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Mechanisms controlling EGF receptor endocytosis and degradation I. Dikic1 Institute of Biochemistry II, Goethe University Medical School, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
Abstract Activated EGF (epidermal growth factor) receptors are removed from the cell surface via endocytosis and subsequent degradation in the lysosome. This ultimately attenuates EGF receptor signals and diminishes the level of cell activation. The mechanisms underlying the EGF receptor down-regulation are beginning to be elucidated at the molecular level. Recent reports have indicated that receptor monoubiquitination and networks of protein–protein interactions control distinct steps in EGF receptor internalization, endosomal trafficking and sorting for lysosomal degradation. The emerging importance of the ubiquitin ligase Cbl and the adaptor molecule CIN85 (Cbl-interacting protein of 85 kDa) in the regulation of these pathways is discussed in detail.
Introduction Growth factor receptors control a wide variety of biological processes including cell proliferation, differentiation, survival and migration [1]. One such example is the EGFR (epidermal growth factor receptor) that plays important roles in physiological and pathological processes in epithelial cells [1]. The mechanisms of EGFR (EGF receptor) signalling and down-regulation are probably the best understood among growth factor receptors in mammalian cells (reviewed in [1,2]). Ligand binding to EGFRs initiates a series of biochemical events, including receptor activation and autophosphorylation, phosphorylation of cellular proteins and the formation of protein–protein networks. In such a way, the external message is converted into a biochemical signature in the cell that eventually determines whether the cell differentiates, moves, divides or dies. The duration and strength of these signals are tightly regulated in the cell by the action of numerous negative regulatory mechanisms [3]. For example, protein phosphatases may interfere with the amplitude and kinetics of the growth factor receptor signals, thereby modulating the biological responses. On the other hand, terminal signal inactivation is accomplished by endocytosis and degradation of activated receptors and associated signalling proteins. These processes are essential to avoid constitutive signalling and tumorigenesis. Following ligand binding, EGFRs are rapidly internalized from the cell surface via several pathways, including clathrin-coated pits [4]. Internalized receptors are initially delivered to early endosomes, which in turn mature into late endosomes and multivesicular bodies [4]. In these structures, EGFRs
Key words: Cbl, CIN85, endocytosis, epidermal growth factor receptor (EGF receptor), ubiquitination. Abbreviations used: EGF, epidermal growth factor; EGFR, EGF receptor; RTK, receptor tyrosine kinase; CIN85, Cbl-interacting protein of 85 kDa; SH3, Src homology 3. 1 e-mail
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undergo sorting, and are either recycled back to the plasma membrane or directed to the lysosome for destruction [4]. A significant body of evidence indicates that endocytosis of EGFRs requires multiple signals, including intrinsic tyrosine kinase activity, endocytic sequence motifs located in the cytoplasmic domain of the receptor, receptor ubiquitination as well as numerous signals in endosomes that direct receptors for lysosomal degradation. These mechanisms have been excellently covered in recent reviews [4–6]. In this report I will discuss in more detail the emerging functions of the ubiquitin ligase Cbl in the regulation of EGFR downmodulation. Cbl directs ligand-dependent EGFR monoubiquitination and also recruits the adaptor molecule CIN85 (Cbl-interacting protein of 85 kDa) to EGFR complexes, thus controlling multiple steps of EGFR trafficking in the endosome (Figure 1).
