Endocytosis: the DUB version

Dynamic modification of endosomal cargo proteins, such as the epidermal growth factor receptor, by ubiquitin can regulate their sorting into the lumen of multivesicular bodies through interactions with a complex protein network incorporating the endosomal sorting complexes required for transport (ESCRTs). Two deubiquitinating enzymes, AMSH and UBPY, interact with ESCRT protein components but exert opposite effects upon the rate of epidermal growth factor receptor downregulation. This might reflect their distinct specificities for different types of polyubiquitin chain linkage. We propose that AMSH might rescue ubiquitinated cargo from lysosomal degradation through disassembly of K63-linked polyubiquitin chains. UBPY function is essential for effective downregulation but is likely to be multifaceted, encompassing activity against both K63-linked and K48-linked polyubiquitin chains and including regulation of the stability of ESCRT-associated proteins such as STAM, by reversing their ubiquitination.     

Endosomal dynamics of Met determine signaling output

Proteasomal activity is required for Met receptor degradation after acute stimulation with hepatocyte growth factor (HGF). Inhibition of proteasomal activity with lactacystin leads to a block in the endocytic trafficking of Met such that the receptor fails to reach late endosomes/lysosomes, where degradation by acid-dependent proteases takes place (). In this article, we have biochemically determined Met internalization rates from the cell surface and shown that lactacystin does not inhibit the initial HGF-dependent internalization step of Met. Instead, it promotes the recycling pathway from early endosomes at the expense of sorting to late endosomes, thereby ensuring rapid return of internalized Met to the cell surface. We have used this perturbation of Met endosomal sorting by lactacystin to examine the consequences for HGF-dependent signaling outputs. In control cells HGF-dependent receptor autophosphorylation reaches a maximal level over 5-10 min but then attenuates over the ensuing 50 min. Furthermore, Met dephosphorylation can be kinetically dissociated from Met degradation. In lactacystin-treated cells, we observe a failure of Met dephosphorylation as well as Met degradation. Elements of the mitogen-activated protein kinase cascade, downstream of receptor activation, show a normal kinetic profile of phosphorylation, indicating that the mitogen-activated protein kinase pathway can attenuate in the face of sustained receptor activation. The HGF-dependent phosphorylation of a receptor substrate that is localized to clathrin-coated regions of sorting endosomes, Hrs, is dramatically reduced by lactacystin treatment. Reduction of cellular Hrs levels by short interfering RNA modestly retards Met degradation and markedly prevents the attenuation of Met phosphorylation. HGF-dependent Hrs phosphorylation and Met dephosphorylation may provide signatures for retention of the receptor in coated regions of the endosome implicated in sorting to lysosomes.     

Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway.

The Met tyrosine kinase receptor is a widely expressed molecule which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). In this communication we demonstrate that significant Met degradation is induced by HGF/SF and that this degradation can be blocked by lactacystin, an inhibitor of proteasome activity. We also show that Met is rapidly polyubiquitinated in response to ligand and that polyubiquitinated Met molecules, which are normally unstable, are stabilized by lactacystin. Both HGF/SF-induced degradation and polyubiquitination of Met were shown to be dependent on the receptor possessing intact tyrosine kinase activity. Finally, we found that a normally highly labile 55-kDa fragment of the Met receptor is stabilized by lactacystin and demonstrate that it represents a cell-associated remnant that is generated following the ligand-independent proteolytic cleavage of the Met receptor in its extracellular domain. This truncated Met molecule encompasses the kinase domain of the receptor and is itself tyrosine phosphorylated. We conclude that the ubiquitin-proteasome pathway plays a significant role in the degradation of the Met tyrosine kinase receptor as directed by ligand-dependent and -independent signals. We propose that this proteolytic pathway may be important for averting cellular transformation by desensitizing Met signaling following ligand stimulation and by eliminating potentially oncogenic fragments generated via extracellular cleavage of the Met receptor.     

Insights into the molecular composition of endogenous unanchored polyubiquitin chains

The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo.     

TrkA receptor endolysosomal degradation is both ubiquitin and proteasome dependent

Gaps in our knowledge exist regarding the degradation of the tropomyosin-regulated kinase A (TrkA) receptor after addition of neurotrophin, nerve growth factor (NGF). TrkA is rapidly and transiently ubiquitinated upon addition of NGF. Here, we demonstrate that the polyubiquitin tag plays a definitive role in receptor sorting. Treatment of PC12 cells with lactacystin prevented NGF-dependent deubiquitination and degradation of TrkA. However, treatment with methylamine, bafilomycin or leupeptin, did not prevent NGF-dependent deubiquitination but blocked the degradation of TrkA. Employing co-immunoprecipitation, biochemical fractionation and confocal microscopy, the kinetics of receptor trafficking post-internalization was observed to occur as a sequel from endosome/multivesicular body, proteasomes, culminating with degradation in the lysosomes. The trafficking of the polyubiquitin-deficient TrkA receptor mutant K485R was impaired and likewise failed to degrade revealing that the receptor escapes degradation. The interaction of TrkA with proteasomes was confirmed by purification and co-immunoprecipitation. We provide evidence that proteasomal deubiquitinating enzymes trim K63-ubiquitin chains from the TrkA receptor prior to its delivery to lysosomes for degradation. Taken together, our results reveal the existence of a novel proteasome-dependent step in receptor degradation.     

