Smad7 Modulates Epidermal Growth Factor Receptor Turnover through Sequestration of c-Cbl

Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration, and tumorigenesis. For the maintenance of homeostasis, EGF signaling should be tightly regulated to prevent the aberrant activation. Smad7 has been known as inhibitory Smad that blocks the signal transduction of transforming growth factor β. In the process of cell proliferation or transformation, Smad7 has been shown the opposite activities as a promoter or suppressor depending on cell types or microenvironments. We found that the overexpression of Smad7 in human HaCaT keratinocyte cells and mouse skin tissues elevated EGF receptor (EGFR) activity by impairing ligand-induced ubiquitination and degradation of activated receptor, which is induced by the E3 ubiquitin ligase c-Cbl. The C-terminal MH2 region but not MH1 region of Smad7 is critical for interaction with c-Cbl to inhibit the ubiquitination of EGFR. Interestingly, wild-type Smad7, but not Smad6 or mutant Smad7, destabilized the EGF-induced complex formation of c-Cbl and EGFR. These data suggest a novel role for Smad7 as a promoter for prolonging the EGFR signal in keratinocyte and skin tissue by reducing its ligand-induced ubiquitination and degradation.     

A Comprehensive,Multi-Scale Dynamical Model of ErbB Receptor Signal Transduction in Human Mammary Epithelial Cells

The non-receptor tyrosine kinase Src and receptor tyrosine kinase epidermal growth factor receptor (EGFR/ErbB1) have been established as collaborators in cellular signaling and their combined dysregulation plays key roles in human cancers, including breast cancer. In part due to the complexity of the biochemical network associated with the regulation of these proteins as well as their cellular functions, the role of Src in EGFR regulation remains unclear. Herein we present a new comprehensive, multi-scale dynamical model of ErbB receptor signal transduction in human mammary epithelial cells. This model, constructed manually from published biochemical literature, consists of 245 nodes representing proteins and their post-translational modifications sites, and over 1,000 biochemical interactions. Using computer simulations of the model, we find it is able to reproduce a number of cellular phenomena. Furthermore, the model predicts that overexpression of Src results in increased endocytosis of EGFR in the absence/low amount of the epidermal growth factor (EGF). Our subsequent laboratory experiments also suggest increased internalization of EGFR upon Src overexpression under EGF-deprived conditions, further supporting this model-generated hypothesis.     

Rab21 attenuates EGF-mediated MAPK signaling through enhancing EGFR internalization and degradation

Epidermal growth factor (EGF) receptor (EGFR) signal transduction is regulated by endocytosis where many Rab proteins play an important role in the determination of the receptor recycle or degradation. In an effort to better understand how EGF signaling is regulated, we examined the role of Rab21 in regulation of the degradation and signal transduction of the EGFR. Using a transient expression protocol in HEK293T and HeLa cells, we found that Rab21 enhanced the degradation of EGFR through accelerating its internalization in both EGF-independent and EGF-dependent manners. We further demonstrated that Rab21 interacted with EGFR by immunoprecipitation experiments. Interestingly, we observed that overexpression of Rab21 attenuated EGF-mediated mitogen-activated protein kinase (MAPK) signaling by inducing EGFR degradation. Taken together, these data suggest that Rab21 plays a negative role in the EGF-mediated MAPK signaling pathway.     

Magnitude of Ubiquitination Determines the Fate of Epidermal Growth Factor Receptor Upon Ligand Stimulation

Receptor tyrosine kinases (RTK) bind growth factors and are critical for cell proliferation and differentiation. Their dysregulation leads to a loss of growth control, often resulting in cancer. Epidermal growth factor receptor (EGFR) is the prototypic RTK and can bind several ligands exhibiting distinct mitogenic potentials. Whereas the phosphorylation on individual EGFR sites and their roles for downstream signaling have been extensively studied, less is known about ligand-specific ubiquitination events on EGFR, which are crucial for signal attenuation and termination. We used a proteomics-based workflow for absolute quantitation combined with mathematical modeling to unveil potentially decisive ubiquitination events on EGFR from the first 30 seconds to 15 minutes of stimulation. Four ligands were used for stimulation: epidermal growth factor (EGF), heparin-binding-EGF like growth factor, transforming growth factor-α and epiregulin. Whereas only little differences in the order of individual ubiquitination sites were observed, the overall amount of modified receptor differed depending on the used ligand, indicating that absolute magnitude of EGFR ubiquitination, and not distinctly regulated ubiquitination sites, is a major determinant for signal attenuation and the subsequent cellular outcomes.     

Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry

Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.     