Ubiquitination and EGFR endocytosis Ubiquitin is an evolutionarily conserved protein that may be covalently attached to another protein, via a process known as ubiquitination [7]. Ubiquitination is mediated in three steps by ubiquitin-activating (E1), -conjugating (E2) and -ligase (E3) enzymes, giving rise to an isopeptide bond between the C-terminal glycine (Gly-76) of ubiquitin and the ε-amino group of a lysine in the substrate [7]. The specificity in the process is mainly mediated by the E3 enzyme, which recognizes and interacts with the substrate. Attachment of a single ubiquitin to a single lysine or to several lysine residues of proteins is referred to as monoubiquitination or multiple monoubiquitination, respectively [8]. On the other hand, the formation of a ubiquitin chain by the iterative addition of single ubiquitin molecules to ubiquitin itself results in polyubiquitination [7,8]. The type of ubiquitin modification largely determines the fate of the ubiquitinated proteins. Monoubiquitination is involved in endocytosis of
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Figure 1 Role of Cbl and CIN85 in EGFR endocytosis Ligand binding to the EGFR leads to its dimerization and autophosphorylation, followed by binding of Cbl to the activated EGFR. Cbl promotes monoubiquitination of the activated EGFR at the plasma membrane and thereby regulates receptor endocytosis. In complex with the EGFR, Cbl becomes phosphorylated at tyrosine residues in the C-terminal part. This leads to a conformational change of Cbl, which enhances the interaction between specific proline-arginine motifs in Cbl/Cbl-b and the three SH3 domains of CIN85. CIN85, in turn, recruits endophilins to EGFR–Cbl complexes, which regulate EGFR endocytosis by changing the curvature of the plasma membrane. Moreover, Cbl mediates monoubiquitination of CIN85 in the complex with activated EGFRs. Thus, Cbl promotes EGFR endocytosis both by mediating receptor monoubiquitination and by recruiting CIN85–endophilin complexes to activated EGFRs.
plasma membrane proteins, histone regulation and budding of retroviruses from the plasma membrane [8], whereas a ubiquitin chain consisting of at least four ubiquitins is the recognition signal for proteasomal degradation [7]. Ligand-induced ubiquitination of EGFRs has been linked to their internalization and endocytosis. The Cbl family of ubiquitin ligases plays pivotal roles in these processes [9]. Cbl can bind directly to phosphorylated EGFRs via its TKB (tyrosine kinase-binding) domain, while the RING finger domain of Cbl recruits ubiquitin-conjugating enzymes (E2, Ubc) and mediates the transfer of ubiquitin to the receptor (reviewed in [6]). Cbl overexpression enhances EGFR ubiquitination and leads to increased degradation of the activated receptors. Recent lines of evidence show that the ubiquitin ligase Cbl mediates monoubiquitination of EGFRs and that ubiquitin carries both internalization and degradation signals that control endocytic trafficking and sorting of receptors for degradation in the lysosome [10,11]. These findings have provided a molecular explanation of why activated RTKs (receptor tyrosine kinases), which were long thought to be polyubiquitinated, do not undergo proteasomal degradation, but rather follow a trafficking route in the endosome. Cblinduced monoubiquitination of EGFRs is likely to take place at the plasma membrane, since Cbl can ubiquitinate EGFRs under conditions where endocytosis is impaired by low temperature or by expression of a GTPase-defective mutant of dynamin [12,13]. It is thus possible that Cbl-directed monoubiquitination of EGFRs provides initial signals
involved in early steps of receptor endocytosis. This is in line with previous studies showing that a monoubiquitin signal is sufficient for internalization of plasma membrane receptors in yeast [14] and mammalian cells [15]. Cbl also remains associated with the EGFR in early and late endosomes [13,16]. Along this pathway, RTKs may be subjected to multiple cycles of Cbl-mediated transfer of monoubiquitin, leading to their multiubiquitination [10]. How many monoubiquitins are attached to the activated EGFR is not known, but it is probable that it is a limited number [10], possibly attached to the lysines present in its kinase domain [11]. In addition to activated EGFRs, receptor-associated proteins may also be ubiquitinated, thus amplifying the ubiquitin network. EGF stimulation induces monoubiquitination of receptor-associated CIN85 adaptor molecules and endocytic accessory proteins Eps15 (EGFR-pathway substrate 15) [17] and Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) [18]. Eps15 and Hrs act as ubiquitin receptors in the endosome and their monoubiquitination can affect the ability of these proteins to function as gating receptors for ubiquitinated cargo [19]. Despite a large number of studies on ubiquitination of EGFRs, many questions still remain to be answered. In particular, what defines the specificity in determining mono-, multi- or poly-ubiquitination? Why are there multiple monoubiquitination signals attached to activated EGFRs and what is the role of monoubiquitination of accessory endocytic proteins in controlling endosomal sorting of EGFRs? The current interest in these topics makes C 2003
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certain that before too long many of these questions will be answered.