The role of atypical ubiquitination in cell regulation

Ubiquitination is a type of intracellular proteins post-translational modification (PTM) characterized by covalent attachment of ubiquitin molecules to target proteins. This includes monoubiquitination (attachment of one ubiquitin molecule), multiple monoubiquitination also known as multiubiquitination (attachment of several monomeric ubiquitin molecules to a target protein), and polyubiquitination (attachment of ubiquitin chains consisting of several, most frequently four ubiquitin monomers to a target protein). In the case of polyubiquitination, linear or branched polyubiquitin chains are formed. Their formation involves various lysine residues of monomeric ubiquitin. The best studied is Lys48-linked polyubiquitination, which targets proteins for proteasomal degradation. In this review we have considered examples of so-called atypical polyubiquitination, which mainly involves other lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) and also N-terminal methionine. The considered examples convincingly demonstrate that polyubiquitination of proteins (not necessarily) targets proteins for their proteolytic degradation in proteasomes. Atypically polyubiquitinated proteins are involved in regulation of various processes including immune response, genome stability, signal transduction, etc. Alterations of ubiquitination machinery is crucial for development of serious diseases.     

The ubiquitin isopeptidase UBPY regulates endosomal ubiquitin dynamics and is essential for receptor down-regulation

UBPY is a ubiquitin-specific protease that can deubiquitinate monoubiquitinated receptor tyrosine kinases, as well as process Lys-48- and Lys-63-linked polyubiquitin to lower denomination forms in vitro. Catalytically inactive UBPY localizes to endosomes, which accumulate ubiquitinated proteins. We have explored the sequelae of short interfering RNA-mediated knockdown of UBPY. Global levels of ubiquitinated protein increase and ubiquitin accumulates on endosomes, although free ubiquitin levels are unchanged. UBPY-depleted cells have more and larger multivesicular endosomal structures that are frequently associated through extended contact areas, characterized by regularly spaced, electron-dense, bridging profiles. Degradation of acutely stimulated receptor tyrosine kinases, epidermal growth factor receptor and Met, is strongly inhibited in UBPY knockdown cells suggesting that UBPY function is essential for growth factor receptor down-regulation. In contrast, stability of the UBPY binding partner STAM is dramatically compromised in UBPY knockdown cells. The cellular functions of UBPY are complex but clearly distinct from those of the Lys-63-ubiquitin-specific protease, AMSH, with which it shares a binding site on the SH3 domain of STAM.     

Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.

The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.     

Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery

AMSH is an endosomal ubiquitin isopeptidase that can limit EGF receptor downregulation . It directly binds to the SH3 domain of STAM, which is constitutively associated with Hrs, a component of clathrin-coated structures on endosomes. This clathrin coat has been implicated in the recruitment of ubiquitinated growth factor receptors prior to their incorporation into internal vesicles of the multivesicular body (MVB) , through the concerted action of ESCRT complexes I, II, and III . We now show that AMSH is embedded within a network of interactions with components of the MVB-sorting machinery. AMSH and STAM, like Hrs , both bind directly to clathrin. AMSH also interacts with mVps24/CHMP3, a component of ESCRT III complex, and this interaction is reinforced through simultaneous STAM binding. We have explored the effect of interacting components on the in vitro enzymatic activity of AMSH. The enzyme shows specificity for K63- over K48-linked polyubiquitin chains in vitro and is markedly stimulated by coincubation with STAM, indicating that activation of AMSH is coupled to its association with the MVB-sorting machinery. Other interacting factors do not directly stimulate AMSH but may serve to orient the enzyme with respect to substrates on the endosomal membrane.     

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.     

Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages

The ubiquitin proteasome system (UPS) directs programmed destruction of key cellular regulators via posttranslational modification of its targets with polyubiquitin chains. These commonly contain Lys-48 (K48)–directed ubiquitin linkages, but chains containing atypical Lys-11 (K11) linkages also target substrates to the proteasome—for example, to regulate cell cycle progression. The ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C) controls mitotic exit. In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis. We tested this idea by correlating quantitative measures of in vivo K11-specific ubiquitination of individual substrates, including Aurora kinases, with their degradation kinetics tracked at the single-cell level. All anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin. Specific examination of substrates depending on the APC/C coactivator Cdh1 for their degradation revealed Cdh1-dependent enrichment of K11 chains on these substrates, whereas other ubiquitin linkages on the same substrates added during mitotic exit were Cdh1-independent. Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a coactivator-specified manner.     

