Ubiquitin crosstalk connecting cellular processes

Cullin 4 (Cul4), a member of the evolutionally conserved cullin protein family, serves as a scaffold to assemble multisubunit ubiquitin E3 ligase complexes. Cul4 interacts with the Ring finger-containing protein ROC1 through its C-terminal cullin domain and with substrate recruiting subunit(s) through its N-terminus. Previous studies have demonstrated that Cul4 E3 ligase ubiquitylates key regulators in cell cycle control and mediates their degradation through the proteasomal pathway, thus contributing to genome stability. Recent studies from several groups have revealed that Cul4 E3 ligase can target histones for ubiquitylation, and importantly, ubiquitylation of histones may facilitate the cellular response to DNA damage. Therefore, histone ubiquitylation by Cul4 E3 ligase constitutes a novel mechanism through which Cul4 regulates chromatin function and maintains genomic integrity. We outline these studies and suggest that histone ubiquitylation might play important roles in Cul4-regualted chromatin function including the cellular response to DNA damage and heterochromatin gene silencing.     

WW domain HECT E3s target Cbl RING finger E3s for proteasomal degradation

Cbl proteins have RING finger-dependent ubiquitin ligase (E3) activity that is essential for down-regulation of tyrosine kinases. Here we establish that two WW domain HECT E3s, Nedd4 and Itch, bind Cbl proteins and target them for proteasomal degradation. This is dependent on the E3 activity of the HECT E3s but not on that of Cbl. Consistent with these observations, in cells expressing the epidermal growth factor receptor, Nedd4 reverses Cbl-b effects on receptor down-regulation, ubiquitylation, and proximal events in signaling. Cbl-b also targets active Src for degradation in cells, and Nedd4 similarly reverses Cbl-mediated Src degradation. These findings establish that RING finger E3s can be substrates, not only for autoubiquitylation but also for ubiquitylation by HECT E3s and suggest an additional level of regulation for Cbl substrates including protein-tyrosine kinases.     

Arabidopsis membrane-anchored ubiquitin-fold (MUB) proteins localize a specific subset of ubiquitin-conjugating (E2) enzymes to the plasma membrane

The covalent attachment of ubiquitin (Ub) to various intracellular proteins plays important roles in altering the function, localization, processing, and degradation of the modified target. A minimal ubiquitylation pathway uses a three-enzyme cascade (E1, E2, and E3) to activate Ub and select target proteins for modification. Although diverse E3 families provide much of the target specificity, several factors have emerged recently that coordinate the subcellular localization of the ubiquitylation machinery. Here, we show that the family of membrane-anchored ubiquitin-fold (MUB) proteins recruits and docks specific E2s to the plasma membrane. Protein interaction screens with Arabidopsis MUBs revealed that interacting E2s are limited to a well defined subgroup that is phylogenetically related to human UbcH5 and yeast Ubc4/5 families. MUBs appear to interact noncovalently with an E2 surface opposite the active site that forms a covalent linkage with Ub. Bimolecular fluorescence complementation demonstrated that MUBs bind simultaneously to the plasma membrane via a prenyl tail and to the E2 in planta. These findings suggest that MUBs contribute subcellular specificity to ubiquitylation by docking the conjugation machinery to the plasma membrane.     

DDB2, the xeroderma pigmentosum group E gene product, is directly ubiquitylated by Cullin 4A-based ubiquitin ligase complex

Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to UV irradiation and high incidence of skin cancer caused by inherited defects in DNA repair. Mutational malfunction of damaged-DNA binding protein 2 (DDB2) causes the XP complementation group E (XP-E). DDB2 together with DDB1 comprises a heterodimer called DDB complex, which is involved in damaged-DNA binding and nucleotide excision repair. Interestingly, by screening for a cellular protein(s) that interacts with Cullin 4A (Cul4A), a key component of the ubiquitin ligase complex, we identified DDB1. Immunoprecipitation confirmed that Cul4A interacts with DDB1 and also associates with DDB2. To date, it has been reported that DDB2 is rapidly degraded after UV irradiation and that overproduction of Cul4A stimulates the ubiquitylation of DDB2 in the cells. However, as biochemical analysis using pure Cul4A-containing E3 is missing, it is still unknown whether the Cul4A complex directly ubiquitylates DDB2 or not. We thus purified the Cul4A-containing E3 complex to near homogeneity and attempted to ubiquitylate DDB2 in vitro. The ubiquitylation of DDB2 was reconstituted using this pure E3 complex, indicating that DDB-Cul4A E3 complex in itself can ubiquitylate DDB2 directly. We also showed that an amino acid substitution, K244E, in DDB2 derived from a XP-E patient did not affect its ubiquitylation.     