Cbl/CIN85 pathways in EGFR endocytosis Cbl was also shown to promote receptor internalization via a pathway that is functionally separable from its ubiquitin ligase activity and is dependent on Cbl interactions with the adaptor protein CIN85 [6] (Figure 1). CIN85 was independently identified by several groups as Cbl-interacting protein of 85 kDa [20], Ruk (regulator of ubiquitous kinase) [21], SETA [SH3 (Src homology 3) domain-containing gene expressed in tumorigenic astrocytes] [22] and SH3KBP1 (SH3-domain kinase-binding protein 1) [23]. CIN85 occurs in different isoforms due to alternative splicing and differential usage of promoters [24,25]. The longest form of CIN85 is composed of three N-terminal SH3 domains, a central proline-rich domain and at the C-terminus a coiledcoil domain [24]. CIN85 interacts with two members of the Cbl family, Cbl and Cbl-b, but not Cbl-3, and the binding is determined by the presence of a proline-arginine motif in their distal C-terminal tails [26]. CIN85 binding to Cbl is mediated via its SH3 domains and is enhanced by EGF-induced tyrosine phosphorylation of Cbl, while the proline-rich region of CIN85 constitutively interacts with endophilins, the regulatory components of clathrin-coated pits [27]. Endophilins can bind to lipid bilayers and induce local changes in membrane curvature [28]. They associate with other accessory proteins such as dynamin, synaptojanin and amphiphysin, participating in the formation of clathrincoated vesicles [28]. CIN85 was proposed to rapidly recruit endophilin to the activated receptor complex, thus controlling receptor internalization [27,29]. Inhibition of the Cbl–CIN85–endophilin interaction was sufficient to block EGFR endocytosis and degradation, without perturbing the ability of Cbl to ubiquitinate activated receptors [27]. This mechanism seems to be common for other RTKs, as the involvement of the Cbl–CIN85–endophilin pathway has been demonstrated in the case of hepatocyte growth factor receptor, platelet-derived growth factor receptor and c-Kit [26,29]. In addition, it has been proposed that CIN85 acts as a scaffolding protein able to recruit signalling proteins into RTK-associated complexes that are critical for RTK trafficking at multiple steps [24]. All three SH3 domains of CIN85 bind to PXXXPR motifs of Cbl/Cbl-b with high specificity and relatively low affinity, thus enabling fullsize CIN85 to interact simultaneously with multiple Cbl molecules, promoting clustering of Cbl–EGFR complexes in mammalian cells [30]. In addition, the PXXXPR motif is found in a number of other CIN85 effectors (K. Kowanetz and I. Dikic, unpublished work). The relatively low affinity of individual CIN85 SH3 domains could allow for rapid exchange of CIN85 binding partners, depending on their local concentration, cellular compartmentalization, or posttranslational modifications in response to changes in the environment, such as tyrosine phosphorylation of Cbl upon receptor activation. In addition, following EGF stimulation C 2003
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Cbl was shown to mediate monoubiquitination of CIN85, which can be important for endocytic sorting of internalized EGFR complexes [31]. Ubiquitinated forms of CIN85, Cbl and EGFR were found in the same complex and were subjected to concomitant degradation in the lysosome [31]. It remains to be investigated further whether CIN85 binding and ubiquitination play additional roles in EGFR downregulation and signalling.
Conclusions EGFR endocytosis and degradation are regulated at various steps. In particular, the Cbl family of ubiquitin ligases plays a critical role in these processes by enhancing ubiqutination of activated EGFRs and recruiting CIN85 adaptor molecules. In this way, Cbl modulates downstream EGFR signalling by controlling receptor levels and trafficking in the cell. Many thanks to Kaisa Haglund and Iwona Szymkiewicz for comments on and critical reading of the manuscript.
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