An acidic loop and cognate phosphorylation sites define a molecular switch that modulates ubiquitin charging activity in Cdc34-like enzymes

E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in β4α2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 μs molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulates opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the β4α2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.     

Mechanisms of mono- and poly-ubiquitination: Ubiquitination specificity depends on compatibility between the E2 catalytic core and amino acid residues proximal to the lysine

Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3) and the ubiquitin-conjugating enzyme (E2), where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/E2 complexes is the Skp1/Cul1/F box protein complex, SCF^Cdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.     

Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase

The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²⁺-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.     

Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34

Ubiquitin chains linked via lysine 48 (K48) of ubiquitin mediate recognition of ubiquitinated proteins by the proteasome. However, the mechanisms underlying polymerization of this targeting signal on a substrate are unknown. Here we dissect this process using the cyclin-dependent kinase inhibitor Sic1 and its ubiquitination by the cullin-RING ubiquitin ligase SCF(Cdc4) and the ubiquitin-conjugating enzyme Cdc34. We show that Sic1 ubiquitination can be separated into two steps: attachment of the first ubiquitin, which is rate limiting, followed by rapid elongation of a K48-linked ubiquitin chain. Mutation of an acidic loop conserved among Cdc34 orthologs has no effect on attachment of the first ubiquitin onto Sic1 but compromises the processivity and linkage specificity of ubiquitin-chain synthesis. We propose that the acidic loop favorably positions K48 of a substrate-linked ubiquitin to attack SCF bound Cdc34 approximately ubiquitin thioester and thereby enables processive synthesis of K48-linked ubiquitin chains by SCF-Cdc34.     

Human Cdc34 employs distinct sites to coordinate attachment of ubiquitin to a substrate and assembly of polyubiquitin chains

The Cdc34 E2 ubiquitin (Ub) conjugating enzyme catalyzes polyubiquitination of a substrate recruited by the Skp1-Cullin 1-F-box protein-ROC1 E3 Ub ligase. Using mutagenesis studies, we now show that human Cdc34 employs distinct sites to coordinate the transfer of Ub to a substrate and the assembly of polyubiquitin chains. Mutational disruption of the conserved charged stretch (residues 143 to 153) or the acidic loop residues D102 and D103 led to accumulation of monoubiquitinated IkappaBalpha while failing to yield polyubiquitin chains, due to a catalytic defect in Ub-Ub ligation. These results suggest an ability of human Cdc34 to position the attacking Ub for assembly of polyubiquitin chains. Analysis of Cdc34N85Q and Cdc34S138A revealed severe defects of these mutants in both poly- and monoubiquitination of IkappaBalpha, supporting a role for N85 in stabilizing the oxyanion and in coordinating, along with S138, the attacking lysine for catalysis. Finally, Cdc34S95D and Cdc34(E108A/E112A) abolished both poly- and monoubiquitination of IkappaBalpha. Unexpectedly, the catalytic defects of these mutants in di-Ub synthesis can be rescued by fusion of a glutathione S-transferase moiety at E2's N terminus. These findings support the hypothesis that human Cdc34 S95 and E108/E112 are required to position the donor Ub optimally for catalysis, in a manner that might depend on E2 dimerization.     

Release of ubiquitin-charged Cdc34-S - Ub from the RING domain is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1

The S. cerevisiae SCF(Cdc4) is a prototype of RING-type SCF E3s, which recruit substrates for polyubiquitination by the Cdc34 ubiquitin-conjugating enzyme. Current models propose that Cdc34 ubiquitinates the substrate while remaining bound to the RING domain. In contrast, we found that the formation of a ubiquitin thiol ester regulates the Cdc34/SCF(Cdc4) binding equilibrium by increasing the dissociation rate constant, with only a minor effect on the association rate. By using a F72VCdc34 mutant with increased affinity for the RING domain, we demonstrate that release of ubiquitin-charged Cdc34-S - Ub from the RING is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1. Release of ubiquitin-charged E2 from E3 prior to ubiquitin transfer is a previously unrecognized step in ubiquitination, which can explain both the modification of multiple lysines on the recruited substrate and the extension of polyubiquitin chains. We discuss implications of this finding for function of other ubiquitin ligases.     