Targeting WW domains linker of HECT-type ubiquitin ligase Smurf1 for activation by CKIP-1

E3 ubiquitin ligases are final effectors of the enzyme cascade controlling ubiquitylation. A central issue in understanding their regulation is to decipher mechanisms of their assembly and activity. In contrast with RING-type E3s, fewer mechanisms are known for regulation of HECT-type E3s. Smad ubiquitylation regulatory factor 1 (Smurf1), a C2-WW-HECT-domain E3, is crucial for bone homeostasis, in which it suppresses osteoblast activity. However, whether and how its activity is regulated remains unclear. Here we show that Smurf1, but not Smurf2, interacts with casein kinase-2 interacting protein-1 (CKIP-1), resulting in an increase in its E3 ligase activity. Surprisingly, CKIP-1 targets specifically the linker region between the WW domains of Smurf1, thereby augmenting its affinity for and promoting ubiquitylation of the substrate. Moreover, CKIP-1-deficient mice undergo an age-dependent increase in bone mass as a result of accelerated osteogenesis and decreased Smurf1 activity. These findings provide evidence that the WW domains linker is important in complex assembly and in regulating activity of HECT-type E3s and that CKIP-1 functions as the first auxiliary factor to enhance the activation of Smurf1.     

Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4

Cse4 is a variant of histone H3 that is incorporated into a single nucleosome at each centromere in budding yeast. We have discovered an E3 ubiquitin ligase, called Psh1, which controls the cellular level of Cse4 via ubiquitylation and proteolysis. The activity of Psh1 is dependent on both its RING and zinc finger domains. We demonstrate the specificity of the ubiquitylation activity of Psh1 toward Cse4 in vitro and map the sites of ubiquitylation. Mutation of key lysines prevents ubiquitylation of Cse4 by Psh1 in vitro and stabilizes Cse4 in vivo. While deletion of Psh1 stabilizes Cse4, elimination of the Cse4-specific chaperone Scm3 destabilizes Cse4, and the addition of Scm3 to the Psh1-Cse4 ubiquitylation reaction prevents Cse4 ubiquitylation, together suggesting Scm3 may protect Cse4 from ubiquitylation. Without Psh1, Cse4 overexpression is toxic and Cse4 is found at ectopic locations. Our results suggest Psh1 functions to prevent the mislocalization of Cse4.     

Histone H2B Ubiquitylation Is Not Required for Histone H3 Methylation at Lysine 4 in Tetrahymena

Ubiquitylation of histone H2B and/or a component of the system that ubiquitylates H2B is required for methylation of histone H3 at lysine 4 (H3K4) in yeasts and probably in humans. In this study, the single ubiquitylation site was mapped to conserved lysine 115 of the C-terminal region of histone H2B in the single-cell model organism Tetrahymena thermophila. In strains lacking H2B ubiquitylation, H3K4 methylation was not detectably affected. As in other organisms, the E2 ubiquitin-conjugating enzyme Ubc2 and the E3 ubiquitin ligase Bre1 were required for H2B ubiquitylation. However, neither enzyme was required for H3K4 methylation. These studies argue that, in T. thermophila, the histone ubiquitylation mechanism is not required for H3K4 methylation, demonstrating that different organisms can speak different languages in the “cross-talk” among post-translational modifications on different histones.     

Regulating the Regulators: Recent Revelations in the Control of E3 Ubiquitin Ligases

Since its discovery as a post-translational signal for protein degradation, our understanding of ubiquitin (Ub) has vastly evolved. Today, we recognize that the role of Ub signaling is expansive and encompasses diverse processes including cell division, the DNA damage response, cellular immune signaling, and even organismal development. With such a wide range of functions comes a wide range of regulatory mechanisms that control the activity of the ubiquitylation machinery. Ub attachment to substrates occurs through the sequential action of three classes of enzymes, E1s, E2s, and E3s. In humans, there are 2 E1s, ∼35 E2s, and hundreds of E3s that work to attach Ub to thousands of cellular substrates. Regulation of ubiquitylation can occur at each stage of the stepwise Ub transfer process, and substrates can also impact their own modification. Recent studies have revealed elegant mechanisms that have evolved to control the activity of the enzymes involved. In this minireview, we highlight recent discoveries that define some of the various mechanisms by which the activities of E3-Ub ligases are regulated.     