A ubiquitin-interacting motif from Hrs binds to and occludes the ubiquitin surface necessary for polyubiquitination in monoubiquitinated proteins

The ubiquitin-interacting motif (UIM) is a short, approximately 20 residue, structural element, which is present in, but not limited to, the proteins involved in endocytotic and proteasomal degradation. UIMs facilitate endocytotic vesicular sorting of the monoubiquitinated proteins and may be important for the targeting of the polyubiquitinated proteins to the proteasome. Using heteronuclear NMR backbone and side-chain chemical shift mapping of the ubiquitin interaction surface, the UIM from the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, specifically binds to the ubiquitin hydrophobic surface using UIM's well-conserved central helical LALAL motif. Molecular modeling of the ubiquitin:UIM_Hrs complex suggests that binding occurs through a specific interaction of Leu263 and Leu267 of the UIM_Hrs with two ubiquitin hydrophobic patches located in close proximity to the ubiquitin major polyubiquitination site, Lys48. Intramolecular binding of ubiquitin to a UIM in monoubiquitinated proteins would render Lys48 unavailable for further ubiquitination, thus, explaining the absolute requirement of UIMs for monoubiquitination. Two leucines, Leu265 and Leu269, located on the opposite face of UIM_Hrs can also interact, albeit less favorably than Leu263 and Leu267, with the ubiquitin hydrophobic patches, suggesting a possible mode for polyubiquitin:UIM binding and apparent preference of UIMs for polyubiquitins.     

The BRCA1/BARD1 heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue K6 of ubiquitin

The BRCA1 tumor suppressor forms a heterodimer with the BARD1 protein, and the resulting complex functions as an E3 ubiquitin ligase that catalyzes the synthesis of polyubiquitin chains. In theory, polyubiquitination can occur by isopeptide bond formation at any of the seven lysine residues of ubiquitin. The isopeptide linkage of a polyubiquitin chain is a particularly important determinant of its cellular function, such that K48-linked chains commonly target proteins for proteasomal degradation, while K63 chains serve non-proteolytic roles in various signaling pathways. To determine the isopeptide linkage formed by BRCA1/BARD1-dependent polyubiquitination, we purified a full-length heterodimeric complex and compared its linkage specificity with that of E6-AP, an E3 ligase known to induce proteolysis of its cellular substrates. Using a comprehensive mutation analysis, we found that E6-AP catalyzes the synthesis of K48-linked polyubiquitin chains. In contrast, however, the BRCA1/BARD1 heterodimer directs polymerization of ubiquitin primarily through an unconventional linkage involving lysine residue K6. Although heterologous substrates of BRCA1/BARD1 are not known, BRCA1 autoubiquitination occurs principally by conjugation with K6-linked polymers. The ability of BRCA1/BARD1 to form K6-linked polyubiquitin chains suggests that it may impart unique cellular properties to its natural enzymatic substrates.     

Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.     

Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains

Ubiquitin‐binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48‐ and K63‐linked polyubiquitin chains, respectively. UBQLN2 comprises self‐associating regions that drive its homotypic liquid–liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11‐Ub4 and K48‐Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63‐Ub4, M1‐Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin‐binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self‐interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin‐binding shuttles and adaptors.     

Mutants of the deubiquitinating enzyme Ubp14 decipher pathway diversity of ubiquitin-proteasome linked protein degradation

Selective proteolysis is an important regulatory mechanism in all cells. In eukaryotes, this process gains specificity by tagging proteins with the small protein ubiquitin. K48 linked polyubiquitin chains of four and more ubiquitin moieties target proteins for hydrolysis by the proteasome. Prior to degradation the polyubiquitin chain is removed from the protein, cleaved into single units, and recycled. The deubiquitinating enzyme Ubp14 is an important catalyst of this process. Mutants of Ubp14 had been shown to accumulate non-cleaved oligo- and polyubiquitin chains, which resulted in inhibition of overall ubiquitin-proteasome linked proteolysis as well as in inhibition of degradation of some known substrates. Here we show that accumulation of ubiquitin chains due to defective Ubp14 does not uniformly lead to inhibition of ubiquitin-proteasome linked protein degradation. Instead, inhibition of degradation depends on the substrate tested. The results indicate the existence of different paths through which proteins enter the proteasome.     

Endocytosis of receptor tyrosine kinases is driven by monoubiquitylation,not polyubiquitylation

Growth factors stimulate specific receptor tyrosine kinases, but subsequent receptor endocytosis terminates signaling. The ubiquitin ligase c-Cbl targets epidermal growth factor receptors (EGFRs) to endocytosis by tagging them with multiple ubiquitin molecules. However, the type of ubiquitylation is unknown; whereas polyubiquitin chains signal proteasomal degradation, ubiquitin monomers control other processes. We report that in isolation c-Cbl mediates monoubiquitylation rather than polyubiquitylation of EGFRs. Consistent with the sufficiency of monoubiquitylation, when fused to the tail of EGFR, a single ubiquitin induces receptor endocytosis and degradation in cells. By using receptor and ubiquitin mutants, we infer that c-Cbl attaches a founder monoubiquitin to the kinase domain of EGFR and this is complemented by the conjugation of additional monoubiquitins. Hence, receptor tyrosine kinases are desensitized through conjugation of multiple monoubiquitins, which is distinct from polyubiquitin-dependent proteasomal degradation.     

Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.