An integrated model of epidermal growth factor receptor trafficking and signal transduction

Endocytic trafficking of many types of receptors can have profound effects on subsequent signaling events. Quantitative models of these processes, however, have usually considered trafficking and signaling independently. Here, we present an integrated model of both the trafficking and signaling pathway of the epidermal growth factor receptor (EGFR) using a probability weighted-dynamic Monte Carlo simulation. Our model consists of hundreds of distinct endocytic compartments and approximately 13,000 reactions/events that occur over a broad spatio-temporal range. By using a realistic multicompartment model, we can investigate the distribution of the receptors among cellular compartments as well as their potential signal transduction characteristics. Our new model also allows the incorporation of physiochemical aspects of ligand-receptor interactions, such as pH-dependent binding in different endosomal compartments. To determine the utility of this approach, we simulated the differential activation of the EGFR by two of its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). Our simulations predict that when EGFR is activated with TGF-alpha, receptor activation is biased toward the cell surface whereas EGF produces a signaling bias toward the endosomal compartment. Experiments confirm these predictions from our model and simulations. Our model accurately predicts the kinetics and extent of receptor downregulation induced by either EGF or TGF-alpha. Our results suggest that receptor trafficking controls the compartmental bias of signal transduction, rather than simply modulating signal magnitude. Our model provides a new approach to evaluating the complex effect of receptor trafficking on signal transduction. Importantly, the stochastic and compartmental nature of the simulation allows these models to be directly tested by high-throughput approaches, such as quantitative image analysis.     

Ubiquitin-binding domains

Ubiquitin-binding domains (UBDs) are a collection of modular protein domains that non-covalently bind to ubiquitin. These recently discovered motifs interpret and transmit information conferred by protein ubiquitylation to control various cellular events. Detailed molecular structures are known for a number of UBDs, but to understand their mechanism of action, we also need to know how binding specificity is determined, how ubiquitin binding is regulated, and the function of UBDs in the context of full-length proteins. Such knowledge will be key to our understanding of how ubiquitin regulates cellular proteins and processes.     

Signaling-mediated control of ubiquitin ligases in endocytosis

Ubiquitin-dependent regulation of endocytosis plays an important part in the control of signal transduction, and a critical issue in the understanding of signal transduction therefore relates to regulation of ubiquitination in the endocytic pathway. We discuss here what is known of the mechanisms by which signaling controls the activity of the ubiquitin ligases that specifically recognize the targets of ubiquitination on the endocytic pathway, and suggest alternative mechanisms that deserve experimental investigation.     

A network model of early events in epidermal growth factor receptor signaling that accounts for combinatorial complexity

We consider a model of early events in signaling by the epidermal growth factor (EGF) receptor (EGFR). The model includes EGF, EGFR, the adapter proteins Grb2 and Shc, and the guanine nucleotide exchange factor Sos, which is activated through EGF-induced formation of EGFR-Grb2-Sos and EGFR-Shc-Grb2-Sos assemblies at the plasma membrane. The protein interactions involved in signaling can potentially generate a diversity of protein complexes and phosphoforms; however, this diversity has been largely ignored in models of EGFR signaling. Here, we develop a model that accounts more fully for potential molecular diversity by specifying rules for protein interactions and then using these rules to generate a reaction network that includes all chemical species and reactions implied by the protein interactions. We obtain a model that predicts the dynamics of 356 molecular species, which are connected through 3749 unidirectional reactions. This network model is compared with a previously developed model that includes only 18 chemical species but incorporates the same scope of protein interactions. The predictions of this model are reproduced by the network model, which also yields new predictions. For example, the network model predicts distinct temporal patterns of autophosphorylation for different tyrosine residues of EGFR. A comparison of the two models suggests experiments that could lead to mechanistic insights about competition among adapter proteins for EGFR binding sites and the role of EGFR monomers in signal transduction.     

FBXW7/hCDC4 controls glioma cell proliferation in vitro and is a prognostic marker for survival in glioblastoma patients

Ubiquitin is a highly versatile post-translational modification that controls virtually all types of cellular events. Over the past ten years we have learned that diverse forms of ubiquitin modifications and of ubiquitin binding modules co-exist in the cell, giving rise to complex networks of protein:protein interactions. A central problem that continues to puzzle ubiquitinologists is how cells translate this myriad of stimuli into highly specific responses. This is a classical signalling problem. Here, we draw parallels with the phosphorylation signalling pathway and we discuss the expanding repertoire of ubiquitin signals, signal tranducers and signalling-regulated E3 enzymes. We examine recent advances in the field, including a new mechanism of regulation of E3 ligases that relies on ubiquitination.     

