A plant‐specific in vitro ubiquitination analysis system

Protein ubiquitination requires the concerted action of three enzymes: ubiquitin‐activating enzyme (E1), ubiquitin‐conjugating enzyme (E2) and ubiquitin ligase (E3). These ubiquitination enzymes belong to an abundant protein family that is encoded in all eukaryotic genomes. Describing their biochemical characteristics is an important part of their functional analysis. It has been recognized that various E2/E3 specificities exist, and that detection of E3 ubiquitination activity in vitro may depend on the recruitment of E2s. Here, we describe the development of an in vitro ubiquitination system based on proteins encoded by genes from Arabidopsis. It includes most varieties of Arabidopsis E2 proteins, which are tested with several RING‐finger type E3 ligases. This system permits determination of E3 activity in combination with most of the E2 sub‐groups that have been identified in the Arabidopsis genome. At the same time, E2/E3 specificities have also been explored. The components used in this system are all from plants, particularly Arabidopsis, making it very suitable for ubiquitination assays of plant proteins. Some E2 proteins that are not easily expressed in Escherichia coli were transiently expressed and purified from plants before use in ubiquitination assays. This system is also adaptable to proteins of species other than plants. In this system, we also analyzed two mutated forms of ubiquitin, K48R and K63R, to detect various types of ubiquitin conjugation.     

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.     

Evidence for a particulate location of ubiquitin conjugates and ubiquitin-conjugating enzymes in rabbit brain.

Conjugate ubiquitin was previously found in the nucleus, cytoplasm, and membranes of eukaryotic cells while the enzymes of the ubiquitin-conjugating system appear to be cytoplasmic. We have prepared the mitochondrial fraction from rabbit brain by discontinuous density gradient ultracentrifugation and by Western blotting, using a specific antibody against conjugate ubiquitin, showing that it contains ubiquitin conjugates in a very wide molecular weight range. Electron microscopy and measurement of specific enzyme markers show that this fraction not only contains mitochondria but also some endoplasmic reticulum vesicles. Immunostaining with anti-ubiquitin IgG followed by immunodecoration with colloidal gold particles provides evidence for the presence of conjugate ubiquitin both in mitochondria and in the endoplasmic reticulum. Furthermore, this "mitochondrial fraction" shows a pronounced ATP-dependent ability to conjugate 125I-ubiquitin into a number of endogenous proteins as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Addition of E1, E2, and E3, the enzymes of the ubiquitin conjugating system purified from rabbit reticulocytes, does not further increase this ubiquitination nor incorporate 125I-ubiquitin into additional protein bands. The same mitochondrial fraction is not able to carry out any ATP-dependent degradation of 125I-albumin; however, it contains an isopeptidase activity able to release the covalently incorporated 125I-ubiquitin and is also able to conjugate 125I-ubiquitin to exogenous proteins as oxidized RNase. By affinity chromatography on ubiquitin-agarose of fraction II of a crude Triton X-100 extract of the mitochondrial fraction, several proteins corresponding in Mr to the E1 and E2s enzymes were obtained. These proteins were also able to form specific ubiquitin-thiol ester bounds on sodium dodecyl sulfate-polyacrylamide gels and to support 125I-ubiquitin conjugation to oxidized RNase. Detergent fractionation of the mitochondrial fraction provided evidence for a possible localization of the ubiquitin conjugating activity in the mitochondrial external membrane and endoplasmic reticulum. The presence of an active ubiquitin protein conjugating system in mitochondria and endoplasmic reticulum may be related to the turnover of organelle proteins as well as to specific cell functions such as import of proteins into mitochondria and ubiquitination of externally oriented membrane-bound proteins.     

High performance liquid chromatography resolution of ubiquitin pathway enzymes from wheat germ

The highly conserved protein ubiquitin is involved in several cellular processes in eukaryotes as a result of its covalent ligation to a variety of target proteins. Here, we describe the purification of several enzymatic activities involved in ubiquitin-protein conjugate formation and disassembly from wheat germ (Triticum vulgare) by a combination of ubiquitin affinity chromatography and anion-exchange high performance liquid chromatography. Using this procedure, ubiquitin activating enzyme (E1), several distinct ubiquitin carrier proteins (E2s) with molecular masses of 16, 20, 23, 23.5, and 25 kilodaltons, and a ubiquitin-protein hydrolase (isopeptidase) were isolated. Purified E1 formed a thiol ester linkage with ¹²⁵I-ubiquitin in an ATP-dependent manner and transferred bound ubiquitin to the various purified E2s. The ubiquitin protein hydrolase fraction was sensitive to hemin, and in an ATP-independent reaction, was capable of removing the ubiquitin moiety from both ubiquitin ¹²⁵I-lysozyme conjugates (ε-amino or isopeptide linkage) and the ubiquitin 52-amino acid extension protein fusion (α-amino or peptide linkage). Using this procedure, wheat germ represents an inexpensive source from which enzymes involved in the ubiquitin pathway may be isolated.     

