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.     

Multimodal Mechanism of Action for the Cdc34 Acidic Loop: A CASE STUDY FOR WHY UBIQUITIN-CONJUGATING ENZYMES HAVE LOOPS AND TAILS*

Together with ubiquitin ligases (E3), ubiquitin-conjugating enzymes (E2) are charged with the essential task of synthesizing ubiquitin chains onto protein substrates. Some 75% of the known E2s in the human proteome contain unique insertions in their primary sequences, yet it is largely unclear what effect these insertions impart on the ubiquitination reaction. Cdc34 is an important E2 with prominent roles in cell cycle regulation and signal transduction. The amino acid sequence of Cdc34 contains an insertion distal to the active site that is absent in most other E2s, yet this acidic loop (named for its four invariably conserved acidic residues) is critical for Cdc34 function both in vitro and in vivo. Here we have investigated how the acidic loop in human Cdc34 promotes ubiquitination, identifying two key molecular events during which the acidic loop exerts its influence. First, the acidic loop promotes the interaction between Cdc34 and its ubiquitin ligase partner, SCF. Second, two glutamic acid residues located on the distal side of the loop collaborate with an invariably conserved histidine on the proximal side of the loop to suppress the pK_a of an ionizing species on ubiquitin or Cdc34 which greatly contributes to Cdc34 catalysis. These results demonstrate that insertions can guide E2s to their physiologically relevant ubiquitin ligases as well as provide essential modalities that promote catalysis.     

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.     

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.     

The CK2 phosphorylation of catalytic domain of Cdc34 modulates its activity at the G1 to S transition in Saccharomyces cerevisiae

The ubiquitin-conjugating enzyme Cdc34 was recently shown to be phosphorylated by CK2 on the C-terminal tail. Here we present novel findings indicating that in budding yeast CK2 phosphorylates Cdc34 within the N-terminal catalytic domain. Specifically, we show, by direct mass spectrometry analysis, that Cdc34 is phosphorylated in vitro and in vivo by CK2 on Ser130 and Ser167, and that the phosphoserines 130 and 167 are not present after CK2 inactivation in a cka1Δcka2-8ts strain. CK2 phosphorylation of Ser130 and Ser167 strongly stimulates Cdc34 ubiquitin charging in vitro. The Cdc34S130AS167A mutant shows a basal ubiquitin charging activity which is indistinguishable from that of wild type but is not activated by CK2 phosphorylation and its expression fails to complement a cdc34-2ts yeast strain, supporting a model in which activation of Cdc34 involves CK2-mediated phosphorylation of its catalytic domain.     

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 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.     

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.     

The E3 Ubiquitin Ligase Atrophin Interacting Protein 4 Binds Directly To The Chemokine Receptor CXCR4 Via a Novel WW Domain-mediated Interaction

The E3 ubiquitin ligase atrophin interacting protein 4 (AIP4) mediates ubiquitination and down-regulation of the chemokine receptor CXCR4. AIP4 belongs to the Nedd4-like homologous to E6-AP carboxy terminus domain family of E3 ubiquitin ligases, which typically bind proline-rich motifs within target proteins via the WW domains. The intracellular domains of CXCR4 lack canonical WW domain binding motifs; thus, whether AIP4 is targeted to CXCR4 directly or indirectly via an adaptor protein remains unknown. Here, we show that AIP4 can interact directly with CXCR4 via a novel noncanonical WW domain-mediated interaction involving serine residues 324 and 325 within the carboxy-terminal tail of CXCR4. These serine residues are critical for mediating agonist-promoted binding of AIP4 and subsequent ubiquitination and degradation of CXCR4. These residues are phosphorylated upon agonist activation and phosphomimetic mutants show enhanced binding to AIP4, suggesting a mechanism whereby phosphorylation mediates the interaction between CXCR4 and AIP4. Our data reveal a novel noncanonical WW domain-mediated interaction involving phosphorylated serine residues in the absence of any proline residues and suggest a novel mechanism whereby an E3 ubiquitin ligase is targeted directly to an activated G protein-coupled receptor.     

Protein kinase CK1delta phosphorylates key sites in the acidic domain of murine double-minute clone 2 protein (MDM2) that regulate p53 turnover

Murine double-minute clone 2 protein (MDM2) is an E3 ubiquitin ligase that regulates the turnover of several cellular factors including the p53 tumor suppressor protein. As part of the mechanism of p53 induction in response to DNA damage, a cluster of serine residues within the central acidic domain of MDM2 become hypophosphorylated, leading to attenuation of MDM2-mediated p53 destruction. In the present study, we identify the protein kinase CK1delta as a major cellular activity that phosphorylates MDM2. Amino acid substitution, coupled with phosphopeptide analyses, indicates that several serine residues in the acidic domain, including Ser-240, Ser-242, and Ser-246, as well as Ser-383 in the C-terminal region, are phosphorylated by CK1delta in vitro. We also show, through expression of a dominant negative mutant of CK1delta or treatment of cells with IC261, a CK1delta-selective inhibitor, that MDM2 is phosphorylated by CK1delta in cultured cells. These data establish the identity of a key signaling molecule that promotes the phosphorylation of a major regulatory region in MDM2 under normal growth conditions.     

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.     

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.     