Loop 7 of E2 enzymes: an ancestral conserved functional motif involved in the E2-mediated steps of the ubiquitination cascade

The ubiquitin (Ub) system controls almost every aspect of eukaryotic cell biology. Protein ubiquitination depends on the sequential action of three classes of enzymes (E1, E2 and E3). E2 Ub-conjugating enzymes have a central role in the ubiquitination pathway, interacting with both E1 and E3, and influencing the ultimate fate of the substrates. Several E2s are characterized by an extended acidic insertion in loop 7 (L7), which if mutated is known to impair the proper E2-related functions. In the present contribution, we show that acidic loop is a conserved ancestral motif in E2s, relying on the presence of alternate hydrophobic and acidic residues. Moreover, the dynamic properties of a subset of family 3 E2s, as well as their binary and ternary complexes with Ub and the cognate E3, have been investigated. Here we provide a model of L7 role in the different steps of the ubiquitination cascade of family 3 E2s. The L7 hydrophobic residues turned out to be the main determinant for the stabilization of the E2 inactive conformations by a tight network of interactions in the catalytic cleft. Moreover, phosphorylation is known from previous studies to promote E2 competent conformations for Ub charging, inducing electrostatic repulsion and acting on the L7 acidic residues. Here we show that these active conformations are stabilized by a network of hydrophobic interactions between L7 and L4, the latter being a conserved interface for E3-recruitment in several E2s. In the successive steps, L7 conserved acidic residues also provide an interaction interface for both Ub and the Rbx1 RING subdomain of the cognate E3. Our data therefore suggest a crucial role for L7 of family 3 E2s in all the E2-mediated steps of the ubiquitination cascade. Its different functions are exploited thank to its conserved hydrophobic and acidic residues in a finely orchestrate mechanism.     

The Human Cdc34 Carboxyl Terminus Contains a Non-covalent Ubiquitin Binding Activity That Contributes to SCF-dependent Ubiquitination

Cdc34 is an E2 ubiquitin-conjugating enzyme that functions in conjunction with SCF (Skp1·Cullin 1·F-box) E3 ubiquitin ligase to catalyze covalent attachment of polyubiquitin chains to a target protein. Here we identified direct interactions between the human Cdc34 C terminus and ubiquitin using NMR chemical shift perturbation assays. The ubiquitin binding activity was mapped to two separate Cdc34 C-terminal motifs (UBS1 and UBS2) that comprise residues 206–215 and 216–225, respectively. UBS1 and UBS2 bind to ubiquitin in the proximity of ubiquitin Lys⁴⁸ and C-terminal tail, both of which are key sites for conjugation. When bound to ubiquitin in one orientation, the Cdc34 UBS1 aromatic residues (Phe²⁰⁶, Tyr²⁰⁷, Tyr²¹⁰, and Tyr²¹¹) are probably positioned in the vicinity of ubiquitin C-terminal residue Val⁷⁰. Replacement of UBS1 aromatic residues by glycine or of ubiquitin Val⁷⁰ by alanine decreased UBS1-ubiquitin affinity interactions. UBS1 appeared to support the function of Cdc34 in vivo because human Cdc34(1–215) but not Cdc34(1–200) was able to complement the growth defect by yeast Cdc34 mutant strain. Finally, reconstituted IκBα ubiquitination analysis revealed a role for each adjacent pair of UBS1 aromatic residues (Phe²⁰⁶/Tyr²⁰⁷, Tyr²¹⁰/Tyr²¹¹) in conjugation, with Tyr²¹⁰ exhibiting the most pronounced catalytic function. Intriguingly, Cdc34 Tyr²¹⁰ was required for the transfer of the donor ubiquitin to a receptor lysine on either IκBα or a ubiquitin in a manner that depended on the neddylated RING sub-complex of the SCF. Taken together, our results identified a new ubiquitin binding activity within the human Cdc34 C terminus that contributes to SCF-dependent ubiquitination.     