UMI, a novel RNF168 ubiquitin binding domain involved in the DNA damage signaling pathway

Ubiquitination regulates important cellular processes, including the DNA damage response (DDR) and DNA repair. The complexity of the ubiquitin-mediated signals is decoded by ubiquitin receptors, which contain protein modules named ubiquitin binding domains (UBDs). We previously identified a new ubiquitin ligase, RNF168, involved in DDR and endowed with two UBDs named MIU (motif interacting with ubiquitin). Here we have provided the identification of a novel UBD, the UMI (UIM- and MIU-related UBD), present in RNF168, and characterized the interaction surface with ubiquitin, centered on two Leu residues. We have demonstrated that integrity of the UMI, in addition to the MIUs, is necessary for the proper localization of RNF168 and for ubiquitination of nuclear proteins, including histone H2A. Finally, we have shown that simultaneous inactivation of UMI and MIUs prevents the recruitment to DDR foci of the crucial downstream mediator 53BP1.     

Roles of various cullin-RING E3 ligases involved in hormonal and stress responses in plants

Post-translational modification plays an important role in the regulation of protein stability, enzyme activity, and the cellular localization of proteins. Ubiquitination is a representative post-translational modification in eukaryotes that is mainly responsible for protein degradation. There have been a number of reports on the role of ubiquitination in various cellular responses in plants, such as regulation of the cell division cycle, stress responses and hormonal signaling. Among the three types of ubiquitination-related enzymes, E3 ubiquitin ligase is critical in determining substrate specificity. The importance of cullin-RING E3 ligase (CRL), a type of E3 ligase, has been emphasized during the recent decade due to its large number and its involvement in various plant cellular processes. Here, we describe how CRL E3 ligase complexes are involved in cellular events mediated by plant hormones and during plant stress adaptation while focusing on their substrate receptors.     

蛋白质的泛素化修饰在细胞应激反应中的作用

泛素是真核细胞内广泛存在的一种高度保守的蛋白质。在特定泛素化酶催化下实现的蛋白质泛素化修饰反应能够高选择性地降解细胞中的特定信号蛋白质,对维持细胞正常的生理功能具有非常重要的作用。另外,某些泛素化修饰反应也能够实现与蛋白质降解无关的功能调控作用。p53、NF-κB和GADD45α是在细胞应激损伤反应中具有广泛调控作用的信号蛋白,发生在这些分子上的泛素化修饰反应是它们发挥相关分子机制的重要基础。     

The RING finger of c-Cbl mediates desensitization of the epidermal growth factor receptor.

Ligand-induced activation of surface receptors, including the epidermal growth factor receptor (EGFR), is followed by a desensitization process involving endocytosis and receptor degradation. c-Cbl, a tyrosine phosphorylation substrate shared by several signaling pathways, accelerates desensitization by recruiting EGFR and increasing receptor polyubiquitination. Here we demonstrate that the RING type zinc finger of c-Cbl is essential for ubiquitination and subsequent desensitization of EGFR. Mutagenesis of a single cysteine residue impaired the ability of c-Cbl to enhance both down-regulation and ubiquitination of EGFR in living cells, although the mutant retained binding to the activated receptor. Consequently, the mutant form of c-Cbl acquired a dominant inhibitory function and lost the ability to inhibit signaling downstream to EGFR. In vitro reconstitution of EGFR ubiquitination implies that the RING finger plays an essential direct role in ubiquitin ligation. Our results attribute to the RING finger of c-Cbl a causative role in endocytic sorting of EGFR and desensitization of signal transduction.     

Interactions between the three CIN85 SH3 domains and ubiquitin: implications for CIN85 ubiquitination

CIN85 is an adaptor protein linking the ubiquitin ligase Cbl and clathrin-binding proteins in clathrin-mediated receptor endocytosis. The SH3 domains of CIN85 bind to a proline-rich region of Cbl. Here we show that all three SH3 domains of CIN85 bind to ubiquitin. We also present a data-based structural model of the CIN85 SH3-C domain in complex with ubiquitin. In this complex, ubiquitin binds to the canonical interaction surface of the SH3 domain for proline-rich ligands and mimics the PPII helix, and we provide evidence that ubiquitin competes with these ligands for binding. We demonstrate that disruption of ubiquitin binding results in constitutive ubiquitination of CIN85 and an increased level of ubiquitination of EGFR in the absence of EGF stimulation. These results suggest that competition between Cbl and ubiquitin binding to CIN85 regulates Cbl function and EGFR endocytosis.     

Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits

Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitin-interacting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.