EGF receptor endocytosis and degradation

The major interest of our current work is to understand the mechanisms involved in negative regulation of EGF receptor signaling pathways (Dikic and Giordano, 2003). Terminal EGFR 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. Ligand-induced ubiquitination of EGF receptors has been linked to their negative regulation by internalization and endocytic targeting to destruction in the lysosome. The Cbl family of ubiquitin ligases plays pivotal roles in these processes (Dikic et al. 2003). Cbl can directly bind to phosphorylated EGF receptors via its tyrosine kinase binding (TKB) domain, while the RING finger domain of Cbl recruits ubiquitin-conjugating enzymes (E2, Ubc) and mediates the transfer of ubiquitin to the receptor.

Figure 1. Members of the Cbl protein family. Domain structure of mammalian Cbl, Cbl-b and Cbl-c, Drosophila melanogaster D-Cbl short and long as well as Caenorhabditis elegans SLI-1 are shown.

Recent lines of evidence show that Cbl mediates monoubiquitination of EGF receptors and that ubiquitin carries both internalization and degradation signals that control trafficking and sorting of receptors for degradation in the lysosome (Haglund et al. 2003). In addition, we have recently discovered a novel pathway by which Cbl and Cbl-b regulate internalization of the EGF receptor (Soubeyran et al, 2002; Szymkiewicz et al., 2002). This pathway is functionally separable from the ubiquitin ligase activity of Cbl and it depends on binding of the adaptor protein CIN85 to Cbl and recruitment of endophilins in complexes with activated EGF receptors (Dikic, 2002; Soubeyran et al, 2002; Szymkiewicz et al., 2002). Subsequently, we have shown that CIN85 associates with Cbl/receptor complexes along the endocytic pathway, where Cbl directs monoubiquitination of CIN85 (Haglund et al, 2002). These events seem to be critical for proper routing of EGF receptor complexes for degradation in the lysosomal compartment (Haglund et al, 2002).

Figure 2. Intracellular trafficking of activated receptors.
The activation of EGF receptors by EGF induces its phosphorylation and the recruitment of Cbl that mediates its multi-monoubiquitination. This process is rapidly followed by internalization of activated receptors/Cbl complexes and their subsenquent trafficking toward various intracellular compartments leading, in fine, to either the recycling of the receptors to the cell surface or their concomitant degradation in the lysosome. This mechanisms are regulated by divers proteins able to interact with ubiquitin moeties via specific domains (UIM:Ubiquitin Interacting Motif; UBA:UBiquitin Associated; UBC:UBiquitin Conjugating enzyme like) and that can also undergo mono-ubiquitination.

Major questions we are currently addressing deal with the role of monoubiquitin signals in endocytic sorting of cargo (i.e EGFR and associated receptor complexes) as well as functions of ubiquitin binding proteins that serve as ubiquitin receptors along the endosome (see Haglund et al. 2003b). In addition, we are particularly interested to understand the mechanisms which define the specificity in determining mono-, multi- or poly-ubiquitination of distinct Cbl-substrates?

Ubiquitin signals in inflammatory pathways

Ubiquitin signals are deeply involved in the inflammatory pathways activated by various stimuli, such as cytokines (TNF-α, IL-1β), bacterial proteins (LPS) and viral infection.

In response to such stimuli, NF-κB, JNK and MAPK pathways are activated in cells. Among these signaling pathways, NF-κB signaling requires various types of ubiquitin signals for its activation.

For example, in the TNFR signaling pathway, there are 3 steps where ubiquitin signals plays a role (see figure). First, adaptor molecules, such as RIPK1 and TRAFs are modified by Lys63-linked ubiquitin chains. This modification attracts TAB2, which binds specifically to Lys63-linked ubiquitin chains, and forms a complex with TAK1. This complex formation is required for downstream activation. Secondly, a subunit of the IKK complex, NEMO, needs to interact with linear ubiquitin chains to activate NF-κB. Thirdly, an inhibitory molecule, IκBα is modified by Lys48-linked ubiquitin chains that leads the protein to proteasomal degradation. This enables p65/p50 to translocate into the nucleus to activate transcription of target genes.

Our recent discovery of linear-ubiquitin chain recognition by NEMO was very striking because it was totally different from the previous textbook knowledge of NF-κB activation mechanisms. This was the first crystal structure of a ubiquitin chain with its specific domain UBAN present in NEMO and ABIN proteins. With this discovery of linear-ubiquitin involvement in NF-κB signaling, our major focus now is to explore how linear-ubiquitin chains regulate the inflammatory signaling pathway in a broader perspective.

The topics that we want to address are,

1. What are the linear ubiquitin specific E3 ligases/DUBs, which control inflammatory signaling?
2. What are the target proteins (substrates) of linear ubiquitylation in the inflammatory signaling pathway?
3. Which proteins bind to linear ubiquitin in regulation of inflammatory signaling?
4. Can we create ubiquitin sensors to detect in vivo linear ubiquitination of substrates?

References:

1. Nicola Crosetto, David Komander, Ivan Dikic. Ubiquitin signalling by ubiquitin-binding domains. Nat Rev Mol Cell Biol. 2010;11(5).
   