The Acidic Tail domain of Human Cdc34 is Required for p27Kip1 Ubiquitination and Complementation of a cdc34 Temperature Sensitive Yeast Strain

Human Cdc34 is an ubiquitin conjugating enzyme or E2 that ubiquitinates substrates including p27Kip1, I?B?, Wee1, and MyoD. Cdc34 possesses a core catalytic domain encoding the active site cysteine and an acidic tail domain within the carboxyl terminal 36 amino acids. Studies suggest that Cdc34 is phosphorylated in mammalian cells at 5 potential residues within the tail domain. In order to study the biological significance of the Cdc34 acidic tail domain and the possible significance of phosphorylation within this region, we tested the ability of human Cdc34 mutants to complement the cdc34-2 temperature sensitive (ts) strain of Saccharomyces cerevisiae. Our studies indicated that complementation of the cdc34-2 ts strain was critically dependent upon the carboxyl-terminal 36 amino acids of human Cdc34, but did not require phosphorylation of human Cdc34 residues S203, S222, S231, T233, and S236. Further studies demonstrated that although a Cdc34 mutant bearing a deletion of the C-terminal 36 amino acids (Cdc34 1-200) was efficiently charged with ubiquitin by E1, it was severely reduced for the ability to ubiquitinate p27Kip1 in vitro compared to wildtype Cdc34. Both in vivo and in vitro binding studies indicated that Cdc34 1-200 bound to the E3-SCF components, Cul1 and Roc1, at levels comparable to the wildtype Cdc34. These studies suggest that the 36 amino acid acidic tail domain of human Cdc34 is critical for its ability to transfer ubiquitin to a substrate and is dispensable for the association of Cdc34 with Cul1 and Roc1. We postulate that the tail domain of Cdc34 may be important for its efficient dissociation from Cul1 and Roc1, an essential requirement for ubiquitination by the budding yeast Cdc34p, or it may be required more directly for ubiquitin transfer to the substrate.     

Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

The attachment of ubiquitin (Ub) to lysines on substrates or itself by ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes results in protein ubiquitination. Lysine selection is important for generating diverse substrate-Ub structures and targeting proteins to different fates; however, the mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines. Evaluation of the relative importance of different residues positioned −2, −1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the −1 and −2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter the native salt-bridge interactions in Ub and Cdc34, resulting in misplacement of Sic1 lysine 50 in the Cdc34 catalytic cleft. During polyubiquitination, Cdc34 showed a strong preference for Ub lysine 48 (K48), with lower activity towards lysine 11 (K11) and lysine 63 (K63). Mutating the −2, −1, +1 and +2 sites surrounding K11 and K63 to mimic those surrounding K48 did not improve their ubiquitination, indicating that further determinants are important for Ub K48 specificity. Modeling the ternary structure of acceptor Ub with the Cdc34~Ub complex as well as in vitro ubiquitination assays unveiled the importance of K6 and Q62 of acceptor Ub for Ub K48 polyubiquitination. These findings provide molecular and structural insight into substrate lysine and Ub K48 specificity by Cdc34.     

The Acidic Tail of the Cdc34 Ubiquitin-conjugating Enzyme Functions in Both Binding to and Catalysis with Ubiquitin Ligase SCFCdc4