Architecture of the catalytic HPN motif is conserved in all E2 conjugating enzymes

E2 conjugating enzymes are the central enzymes in the ubiquitination pathway and are responsible for the transfer of ubiquitin and ubiquitin-like proteins on to target substrates. The secondary structural elements of the catalytic domain of these enzymes is highly conserved, including the sequence conservation of a three-residue HPN (His-Pro-Asn) motif located upstream of the active-site cysteine residue used for ubiquitin conjugation. Despite the vast structural knowledge of E2 enzymes, the catalytic mechanism of these enzymes remains poorly understood, in large part due to variation in the arrangements of the residues in the HPN motif in existing E2 structures. In the present study, we used the E2 enzyme HIP2 to probe the structures of the HPN motif in several other E2 enzymes. A combination of chemical-shift analysis, determination of the histidine protonation states and amide temperature coefficients were used to determine the orientation of the histidine ring and hydrogen-bonding arrangements within the HPN motif. Unlike many three-dimensional structures, we found that a conserved hydrogen bond between the histidine imidazole ring and the asparagine backbone amide proton, a common histidine protonation state, and a common histidine orientation exists for all E2 enzymes examined. These results indicate that the histidine within the HPN motif is orientated to structurally stabilize a tight turn motif in all E2 enzymes and is not orientated to interact with the asparagine side chain as proposed in some mechanisms. These results suggest that a common catalysis mechanism probably exists for all E2 conjugating enzymes to facilitate ubiquitin transfer.     

HIF-1α的泛素化和SUMO化修饰

低氧诱导因子-1（hypoxia-inducible factor-1,HIF-1）是异二聚体的转录因子,由氧敏感的α亚基和在细胞内稳定表达的β亚基组成,在细胞低氧应答反应中起核心作用,能调节100多种涉及低氧应激下细胞适应和存活的靶基因.泛素是一种由76个氨基酸残基组成的保守性多肽,广泛存在真核生物中.SUMO是泛素样蛋白家族成员,分子量约为12 kD的小蛋白,从拟南芥到人类普遍存在.泛素和SUMO可共价结合许多靶底物蛋白,对其进行翻译后修饰,该过程分别称为泛素化与SUMO化.近来研究显示,HIF-1α的翻译后修饰如泛素化、SUMO化可调节其的稳定性,从而改变HIF 1α的转录激活活性.本文主要就HIF-1α泛素化及SUMO化修饰等问题作一综述.     

Reciprocally interacting domains of protein phosphatase 1 and focal adhesion kinase

The Murine double-minute clone 2 (Mdm2) onco-protein is the principal regulator of the tumour suppressor, p53. Mdm2 acts as an E3-type ubiquitin ligase that mediates the ubiquitylation and turnover of p53 under normal, unstressed circumstances. In response to cellular stress, such as DNA damage, the Mdm2–p53 interaction is disrupted. Part of the mechanism of uncoupling p53 from Mdm2-mediated degradation involves hypo-phosphorylation of a cluster of phosphorylated serine residues in the central acidic domain of Mdm2. Here, we show that two of the residues within this domain that are phosphorylated in vivo, Ser-260 and Ser-269, are phosphorylated by CK2 in vitro. Treatment of cells with the CK2 inhibitor, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), leads to the induction of p53 and downstream targets of p53 including Mdm2 itself and p21. These data are consistent with the idea that CK2-mediated phosphorylation of Mdm2 may regulate Mdm2-mediated p53 turnover.     

CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37

Cdc37 is a kinase-associated molecular chaperone whose function in concert with Hsp90 is essential for many signaling protein kinases. Here, we report that mammalian Cdc37 is a pivotal substrate of CK2 (casein kinase II). Purified Cdc37 was phosphorylated in vitro on a conserved serine residue, Ser13, by CK2. Moreover, Ser13 was the unique phosphorylation site of Cdc37 in vivo. Crucially, the CK2 phosphorylation of Cdc37 on Ser13 was essential for the optimal binding activity of Cdc37 toward various kinases examined, including Raf1, Akt, Aurora-B, Cdk4, Src, MOK, MAK, and MRK. In addition, nonphosphorylatable mutants of Cdc37 significantly suppressed the association of Hsp90 with protein kinases, while the Hsp90-binding activity of the mutants was unchanged. The treatment of cells with a specific CK2 inhibitor suppressed the phosphorylation of Cdc37 in vivo and reduced the levels of Cdc37 target kinases. These results unveil a regulatory mechanism of Cdc37, identify a novel molecular link between CK2 and many crucial protein kinases via Cdc37, and reveal the molecular basis for the ability of CK2 to regulate pleiotropic cellular functions.     

E1-E2 interactions in ubiquitin and Nedd8 ligation pathways

Initial rates of E1-catalyzed E2 transthiolation have been used as a reporter function to probe the mechanism of 125I-ubiquitin transfer between activation and ligation half-reactions of ubiquitin conjugation. A functional survey of 11 representative human E2 paralogs reveals similar Km for binding to human Uba1 ternary complex (Km(ave)=121±72 nm) and kcat for ubiquitin transfer (kcat(ave)=4.0±1.2 s(-1)), suggesting that they possess a conserved binding site and transition state geometry and that they compete for charging through differences in intracellular concentration. Sequence analysis and mutagenesis localize this binding motif to three basic residues within Helix 1 of the E2 core domain, confirmed by transthiolation kinetics. Partial conservation of the motif among E2 paralogs not recognized by Uba1 suggests that another factor(s) account for the absolute specificity of cognate E2 binding. Truncation of the Uba1 carboxyl-terminal β-grasp domain reduces cognate Ubc2b binding by 31-fold and kcat by 3.5×10(4)-fold, indicating contributions to E2 binding and transition state stabilization. Truncation of the paralogous domain from the Nedd8 activating enzyme has negligible effect on cognate Ubc12 transthiolation but abrogates E2 specificity toward non-cognate carrier proteins. Exchange of the β-grasp domains between ubiquitin and Nedd8 activating enzymes fails to reverse the effect of truncation. Thus, the conserved Helix 1 binding motif and the β-grasp domain direct general E2 binding, whereas the latter additionally serves as a specificity filter to exclude charging of non-cognate E2 paralogs in order to maintain the fidelity of downstream signaling.     

PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity

PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin’s ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.