Physiological functions of FBW7 in cancer and metabolism

FBW7 is one of the most well characterized F-box proteins that serve as substrate recognition subunits of SCF (Skp1-Cullin 1-F-box proteins) E3 ubiquitin ligase complexes. SCF^FBW7 plays key roles in regulating cell cycle progression, differentiation, and stem cell maintenance largely through targeting a broad range of oncogenic substrates for proteasome-dependent degradation. The identification of an increasing number of FBW7 substrates for ubiquitination, and intensive in vitro and in vivo studies have revealed a network of signaling components controlled by FBW7 that contributes to metabolic regulation as well as its tumor suppressor role. Here we mainly focus on recent findings that highlight a critical role for FBW7 in cancer and metabolism.     

Identification of TRAF6 as a ubiquitin ligase engaged in the ubiquitination of SopB,a virulence effector protein secreted by Salmonella typhimurium

The phosphoinositide phosphatase SopB is one of the effectors injected by Salmonellatyphimurium (S.typhimurium) that diversifies its function through a ubiquitin-dependent differential localization. However, it is unclear which E3 ubiquitin ligase is responsible for ubiquitination of SopB. Based on the E1-E2-E3 trio of enzymes responsible for the ubiquitin activation and translocation to substrate proteins, we constructed an in vitro assay of SopB ubiquitination. Using this assay, we purified an E3 ubiquitin ligase, TRAF6, from the Henle-407 S100 extraction that may be responsible for the ubiquitination of SopB. To investigate the functional correlation of TRAF6, we showed that recombinant TRAF6 specifically ubiquitinates SopB in a dose-dependent manner in vitro. Upon infection, the ubiquitination of SopB was absolutely blocked by TRAF6 deletion, as shown in Traf6^−/− mouse embryonic fibroblasts (MEFs) compared with Traf6^+/+ MEFs. However, the ectopic expression of TRAF6 in Traf6^−/− MEFs rescued the two species of ubiquitin-conjugated SopB, which strengthens the role of TRAF6 in SopB ubiquitination. The analysis of E2 revealed that UbcH5c and not other E2 conjugating enzymes are required for TRAF6-mediated SopB ubiquitination both in vitro and in vivo. In summary, these results suggest the relevance of UbcH5c/TRAF6 in SopB during S.typhimurium infection and thereby imply that S.typhimurium has evolved a mechanism of utilizing the host’s E3 ubiquitin ligase to modify and modulate the function of its effector protein in order to ensure pathogen and host cell survival.     

SCFFBXO28-mediated self-ubiquitination of FBXO28 promotes its degradation

The F-box protein is the substrate recognition subunit of SCF (SKP1/CUL1/F-box) E3 ubiquitin ligase complex, a multicomponent RING-type E3 ligase involved in the regulation of numerous cellular processes by targeting critical regulatory proteins for ubiquitination. However, whether and how F-box proteins are regulated is largely unknown. Here we report that FBXO28, a poorly characterized F-box protein, is a novel substrate of SCF E3 ligase. Pharmaceutical or genetic inhibition of neddylation pathway that is required for the activation of SCF stabilizes FBXO28 and prolongs its half-life. Meanwhile, FBXO28 is subjected to ubiquitination and cullin1-based SCF complex promotes FBXO28 degradation. Moreover, deletion of F-box domain stabilizes FBXO28 and knockdown of endogenous FBXO28 strongly upregulates exogenous FBXO28 expression. Taken together, these data reveal that SCF^FBXO28 is the E3 ligase responsible for the self-ubiquitination and proteasomal degradation of FBXO28, providing a new clue for the upstream signaling regulation for F-box proteins.     

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.     

Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity

Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF^Cdc4 and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF^Cdc4 ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF^Cdc4 and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF^Cdc4 is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK.     