Ubiquitin ligases, together with their cognate ubiquitin-conjugating enzymes, are responsible for the ubiquitylation of proteins, a process that regulates a myriad of eukaryotic cellular functions. The first cullin-RING ligase discovered, yeast SCF^Cdc4, functions with the conjugating enzyme Cdc34 to regulate the cell cycle. Cdc34 orthologs are notable for their highly acidic C-terminal extension. Here we confirm that the Cdc34 acidic C-terminal tail has a role in Cdc34 binding to SCF^Cdc4 and makes a major contribution to the submicromolar K_m of Cdc34 for SCF^Cdc4. Moreover, we demonstrate that a key functional property of the tail is its acidity. Our analysis also uncovers an unexpected new function for the acidic tail in promoting catalysis. We demonstrate that SCF is functional when Cdc34 is fused to the C terminus of Cul1 and that this fusion retains partial function even when the acidic tail has been deleted. The Cdc34-SCF fusion proteins that lack the acidic tail must interact in a fundamentally different manner than unfused SCF and wild type Cdc34, demonstrating that distinct mechanisms of E2 recruitment to E3, as is seen in nature, can sustain substrate ubiquitylation. Finally, a search of the yeast proteome uncovered scores of proteins containing highly acidic stretches of amino acids, hinting that electrostatic interactions may be a common mechanism for facilitating protein assembly.     

The Human COP9 Signalosome Protects Ubiquitin-conjugating Enzyme 3 (UBC3/Cdc34) from β-Transducin Repeat-containing Protein (βTrCP)-mediated Degradation

The COP9 signalosome (CSN) is an essential multisubunit complex that regulates the activity of cullin-RING ubiquitin ligases by removing the ubiquitin-like peptide NEDD8 from cullins. Here, we demonstrate that the CSN can affect other components of the ubiquitination cascade. Down-regulation of human CSN4 or CSN5 induced proteasome-mediated degradation of the ubiquitin-conjugating enzyme UBC3/Cdc34. UBC3 was targeted for ubiquitination by the cullin-RING ubiquitin ligase SCF^βTrCP. This interaction required the acidic C-terminal extension of UBC3, which is absent in ubiquitin-conjugating enzymes of the UBCH5 family. Conversely, the UBC3 acidic domain was sufficient to impart sensitivity to SCF^βTrCP-mediated ubiquitination to UBCH5 enzymes. Our work indicates that the CSN is necessary to ensure the stability of selected ubiquitin-conjugating enzymes and uncovers a novel pathway of regulation of ubiquitination processes.     

Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin–proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2^S17D) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg⁹⁷/Lys⁹⁸, and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.     

Functional interaction of 13 yeast SCF complexes with a set of yeast E2 enzymes in vitro

SCF complexes are multi-subunit ubiquitin ligases that, in concert with the E1 and E2 ubiquitination enzymes, catalyze the ubiquination of specific target proteins. Only three yeast SCFs have been reconstituted and characterized to date; each of these ubiquitinates its target protein with the E2 Cdc34. We have reconstituted and purified 1 known and 12 novel yeast SCF complexes, and explored the ability of these complexes to function with 5 different purified E2 enzymes; Ubc1, Cdc34, Ubc4, Ubc8 and Ubc11. We have found that the ubiquitination of Sic1 by the reconstituted SCF(Cdc4) complex was specifically catalyzed by two of the five E2 enzymes tested in vitro; Cdc34 and Ubc4. We also show that at least eight of the purified SCF complexes clearly ubiquitinated their F-box proteins in vitro, lending support for a regulatory mechanism in which F-box proteins catalyze their own destruction. The autoubiquitination of each F-box was in some cases catalyzed only by Cdc34, and in other cases preferentially catalyzed by Ubc4. Ubc4 thus interacts with multiple SCFs in vitro, and the interactions among SCF and E2 components of the ubiquitination machinery may allow further diversification of the roles of SCFs in vivo.     