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2. Rahighi S, Ikeda F, Kawasaki M, Akutsu M, Suzuki N, Kato R, Kensche T, Uejima T, Bloor S, Komander D, Randow F, Wakatsuki S, Dikic I. Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation. Cell. 2009;136(6):1098-109.
3. Dikic I, Wakatsuki S, Walters KJ. Ubiquitin-binding domains - from structures to functions. Nat Rev Mol Cell Biol. 2009;10(10):659-71.
4. Nagy V, Dikic I. Ubiquitin ligase complexes: from substrate selectivity to conjugational specificity Biol Chem. 2010;391(2-3):163-9.
5. Ikeda F, Dikic I. Atypical ubiquitin chains: new molecular signals. 'Protein Modifications: Beyond the Usual Suspects' review series. EMBO Rep. 2008;9(6):536-42.
6. Terzic J, Marinovic-Terzic I, Ikeda F, Dikic I. Ubiquitin signals in the NF-kappaB pathway. Biochem Soc Trans. 2007;35(Pt 5):942-5.

Specificity in Ubiquitin signaling

Ubiquitin is a highly conserved 76-amino-acid polypeptide that is covalently attached to target proteins via an isopeptide bond between the carboxyl-terminal glycine of ubiquitin and the e-amino group of a lysine in substrate proteins. This occurs through a three-step process involving ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-ligating (E3) enzymes (Hofmann and Pickart, 2001). The addition of a single ubiquitin molecule to a substrate is defined as monoubiquitination (Figure 1) (Dunn and Hicke, 2001). Alternatively, several lysine residues in the substrate can be tagged with single ubiquitin molecules, giving rise to multiple monoubiquitination, also referred to as multiubiquitination (Figure 1) (Dikic et al., 2003; Haglund et al., 2003; Mosesson et al., 2003). Moreover, ubiquitin contains seven lysine residues, which can also be targeted by another ubiquitin in an iterative process, known as polyubiquitination, that leads to the formation of a ubiquitin chain attached to a single lysine of a protein substrate (Figure 1) (Hofmann and Pickart, 2001). It is now clear that different types of ubiquitin conjugates are involved in the regulation of different cellular processes (Figure 1). Monoubiquitination is implicated in endocytosis of plasma membrane proteins, sorting of proteins to multivesicular bodies (MVB), budding of retroviruses, DNA repair, histone activity and transcriptional regulation (Dunn and Hicke, 2001). On the other hand, polyubiquitin chains formed via lysine 48 of two ubiquitins have a well characterized role in targeting proteins to degradation by the 26S proteasome, while ubiquitin chains formed through lysine 63 are involved in endocytosis processes and DNA repair (Ettenberg et al., 2001; Hofmann and Pickart, 2001).

The wide variety of proteins known to be modified by Ub in cells points to the existence of a large Ub-linked network participating in numerous cellular processes. The recent identification of Ub-binding domains found in proteins with known function in diverse biological processes has provided some insights in how specific Ub-interactors may regulate Ub functions in cells. Currently known domains able to directly interact with Ub include UIM (ubiquitin-interacting motif), UBA (ubiquitin-associated domain), UBC (ubiquitin-conjugating enzyme-like)/UEV (ubiquitin E2 variant), CUE (Cue1-homologous), PAZ (polyubiquitin-associated zinc finger) and NZF (novel zinc finger) domain (recently reviewed in DiFiore et al 2003 and Schnell and Hicke, 2003).

Ub-binding domains have different structural folds recognizing distinct patches on the Ub surface. They are thus able to bind Ub with different affinities and/or may specifically recognize different Ub modification such as Ub-monomer versus Ub-chains (Di Fiore et al., 2003; Schnell and Hicke, 2003). Several recent studies have suggested that Ub-binding proteins may participate in the control of Ub-dependent physiological and pathological processes, including intracellular transport, DNA repair, gene transcription or silencing, HIV budding, and neurological disorders, like Alzheimer and Parkinson (Di Fiore et al., 2003; Schnell and Hicke, 2003). However, much more work remains to be done to prove that interactions between ubiquitinated substrates and Ub-binding proteins are essential for regulation of these processes in particular in vivo. We are currently analyzing functional roles of several Ub-binding proteins by inducible expression of small interfering RNA in embryonic stem (ES) cells. The siRNA target vectors will be stably integrated into the genome of ES cells that can be differentiated into neuronal and hematopoietic cell types, and following induction of siRNA the effects on processes like apoptosis, proliferation, cell cycle, protein degradation and gene transcription will be examined. Taking advantage of the totipotency of ES cells, we will be able to generate transgenic mice from those cell lines that exhibit the most significant phenotypes.

Regulation of translesion DNA synthesis by ubiquitin

Genome stabilty relies on ubiquitin (Ub) signalling. Many pathways responsible for the maintanance of the stability use Ub as means to initiate and/or propagate signalling. We are interested in understanding the roles of Ub networks in DNA damage response
and so far we have been focusing on a pathway of damage tolerance called translesion DNA synthesis (TLS) as a model. As we are far from understanding all the functions of Ub in DNA damage responses, a lot of challenges stand in front of us and we would like
to contribute to solve some of them. Below are examples of the currently known functions of Ub and Ub-binding domains (UBD) in DNA damage responses, with some of the questions that intrigue us most in red.