Engineering a Ubiquitin Ligase Reveals Conformational Flexibility Required for Ubiquitin Transfer

Protein ubiquitination regulates numerous cellular functions in eukaryotes. The prevailing view about the role of RING or U-box ubiquitin ligases (E3) is to provide precise positioning between the attached substrate and the ubiquitin-conjugating enzyme (E2). However, the mechanism of ubiquitin transfer remains obscure. Using the carboxyl terminus of Hsc70-interacting protein as a model E3, we show herein that although U-box binding is required, it is not sufficient to trigger the transfer of ubiquitin onto target substrates. Furthermore, additional regions of the E3 protein that have no direct contact with E2 play critical roles in mediating ubiquitin transfer from E2 to attached substrates. By combining computational structure modeling and protein engineering approaches, we uncovered a conformational flexibility of E3 that is required for substrate ubiquitination. Using an engineered version of the carboxyl terminus of Hsc70-interacting protein ubiquitin ligase as a research tool, we demonstrate a striking flexibility of ubiquitin conjugation that does not affect substrate specificity. Our results not only reveal conformational changes of E3 during ubiquitin transfer but also provide a promising approach to custom-made E3 for targeted proteolysis.Protein modification by ubiquitin and ubiquitin-like proteins is a common mechanism through which numerous cellular pathways are regulated (1). The canonical cascade of ubiquitination involves the action of three enzymes, termed E1, E2, and E3, which activate and then conjugate ubiquitin to its substrates (2, 3). The E3 ligase catalyzes the final step in ubiquitin transfer in a substrate-specific manner. Despite advances in understanding the enzymatic cascade of ubiquitination, the mechanism of ubiquitin transfer to the substrate remains an outstanding issue (4). In particular, the role of E3 ubiquitin ligases and how they adapt to progressively modified substrates to maintain specific ubiquitin chain topology is still a mystery.The known E3s belong to three protein families: HECT, RING, and U-box. HECT domain enzymes form a covalent intermediate with ubiquitin before the final transfer of ubiquitin to substrates. In contrast, RING and U-box E3s have been suggested to function as adaptors that position the substrate in close proximity to the E2-ubiquitin thioester (E2-Ub) (5). It has become common “wisdom” that the substrate has to be precisely positioned to get ubiquitinated (6). The positioning hypothesis originally predicted that E3 substrates would have a specific ubiquitination site. However, the absence of “consensus” ubiquitination sites has become apparent in an increasing list of E3 substrates (7–9). In addition, the crystal structures of several ubiquitination machinery components have revealed a puzzling gap (∼50 Å) between the substrate binding sites and the E2 active sites (10, 11). This raises a fundamental question in ubiquitin transfer. How does the ubiquitin molecule shuttle from the E2 to substrates? Though several interesting models for ubiquitin transfer have been proposed, only limited explicit experimental evidence support these models (4).We used carboxyl terminus of Hsc70-interacting protein (CHIP)³ as a model E3 system to investigate the role of substrate positioning in its ubiquitination. CHIP is a protein quality control E3 that consists of an NH₂-terminal tetratricopeptide repeat (TPR) domain, a helical linker domain, and a COOH-terminal U-box domain (12, 13). The TPR domain of CHIP binds directly to EEVD motifs located at the COOH termini of Hsc/Hsp70 and Hsp90, whereas the U-box domains possess ubiquitin ligase activity. CHIP recruits E2 enzymes of the Ubc4/5 family to ubiquitinate misfolded proteins that occupy the chaperone substrate-binding sites, thus remodeling the chaperones from protein-refolding complexes to complexes that promote degradation (14). Using the chaperone as an adaptor, CHIP targets a variety of substrates for ubiquitination (15). In the absence of substrates, CHIP is also able to ubiquitinate the bound chaperones (16). Thus, there is apparent substrate diversity for CHIP-mediated ubiquitination. Insights into the mechanism of action of CHIP have been provided by an x-ray crystal structure which reveals a remarkable, highly asymmetric dimer (25). Here, we demonstrate the existence of intrinsic structural flexibility in the CHIP homodimer that is required for substrate polyubiquitination. The flexible orientation allows CHIP to accommodate substrates with different sizes and structures. Mutations that restrict the flexibility of CHIP markedly decrease substrate ubiquitination, whereas maintaining flexibility enables us to rebuild a functional ubiquitin ligase with altered substrate specificity. Our results provide evidence for the importance of structural flexibility in E3 ligases, which we propose is of general importance to orchestrate progressive ubiquitin conjugation on substrates.     