The conserved RING-H2 finger of ROC1 is required for ubiquitin ligation

ROC1 is a common component of a large family of ubiquitin E3 ligases that regulate cell cycle progression and signal transduction pathways. Here we present evidence suggesting that a conserved RING-H2 structure within ROC1 is critical for its ubiquitin ligation function. Mercury-containing sulfhydryl modification agents (rho-hydroxymercuribenzoate and mercuric chloride) irreversibly inhibit the ROC1-CUL1 ubiquitin ligase activity without disrupting the complex. Consistent with this, these reagents also eliminate the ability of the Skp1-CUL1-HOS-ROC1 E3 ligase complex to support the ubiquitination of IkappaBalpha. Site-directed mutagenesis analysis identifies RING-H2 finger residues Cys(42), Cys(45), Cys(75), His(77), His(80), Cys(83), Cys(94), and Asp(97) as being essential for the ROC1-dependent ubiquitin ligase activity. Furthermore, C42S/C45S and H80A mutations reduce the ability of ROC1 to interact with CUL1 in transfected cells and diminish the capacity of ROC1-CUL1 to form a stable complex with Cdc34 in vitro. However, C75S, H77A, C94S, and D97A substitutions have no detectable effect on ROC1 binding activities. Thus, the ROC1 RING-H2 finger may possess multiple biochemical properties that include stabilizing an interaction with CUL1 and recruiting Cdc34. A possible role of the RING finger in facilitating the Ub transfer reaction is discussed.     

The F-box: a new motif for ubiquitin dependent proteolysis in cell cycle regulation and signal transduction

The ubiquitin system of intracellular protein degradation controls the abundance of many critical regulatory proteins. Specificity in the ubiquitin system is determined largely at the level of substrate recognition, a step that is mediated by E3 ubiquitin ligases. Analysis of the mechanisms of phosphorylation directed proteolysis in cell cycle regulation has uncovered a new class of E3 ubiquitin ligases called SCF complexes, which are composed of the subunits Skp1, Rbx1, Cdc53 and any one of a large number of different F-box proteins. The substrate specificity of SCF complexes is determined by the interchangeable F-box protein subunit, which recruits a specific set of substrates for ubiquitination to the core complex composed of Skp1, Rbx1, Cdc53 and the E2 enzyme Cdc34. F-box proteins have a bipartite structure--the shared F-box motif links F-box proteins to Skp1 and the core complex, whereas divergent protein-protein interaction motifs selectively bind their cognate substrates. To date all known SCF substrates are recognised in a strictly phosphorylation dependent manner, thus linking intracellular signalling networks to the ubiquitin system. The plethora of different F-box proteins in databases suggests that many pathways will be governed by SCF-dependent proteolysis. Indeed, genetic analysis has uncovered roles for F-box proteins in a variety of signalling pathways, ranging from nutrient sensing in yeast to conserved developmental pathways in plants and animals. Moreover, structural analysis has revealed ancestral relationships between SCF complexes and two other E3 ubiquitin ligases, suggesting that the combinatorial use of substrate specific adaptor proteins has evolved to allow the regulation of many cellular processes. Here, we review the known signalling pathways that are regulated by SCF complexes and highlight current issues in phosphorylation dependent protein degradation.     

RNF220, an E3 ubiquitin ligase that targets Sin3B for ubiquitination

Modification of proteins by ubiquitination plays important roles in various cellular processes. During this process, the target specificity is determined by ubiquitin ligases. Here we identify RNF220 (RING finger protein 220) as a novel ubiquitin ligase for Sin3B. As a conserved RING protein, RNF220 can bind E2 and mediate auto-ubiquitination of itself. Through a yeast two-hybrid screen, we isolated Sin3B as one of its targets, which is a scaffold protein of the Sin3/HDAC (histone deacetylase) corepressor complex. RNF220 specifically interacts with Sin3B both in vitro and in vivo. Sin3B can be regulated by the ubiquitin-proteasome system. Co-expression of RNF220 promotes the ubiquitination and proteasomal degradation of Sin3B. Taken together, these results reveal a new mechanism for regulating the Sin3/HDAC complex.     

SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis

Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.     

