Parkin phosphorylation and modulation of its E3 ubiquitin ligase activity

Mutations in the PARKIN gene are the most common cause of hereditary parkinsonism. The parkin protein comprises an N-terminal ubiquitin-like domain, a linker region containing caspase cleavage sites, a unique domain in the central portion, and a special zinc finger configuration termed RING-IBR-RING. Parkin has E3 ubiquitin-protein ligase activity and is believed to mediate proteasomal degradation of aggregation-prone proteins. Whereas the effects of mutations on the structure and function of parkin have been intensely studied, post-translational modifications of parkin and the regulation of its enzymatic activity are poorly understood. Here we report that parkin is phosphorylated both in human embryonic kidney HEK293 cells and human neuroblastoma SH-SY5Y cells. The turnover of parkin phosphorylation was rapid, because inhibition of phosphatases with okadaic acid was necessary to stabilize phosphoparkin. Phosphoamino acid analysis revealed that phosphorylation occurred mainly on serine residues under these conditions. At least five phosphorylation sites were identified, including Ser101, Ser131, and Ser136 (located in the linker region) as well as Ser296 and Ser378 (located in the RING-IBR-RING motif). Casein kinase-1, protein kinase A, and protein kinase C phosphorylated parkin in vitro, and inhibition of casein kinase-1 caused a dramatic reduction of parkin phosphorylation in cell lysates. Induction of protein folding stress in cells reduced parkin phosphorylation, and unphosphorylated parkin had slightly but significantly elevated autoubiquitination activity. Thus, complex regulation of the phosphorylation state of parkin may contribute to the unfolded protein response in stressed cells.     

Drosophila melanogaster Parkin ubiquitinates peanut and septin1 as an E3 ubiquitin-protein ligase

Autosomal recessive juvenile parkinsonism (AR-JP), a common familial form of Parkinson's disease, is caused by mutations of human Parkin. To deepen the understanding of Parkin biology in an in vivo model of Drosophila, we attempted to characterize the function of Drosophila melanogaster Parkin and found that D. melanogaster Parkin exhibited UbcH8-dependent E3 ubiquitin-protein ligase activity. Using E2 binding and in vitro ubiquitination assays, UbcH8 preferentially was found to bind to Parkin mutants harboring functional RING1 domains, but failed to bind to mutants harboring point mutants with complete loss of function. This inability of UbcH8 binding to such mutants was accompanied by abrogation of an E3 ligase activity, indicating that D. melanogaster Parkin as an E3 ligase interacts with UbcH8 through its RING1 domain. An in vivo ubiquitination assay revealed that D. melanogaster Parkin existed in ubiquitinated form in vivo. Moreover, peanut and septin1, D. melanogaster septin proteins, were also ubiquitinated by D. melanogaster Parkin. Co-immunoprecipitation with membrane protein Syntaxin indicated direct binding of septin proteins to syntaxin, implicating their relevance in the exocytosis of dopamine in cells. Western blot analysis and DNA fragmentation indicated that the rate and efficiency of p53-dependent apoptosis were significantly higher in the presence of dopamine than without the septin proteins. Therefore, our findings in the present study demonstrate that Parkin possibly influences septin protein effects on p53-mediated apoptosis, helping to extend the utility of Drosophila as a model system for the study of neurodegeneration.     

E3 ubiquitin-protein ligase activity of Parkin is dependent on cooperative interaction of RING finger (TRIAD) elements

The parkin gene codes for a 465-amino acid protein which, when mutated, results in autosomal recessive juvenile parkinsonism (AR-JP). Symptoms of AR-JP are similar to those of idiopathic Parkinson's disease, with the notable exception being the early onset of AR-JP. We have cloned and expressed human Parkin in Escherichia coli and have examined Parkin-mediated ubiquitination in an in vitro ubiquitination assay using purified recombinant proteins. We found that Parkin has E3 ubiquitin ligase activity in this system, demonstrating for the first time that the E3 activity is an intrinsic function of the Parkin protein and does not require posttranslational modification or association with cellular proteins other than an E2 (human Ubc4 E2 was utilized in this ubiquitination assay). Mutagenesis of individual elements of the conserved RING TRIAD domain indicated that at least two elements were required for ubiquitin ligase activity and suggested a functional cooperation between the RING finger elements. Since the activity assays were conducted with recombinant proteins purified from E. coli, this is the first time TRIAD element interaction has been demonstrated as an intrinsic feature of Parkin E3 activity.     

CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein

The ubiquitin–proteasome system catalyses the immediate destruction of misfolded or impaired proteins generated in cells, but how this proteolytic machinery recognizes abnormality of cellular proteins for selective elimination remains elusive. Here, we report that the C-terminus of Hsc70-interacting protein (CHIP) with a U-box domain is an E3 ubiquitin-ligase collaborating with molecular chaperones Hsp90 and Hsc70. Thermally denatured firefly luciferase was multiubiquitylated by CHIP in the presence of E1 and E2 (Ubc4 or UbcH5c) in vitro, only when the unfolded substrate was captured by Hsp90 or Hsc70 and Hsp40. No ubiquitylating activity was detected in CHIP lacking the U-box region. CHIP efficiently ubiquitylated denatured luciferase trapped by the C-terminal region of Hsp90, which contains a CHIP binding site. CHIP also showed self-ubiquitylating activity independent of target ubiquitylation. Our results indicate that CHIP can be regarded as ‘a quality-control E3’ that selectively ubiquitylates unfolded protein(s) by collaborating with molecular chaperones.     

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.     

