of ubiquitinated cargos

Clathrin-independent endocytosis of ubiquitinated cargos

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

Ubiquitin-Dependent Sorting in Endocytosis

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