The loop-less tmCdc34 E2 mutant defective in polyubiquitination in vitro and in vivo supports yeast growth in a manner dependent on Ubp14 and Cka2

Background

The S73/S97/loop motif is a hallmark of the Cdc34 family of E2 ubiquitin-conjugating enzymes that together with the SCF E3 ubiquitin ligases promote degradation of proteins involved in cell cycle and growth regulation. The inability of the loop-less ^Δ12Cdc34 mutant to support growth was linked to its inability to catalyze polyubiquitination. However, the loop-less triple mutant (tm) Cdc34, which not only lacks the loop but also contains the S73K and S97D substitutions typical of the K73/D97/no loop motif present in other E2s, supports growth. Whether ^tmCdc34 supports growth despite defective polyubiquitination, or the S73K and S97D substitutions, directly or indirectly, correct the defect caused by the loop absence, are unknown.

Results

^tmCdc34 supports yeast viability with normal cell size and cell cycle profile despite producing fewer polyubiquitin conjugates in vivo and in vitro. The in vitro defect in Sic1 substrate polyubiquitination is similar to the defect observed in reactions with ^Δ12Cdc34 that cannot support growth. The synthesis of free polyubiquitin by ^tmCdc34 is activated only modestly and in a manner dependent on substrate recruitment to SCF^Cdc4. Phosphorylation of C-terminal serines in ^tmCdc34 by Cka2 kinase prevents the synthesis of free polyubiquitin chains, likely by promoting their attachment to substrate. Nevertheless, ^tm CDC34 yeast are sensitive to loss of the Ubp14 C-terminal ubiquitin hydrolase and DUBs other than Ubp14 inefficiently disassemble polyubiquitin chains produced in ^tm CDC34 yeast extracts, suggesting that the free chains, either synthesized de novo or recycled from substrates, have an altered structure.

Conclusions

The catalytic motif replacement compromises polyubiquitination activity of Cdc34 and alters its regulation in vitro and in vivo, but either motif can support Cdc34 function in yeast viability. Robust polyubiquitination mediated by the S73/S97/loop motif is thus not necessary for Cdc34 role in yeast viability, at least under typical laboratory conditions.     

Cdc34 C-terminal tail phosphorylation regulates Skp1/cullin/F-box (SCF)-mediated ubiquitination and cell cycle progression

The ubiquitin-conjugating enzyme Cdc34 (cell division cycle 34) plays an essential role in promoting the G1-S-phase transition of the eukaryotic cell cycle and is phosphorylated in vivo. In the present study, we investigated if phosphorylation regulates Cdc34 function. We mapped the in vivo phosphorylation sites on budding yeast Cdc34 (yCdc34; Ser207 and Ser216) and human Cdc34 (hCdc34 Ser203, Ser222 and Ser231) to serine residues in the acidic tail domain, a region that is critical for Cdc34's cell cycle function. CK2 (protein kinase CK2) phosphorylates both yCdc34 and hCdc34 on these sites in vitro. CK2-mediated phosphorylation increased yCdc34 ubiquitination activity towards the yeast Saccharomyces cerevisiae Sic1 in vitro, when assayed in the presence of its cognate SCFCdc4 E3 ligase [where SCF is Skp1 (S-phase kinase-associated protein 1)/cullin/F-box]. Similarly, mutation of the yCdc34 phosphorylation sites to alanine, aspartate or glutamate residues altered Cdc34-SCFCdc4-mediated Sic1 ubiquitination activity. Similar results were obtained when yCdc34's ubiquitination activity was assayed in the absence of SCFCdc4, indicating that phosphorylation regulates the intrinsic catalytic activity of Cdc34. To evaluate the in vivo consequences of altered Cdc34 activity, wild-type yCdc34 and the phosphosite mutants were introduced into an S. cerevisiae cdc34 deletion strain and, following synchronization in G1-phase, progression through the cell cycle was monitored. Consistent with the increased ubiquitination activity in vitro, cells expressing the phosphosite mutants with higher catalytic activity exhibited accelerated cell cycle progression and Sic1 degradation. These studies demonstrate that CK2-mediated phosphorylation of Cdc34 on the acidic tail domain stimulates Cdc34-SCFCdc4 ubiquitination activity and cell cycle progression.