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Reference:
1. Bienko M, Green CM, Crosetto N, Rudolf F, Zapart G, Coull B, Kannouche P, Wider G, Peter M, Lehmann AR, Hofmann K, Dikic I. Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science. 2005 Dec

2. Guo C, Tang TS, Bienko M, Parker JL, Bielen AB, Sonoda E, Takeda S, Ulrich HD, Dikic I, Friedberg EC. Ubiquitin-binding motifs in REV1 protein are required for its role in the tolerance of DNA damage. Mol Cell Biol. 2006 Dec

3. Crosetto N, Bienko M, Dikic I. Ubiquitin hubs in oncogenic networks. Mol Cancer Res. 2006 Dec

4. Guo C, Tang TS, Bienko M, Dikic I, Friedberg EC. Requirements for the interaction of mouse Pol kappa with ubiquitin and its biological signicance. J Biol Chem 2008 Feb

5. Crosetto N, Bienko M, Hibbert RG, Perica T, Ambrogio C, Kensche T, Hofmann K, Sixma TK, Dikic I. Human Wrnip1 is localized in replication factories in a ubiquitin-binding zinc nger-dependent manner. J Biol Chem 2008 Dec

6. Sabbioneda S, Green CM, Bienko M, Kannouche P, Dikic I, Lehmann AR. Ubiquitin-binding motif of human DNA polymerase eta is required for correct localization. PNAS 2009 Feb.

7. Bomar MG, D’Souza S, Bienko M, Dikic I, Walker G, Zhou P. Unconventional Ubiquitin Recognition by the Ubiquitin-Binding Motif within the Y-Family DNA Polymerases iota and Rev1. Mor Cell 2010 Feb

8. Bienko M, Green CM, Sabbioneda S, Crosetto N, Matic I, Hibbert RG, Begovic T, Niimi A, Mann M, Lehmann AR, Dikic I. Regulation of translesion synthesis DNA polymerase η by Monoubiquitination. Mol Cell 2010 Feb

Selectivity and specificity in autophagy

Autophagy was first described as cellular response to starvation and describes the general process of delivery of cargo to lysosomes for degradation.Macroautophagy (henceforth "autophagy"), is the most prevalent form and involves the formation of double-membrane vesicles (autophagosomes) that sequester portions of the cytoplasm and eventually fuse with the lysosome, where their cargo is degraded (Klionsky, 2005)

Our group is currently interested in the discovery of novel autophagy receptors important for selectivity of cargo delivery, as well as delivery and fusion of mature autophagosomes to the lysosomes. Using the ubiquitin-like autophagy proteins, Atg8 (GABARAP, GATE-16 and LC3 in mammals), which can be conjugated to phosphatidylethanolamine (PE) in a process similar to ubiquitin conjugation, we carried out an extensive yeast-2-hybrid screen to identify novel receptors. Since Atg8 proteins are present on both the inner and outer leaflets of the isolation membranes, they are in a unique position to act as a platform for the recruitment of adaptor proteins that are important for the process of autophagy.

Previous work in our lab has identified NBR1 (Neighbour of BRCA1) as a novel autophagy receptor that can bind both Atg8-like proteins as well as ubiquitinated substrates. NBR1 has a similar domain structure to another known autophagy receptor, p62/SQSTM1; i.e. both contain a N-terminal PB1, a central Zinc finger and C-terminal UBA domain. We have shown that NBR1 can bind Atg8-like proteins, both in vitro and in vivo, which is dependent on a C-terminal LC3 interacting region (LIR) and NBR1 accumulates in an autophagy and LIR dependent manner (Kirkin et al. Mol. Cell. 2009).

Elimination of damaged mitochondria, as well as clearance of mitochondria during differentiation is regulated by a specialized type of autophagy, i.e. mitophagy (Shweers et al. 2007; Kundu et al, 2008; Sandoval et al, 2008; Zhang et al, 2009). Previously, Nix/BNIP3L protein has been reported to be important for the process of mitochondrial clearance and terminal differentiation of reticulocytes (Zhang & Ney, 2009).

In addition to NBR1, we identified Nix/BNIP3L as an interaction partner for autophagy specific UBL proteins LC3/GABARAP and implicated in mitophagy (Novak et al. 2009). We show that Nix binds to LC3 and GABARAP through two potential LIRs. One of them is particularly important since mutation in W35 was able to abolish Nix:LC3/GABARAP binding in vitro and mitochondrial clearance in vivo.

Dysfunction of autophagy contributes or can be a cause of many diseases and failure of autophagy can promote cancer, neurodegenerative disorders, liver disease, premature aging, inflammatory diseases, such as Crohn´s, and compromises host defense against pathogens. The identification of novel autophagy receptors, such as NBR1 and Nix, will allow us to gain a deeper understanding of the overall process of autophagy and in particular, the mechanisms governing selective autophagy and how it may be deregulated in neurodegenerative, inflammatory diseases as well as cancer.