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.     

A new general method for the biosynthesis of stable isotope-enriched peptides using a decahistidine-tagged ubiquitin fusion system: An application to the production of mastoparan-X uniformly enriched with 15N and 15N/13C

A new strategy is described for the production of peptides enriched with stable isotopes. Peptides of interest are expressed in Escherichia coli (E. coli) cells as recombinant fusion proteins with Saccharomyces cerevisiae ubiquitin. This method yields as much as 30–100 mg/l of isotope-enriched fusion proteins in minimal media. A decahistidine tag attached to the N-terminus of ubiquitin enables a one-step purification of the fusion protein via Ni²⁺-chelating affinity chromatography. The ubiquitin moiety is then easily and specifically cleaved off by a protease, yeast ubiquitin hydrolase. Since this enzyme is also expressed at a high level in E. coli cells and can be purified in one step, the presented strategy has an advantage in view of costs over others that use commercially available proteases. In addition, since ubiquitin fusion proteins easily refold, the fusion protein can be expressed either in a soluble form or as inclusion bodies. This flexibility enables us to prepare peptides that are unstable in a soluble state in E. coli cells. As an example, the expression and the uniform stable isotope enrichment with ¹⁵N and/or¹³ C are described for mastoparan-X, a tetradecapeptide known to activate GTP-binding regulatory proteins. An amide group at the C-terminus of this peptide can also be formed by our method. The presented system is considered powerful for the stable isotope enrichment of short peptides with proton resonances that are too severely overlapped to be analyzed solely by proton NMR.     

Activation of UBC5 ubiquitin-conjugating enzyme by the RING finger of ROC1 and assembly of active ubiquitin ligases by all cullins

Protein ubiquitination plays an important role in regulating the abundance and conformation of a broad range of eukaryotic proteins. This process involves a cascade of enzymes including ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). E1 and E2 represent two families of structurally related proteins and are relatively well characterized. In contrast, the nature and mechanism of E3, proposed to contain activities in catalyzing isopeptide bond formation (ubiquitin ligation) and substrate targeting, remains inadequately understood. Two major families of E3 ubiquitin ligases, the HECT (for homologous to E6-AP C terminus) family and the RING family, have been identified that utilize distinct mechanisms in promoting isopeptide bond formation. Here, we showed that purified RING finger domain of ROC1, an essential subunit of SKP1-cullin/CDC53-F box protein ubiquitin ligases, was sufficient to activate UBCH5c to synthesize polyubiquitin chains. The sequence flanking the RING finger in ROC1 did not contribute to UBCH5c activation, but was required for binding with CUL1. We demonstrated that all cullins, through their binding with ROC proteins, constituted active ubiquitin ligases, suggesting the existence in vivo of a large number of cullin-RING ubiquitin ligases. These results are consistent with the notion that the RING finger domains allosterically activate E2. We suggest that RING-E2, rather than cullin-RING, constitutes the catalytic core of the ubiquitin ligase and that one major function of the cullin subunit is to assemble the RING-E2 catalytic core and substrates together.     

Plant F-box Proteins – Judges between Life and Death

Selective protein degradation through the ubiquitin–26S proteasome system is a key mechanism for post-translational control of regulatory proteins in all eukaryotes. The pivotal components in this system are the multi-subunit E3 Ub-ligase enzymes responsible for specific recognition and ubiquitination of degradation targets. In this review, we focus on plant F-box proteins which confer specificity to the SCF-type E3 enzyme complexes. F-box proteins represent one of the largest and most heterogeneous superfamilies in plants, with hundreds of different representatives exposing an extensive variability of C-terminal target-binding domains, and as such, modulating almost every aspect of plant growth and development. Since the first reports on plant F-box proteins over a decade ago, a lot of progress has been made in our understanding of their relevance for plant physiology. In this review, we combine well-established knowledge with the most recent advances related to plant F-box proteins and their role in plant development, hormone signaling and defense pathways. We also elaborate on the yet poorly described carbohydrate-binding plant F-box proteins presumably targeting glycoproteins for proteasomal degradation.     

Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein)

The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca²⁺/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca²⁺/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca²⁺/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca²⁺/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca²⁺-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway.     

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.