The N-Terminal Extension of UBE2E Ubiquitin-Conjugating Enzymes Limits Chain Assembly

Protein ubiquitylation depends upon the concerted action of ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). All E2s have a conserved ubiquitin-conjugating (UBC) domain but many have variable extensions N- and C-terminal to the UBC domain. For many E2s, the function of the extension is not well understood. Here, we show that the N-terminal extension of the UBE2E proteins regulates formation of polyubiquitin chains by the processive UBC domain. Target proteins are therefore monoubiquitylated by full-length UBE2E, whereas the UBC domain alone polyubiquitylates proteins. Although the N-terminal extension of UBE2E1 is largely disordered in solution, these residues have a critical role in limiting chain building, and when fused to the highly processive E2, UBE2D2, ubiquitylation is limited. For some E2s, interaction of ubiquitin with the ‘backside’ of the UBC domain promotes polyubiquitylation. However, interaction of ubiquitin with the backside of the UBC domain of UBE2E1 does not appear to be important for processivity. This study underscores the importance of studying full-length E2 proteins and not just the highly conserved core domain.     

The emerging family of CULLIN3‐RING ubiquitin ligases (CRL3s): cellular functions and disease implications

Protein ubiquitylation is a post‐translational modification that controls all aspects of eukaryotic cell functionality, and its defective regulation is manifested in various human diseases. The ubiquitylation process requires a set of enzymes, of which the ubiquitin ligases (E3s) are the substrate recognition components. Modular CULLIN‐RING ubiquitin ligases (CRLs) are the most prevalent class of E3s, comprising hundreds of distinct CRL complexes with the potential to recruit as many and even more protein substrates. Best understood at both structural and functional levels are CRL1 or SCF (SKP1/CUL1/F‐box protein) complexes, representing the founding member of this class of multimeric E3s. Another CRL subfamily, called CRL3, is composed of the molecular scaffold CULLIN3 and the RING protein RBX1, in combination with one of numerous BTB domain proteins acting as substrate adaptors. Recent work has firmly established CRL3s as major regulators of different cellular and developmental processes as well as stress responses in both metazoans and higher plants. In humans, functional alterations of CRL3s have been associated with various pathologies, including metabolic disorders, muscle, and nerve degeneration, as well as cancer. In this review, we summarize recent discoveries on the function of CRL3s in both metazoans and plants, and discuss their mode of regulation and specificities.     

Ubiquitin ligases, critical mediators of endoplasmic reticulum-associated degradation

Endoplasmic reticulum-associated degradation (ERAD) represents the primary means of quality control within the secretory pathway. Critical to this process are ubiquitin protein ligases (E3s) which, together with ubiquitin conjugating enzymes (E2s), mediate the ubiquitylation of proteins targeted for degradation from the ER. In this chapter we review our knowledge of both Saccharomyces cerevisiae and mammalian ERAD ubiquitin ligases. We focus on recent insights into these E3s, their associated proteins and potential mechanisms of action.     

Phosphorylation-dependent ubiquitylation and degradation of androgen receptor by Akt require Mdm2 E3 ligase

The androgen receptor (AR) controls several biological functions including prostate cell growth and apoptosis. However, the mechanism by which AR maintains its stability to function properly remains largely unknown. Here we show that Akt and Mdm2 form a complex with AR and promote phosphorylation-dependent AR ubiquitylation, resulting in AR degradation by the proteasome. The effect of Akt on AR ubiquitylation and degradation is markedly impaired in a Mdm2-null cell line compared with the wild-type cell line, suggesting that Mdm2 is involved in Akt-mediated AR ubiquitylation and degradation. Furthermore, we demonstrate that the E3 ligase activity of Mdm2 and phosphorylation of Mdm2 by Akt are essential for Mdm2 to affect AR ubiquitylation and degradation. These results suggest that phosphorylation-dependent AR ubiquitylation and degradation by Akt require the involvement of Mdm2 E3 ligase activity, a novel mechanism that provides insight into how AR is targeted for degradation.