Ivan Dikic

A role for ubiquitin in selective autophagy.

Ubiquitination is the hallmark of protein degradation by the 26S proteasome. However, the proteasome is limited in its capacity to degrade oligomeric and aggregated proteins. Removal of harmful protein aggregates is mediated by autophagy, a mechanism by which the cell sequesters cytosolic cargo and delivers it for degradation by the lysosome. Identification of autophagy receptors, such as p62/SQSTM1 and NBR1, which simultaneously bind both ubiquitin and autophagy-specific ubiquitin-like modifiers, LC3/GABARAP, has provided a molecular link between ubiquitination and autophagy. This review explores the hypothesis that ubiquitin represents a selective degradation signal suitable for targeting various types of cargo, ranging from protein aggregates to membrane-bound organelles and microbes.

Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation

Many cellular proteins are post-translationally modified by the addition of a single ubiquitin or a polyubiquitin chain. Among these are receptor tyrosine kinases (RTKs), which undergo ligand-dependent ubiquitination. The ubiquitination of RTKs has become recognized as an important signal for their endocytosis and degradation in the lysosome; however, it is not clear whether ubiquitination itself is sufficient for this process or simply participates in its regulation. The issue is further complicated by the fact that RTKs are thought to be polyubiquitinated — a modification that is linked to protein degradation by the proteasome. By contrast, monoubiquitination has been associated with diverse proteasome-independent cellular functions including intracellular protein movement. Here we show that the epidermal growth factor and platelet-derived growth factor receptors are not polyubiquitinated but rather are monoubiquitinated at multiple sites after their ligand-induced activation. By using different biochemical and molecular genetics approaches, we show that a single ubiquitin is sufficient for both receptor internalization and degradation. Thus, monoubiquitination is the principal signal responsible for the movement of RTKs from the plasma membrane to the lysosome.

A Role for NBR1 in Autophagosomal Degradation of Ubiquitinated Substrates

Autophagy is a catabolic process where cytosolic cellular components are delivered to the lysosome for degradation. Recent studies have indicated the existence of specific receptors, such as p62, which link ubiquitinated targets to autophagosomal degradation pathways. Here we show that NBR1 (neighbor of BRCA1 gene 1) is an autophagy receptor containing LC3- and ubiquitin (Ub)-binding domains. NBR1 is recruited to Ub-positive protein aggregates and degraded by autophagy depending on an LC3-interacting region (LIR) and LC3 family modifiers. Although NBR1 and p62 interact and form oligomers, they can function independently, as shown by autophagosomal clearance of NBR1 in p62-deficient cells. NBR1 was localized to Ub-positive inclusions in patients with liver dysfunction, and depletion of NBR1 abolished the formation of Ub-positive p62 bodies upon puromycin treatment of cells. We propose that NBR1 and p62 act as receptors for selective autophagosomal degradation of ubiquitinated targets.

Nix is a selective autophagy receptor for mitochondrial clearance

Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin‐like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP‐L1 to damaged mitochondria through its amino‐terminal LC3‐interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.

Atypical ubiquitin chains: new molecular signals

Ubiquitin (Ub) is a small protein modifier that regulates many biological processes, including gene transcription, cell‐cycle progression, DNA repair, apoptosis, virus budding and receptor endocytosis. Ub can be conjugated to target proteins either as a monomer or as Ub chains that vary in length and linkage type. The various types of Ub modification are linked to distinct physiological functions in cells. MonoUb, for example, regulates DNA repair and receptor endocytosis, whereas lysine 48‐linked Ub chains label proteins for proteasomal degradation. More recently, the importance of chains conjugated through the other six lysines in Ub, known as atypical Ub chains, has been revealed. Atypical chains can be homotypic, sequentially using the same lysine residue in Ub for conjugation; mixed‐linkage, utilizing several distinct lysines to connect consecutive Ub moieties; or heterologous, connecting Ub with other Ub‐like modifiers. Here, we describe recent progress in the understanding of atypical Ub chain assembly and their recognition by Ub‐binding domains, and we discuss further their functional roles in vivo.

Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation

Heparin is required for fibroblast growth factor (FGF) stimulation of biological responses. Using isothermal titration calorimetry, we show that acidic FGF (aFGF) forms a 1:1 complex with the soluble extracellular domain of FGF receptor (FGFR). Heparin exerts its effect by binding to many molecules of aFGF. The resulting aFGF-heparin complex can bind to several receptor molecules, leading to FGFR dimerization. In two cell lines lacking endogenous heparan sulfate, exogenous heparin is required for FGFR dimerization, tyrosine kinase activation, c-fos mRNA transcription, and cell proliferation. Moreover, a synthetic heparin analog that binds monovalently to aFGF blocks FGFR dimerization, activation, and signaling via FGFR. We propose that heparin causes oligomerization of aFGF such that its binding to FGFR results in dimerization and activation. This represents a novel mechanism for transmembrane signaling and may account for the action of many heparin-bound growth factors.

Ubiquitylation and cell signaling

Ubiquitylation is an emerging mechanism implicated in a variety of nonproteolytic cellular functions. The attachment of a single ubiquitin (Ub) or poly‐Ub (lysine 63) chains to proteins control gene transcription, DNA repair and replication, intracellular trafficking and virus budding. In these processes, protein ubiquitylation exhibits inducibility, reversibility and recognition by specialized domains, features similar to protein phosphorylation, which enable Ub to act as a signaling device. Here, we highlight several recent examples on how Ub regulates signaling and how signaling regulates ubiquitylation during physiological and pathological cellular processes.

Ubiquitin-binding domains — from structures to functions

Ubiquitin-binding domains (UBDs) are modular elements that bind non-covalently to the protein modifier ubiquitin. Recent atomic-level resolution structures of ubiquitin–UBD complexes have revealed some of the mechanisms that underlie the versatile functions of ubiquitin in vivo. The preferences of UBDs for ubiquitin chains of specific length and linkage are central to these functions. These preferences originate from multimeric interactions, whereby UBDs synergistically bind multiple ubiquitin molecules, and from contacts with regions that link ubiquitin molecules into a polymer. The sequence context of UBDs and the conformational changes that follow their binding to ubiquitin also contribute to ubiquitin signalling. These new structure-based insights provide strategies for controlling cellular processes by targeting ubiquitin–UBD interfaces.

Ubiquitin-Binding Domains in Y-Family Polymerases Regulate Translesion Synthesis

Translesion synthesis (TLS) is the major pathway by which mammalian cells replicate across DNA lesions. Upon DNA damage, ubiquitination of proliferating cell nuclear antigen (PCNA) induces bypass of the lesion by directing the replication machinery into the TLS pathway. Yet, how this modification is recognized and interpreted in the cell remains unclear. Here we describe the identification of two ubiquitin (Ub)–binding domains (UBM and UBZ), which are evolutionarily conserved in all Y-family TLS polymerases (pols). These domains are required for binding of polη and polι to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of polη is essential to efficiently restore a normal response to ultraviolet irradiation in xeroderma pigmentosum variant (XP-V) fibroblasts. Our results indicate that Ub-binding domains of Y-family polymerases play crucial regulatory roles in TLS.

Cbl–CIN85–endophilin complex mediates ligand-induced downregulation of EGF receptors

Cbl is a multi-adaptor protein involved in ligand-induced downregulation of receptor tyrosine kinases. It is thought that Cbl-mediated ubiquitination of active receptors is essential for receptor degradation and cessation of receptor-induced signal transduction. Here we demonstrate that Cbl additionally regulates epidermal growth factor (EGF) receptor endocytosis. Cbl rapidly recruits CIN85 (Cbl-interacting protein of 85K; ref. 6) and endophilins (regulatory components of clathrin-coated vesicles) to form a complex with activated EGF receptors, thus controlling receptor internalization. CIN85 was constitutively associated with endophilins, whereas CIN85 binding to the distal carboxy terminus of Cbl was increased on EGF stimulation. Inhibition of these interactions was sufficient to block EGF receptor internalization, delay receptor degradation and enhance EGF-induced gene transcription, without perturbing Cbl-directed receptor ubiquitination. Thus, the evolutionary divergent C terminus of Cbl uses a mechanism that is functionally separable from the ubiquitin ligase activity of Cbl to mediate ligand-dependent downregulation of receptor tyrosine kinases.