Conjugation to Nedd8 Instigates Ubiquitylation and Down-regulation of Activated Receptor Tyrosine Kinases

Abstract

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.Growth factors and their transmembrane receptors, harboring intrinsic tyrosine kinase activity, play essential roles in cell fate determination. Whereas the process leading to growth factor-induced activation of signaling pathways is relatively well understood, mechanisms that initiate signal desensitization are only beginning to be unraveled (reviewed in Ref. 1). In the case of the epidermal growth factor receptor (EGFR) 2 and related receptor tyrosine kinases, the major signal attenuation process involves ligand-induced internalization of activated receptors and their sorting to degradation in lysosomes (reviewed in Ref.2). In analogy to the pivotal role played by phosphotyrosine and respective binding domains (e.g. Src homology domain 2) in positive signaling pathways, ubiquitin and ubiquitin-binding domains (e.g. ubiquitin-interacting motifs (UIMs)) regulate receptor endocytosis and sorting for lysosomal degradation (3). For example, recruitment of an E3 ubiquitin ligase, called c-Cbl (4 -7), enables subsequent conjugation of ubiquitin to multiple lysines of EGFR (8 -10). The appended ubiquitins are thought to recruit a set of endocytic proteins (e.g. Eps15) through their UIMs (reviewed in Ref. 11).In contrast to the well understood cellular functions of protein ubiquitylation, the roles played by ubiquitin-like proteins such as Nedd8 and SUMO are less characterized (12). Nedd8 is the closest kin of ubiquitin and it is linked to other proteins by an amide bond linking the carboxyl-terminal carboxylate to lysine residues. Neddylation is initiated by a heterodimeric complex comprising Uba3 and the amyloid precursor proteinbinding protein (APP-BP1). Ubc12, an E2-like component, then mediates conjugation of Nedd8 (13), but the identity and function of Nedd8-specific E3 ligases are less understood. Modification of Cullins, the first discovered neddylation substrate, is promoted by Roc1/Rbx, a RING finger protein (14, 15), which recruits Ubc12 (16, 17). Cullins are shared subunits of SCF (Skp1-Cdc53/CUL-F-box) complexes, assembled E3 ubiquitin ligases that regulate ubiquitylation of proteins involved primarily in cell cycle control. Neddylation of Cullin1 and Cullin2 activates their E3 ubiquitin ligase activity toward subs...

MLN4924 Is an Efficient Inhibitor of NEDD8 Conjugation in Plants

“…Neddylation was also reported for ribosomal proteins , and specifically L11 was shown to be deneddylated in response to nucleolar stress and to then relocalize from the nucleolus to the cytoplasm. In addition, other mammalian proteins such as APP1 (Lee et al, 2008), BCA3 (Gao et al, 2006), EGFR (Oved et al, 2006), and pVHL (Stickle et al, 2004;Russell and Ohh, 2008) have been described to be neddylated, and regulatory roles have been attributed to the NEDD8 modification. Future research will now allow for the confirmation and examination of the role of NEDD8 as a modifier of the set of putative NEDD8-modified proteins presented in this study.…”

A Targeted Proteomic Analysis of the Ubiquitin-Like Modifier Nedd8 and Associated Proteins

“…In contrast, multiple lysines of p53 and EGFR seem to be involved in neddylation. 16,18 Interestingly, in vitro neddylation of Cul-1 resulted in hyperneddylation by immunoblotting analysis, 19 although it is not known whether the hyperneddylation of Cul-1 is due to the formation of multiple mononeddylation or polyneddylation on Cul-1. Nevertheless, whether and how Nedd8 can form chain assemblies needs to be explored.…”

“…Recently, several noncullin proteins have been identified as Nedd8 substrates such as pVHL, p53, BCA3, and EGF receptor. [15][16][17][18] Neddylation of pVHL affects the activity of pVHL-containing ubiquitin ligases, whereas neddylation of p53 and BCA3 has an impact on transcriptional activities, and neddylation of EGFR is essential for ligand-induced down-regulation and degradation of EGFR.…”

The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis

“…Modification of CRLs by the ubiquitin-like Nedd8 (neural precursor cell-expressed developmentally downregulated protein 8), called neddylation, has been demonstrated to be essential for their activation 1,5 . In addition, non-cullin proteins such as p53, EGFR, pVHL, ribosomal proteins, Parkin, E2F-1 and TGFb II receptor have been shown to be neddylated as well [6][7][8][9][10][11][12][13] .…”