Small, N-terminal tags activate Parkin E3 ubiquitin ligase activity by disrupting its autoinhibited conformation

Parkin is an E3 ubiquitin ligase, mutations in which cause Autosomal Recessive Parkinson's Disease. Many studies aimed at understanding Parkin function, regulation and dysfunction are performed using N-terminal epitope tags. We report here that the use of small tags such as FLAG, cMyc and HA, influence the physical stability and activity of Parkin in and out of cells, perturbing the autoinhibited native state of Parkin, resulting in an active-for-autoubiquitination species.     

CARD-mediated autoinhibition of cIAP1's E3 ligase activity suppresses cell proliferation and migration

E3 ligases mediate the covalent attachment of ubiquitin to target proteins thereby enabling ubiquitin-dependent signaling. Unraveling how E3 ligases are regulated is important because miscontrolled ubiquitylation can lead to disease. Cellular inhibitor of apoptosis (cIAP) proteins are E3 ligases that modulate diverse biological processes such as cell survival, proliferation, and migration. Here, we have solved the structure of the caspase recruitment domain (CARD) of cIAP1 and identified that it is required for cIAP1 autoregulation. We demonstrate that the CARD inhibits activation of cIAP1's E3 activity by preventing RING dimerization, E2 binding, and E2 activation. Moreover, we show that the CARD is required to suppress cell proliferation and migration. Further, CARD-mediated autoregulation is also necessary to maximally suppress caspase-8-dependent apoptosis and vascular tree degeneration in vivo. Taken together, our data reveal mechanisms by which the E3 ligase activity of cIAP1 is controlled, and how its deregulation impacts on cell proliferation, migration and cell survival.     

The serine protease HtrA2/Omi cleaves Parkin and irreversibly inactivates its E3 ubiquitin ligase activity

The serine protease HtrA2 is important in regulating not only apoptosis but also cellular homeostasis. Recently, several lines of evidence suggest that HtrA2 may be intimately associated with Parkin; however, little is known about the functional relationships between HtrA2 and Parkin. Here we have shown that HtrA2 is co-localized with Parkin in the cytosol through the release of HtrA2 from the mitochondria upon cellular stresses. Moreover, endogenous levels of Parkin were significantly decreased in wild-type (HtrA2^+/+) mouse embryonic fibroblasts (MEF) compared with those in HtrA2-knockout (HtrA2^−/−) MEF under the same stress conditions. Using cleavage and binding assays, we have demonstrated that HtrA2 specifically binds to and directly cleaves the E3 ubiquitin (Ub) ligase Parkin. Interestingly, the HtrA2-mediated Parkin cleavage irreversibly disrupts Parkin-mediated synphilin-1 ubiquitination and autoubiquitination, indicating that HtrA2 may play a critical role in the Parkin-related pathway involved in the ubiquitin proteasome system.     

The interferon-inducible ubiquitin-protein isopeptide ligase (E3) EFP also functions as an ISG15 E3 ligase

The expression of the ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) are strongly activated by interferons. Accordingly, ISG15 expression and protein ISGylation are strongly activated upon viral and bacterial infections and during other stress conditions, suggesting important roles for the ISG15 system in innate immune responses. Here, we report the identification of the ubiquitin-protein isopeptide ligase (E3) EFP (estrogen-responsive finger protein) as the ISG15 E3 ligase for 14-3-3sigma protein. Like other known components of the protein ISGylation system (ISG15, UBE1L, UBP43, and UBC8), EFP is also an interferon-inducible protein. Expression of EFP small interfering RNA decreased the ISGylation of 14-3-3sigma in the 293T cell ISGylation system as well as in MCF-7 cells upon interferon treatment. Furthermore, the ISGylation enzyme activity of EFP was RING domain-dependent. These findings indicate that EFP is an ISG15 E3 ligase for 14-3-3sigma in vivo. The fact that both UBC8 and EFP are common components in the ubiquitin and ISG15 conjugation pathways suggests a mechanism whereby a limited set of enzymes accomplishes diverse post-translational modifications of their substrates in response to changes in environmental stimulations.     

Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein

Ubiquitin- and proteasome-dependent outer mitochondrial membrane (OMM)-associated degradation (OMMAD) is critical for mitochondrial and cellular homeostasis. However, the scope and molecular mechanisms of the OMMAD pathways are still not well understood. We report that the OMM-associated E3 ubiquitin ligase MARCH5 controls dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and cell sensitivity to stress-induced apoptosis. MARCH5 knockout selectively inhibited ubiquitination and proteasomal degradation of MiD49, a mitochondrial receptor of Drp1, and consequently led to mitochondrial fragmentation. Mitochondrial fragmentation in MARCH5^−/− cells was not associated with inhibition of mitochondrial fusion or bioenergetic defects, supporting the possibility that MARCH5 is a negative regulator of mitochondrial fission. Both MARCH5 re-expression and MiD49 knockout in MARCH5^−/− cells reversed mitochondrial fragmentation and reduced sensitivity to stress-induced apoptosis. These findings and data showing MARCH5-dependent degradation of MiD49 upon stress support the possibility that MARCH5 regulation of MiD49 is a novel mechanism controlling mitochondrial fission and, consequently, the cellular response to stress.     

Inactivating the ubiquitin ligase Parkin suppresses cell proliferation and induces apoptosis in human keloids

Keloids are a common type of pathological skin healing, characterized by the destruction of the vascular network. Thus, keloids often exhibit anoxic conditions. Hypoxia-inducible factor-1α (HIF-1α) is a core factor that mediates hypoxia stress responses and allows the cells to adapt to low-oxygen conditions. In the current study, we identified that Parkin acted as an E3 ubiquitin ligase, contributing to the degradation of HIF-1α in keloid fibroblasts (KFs). Silencing of Parkin in KFs upregulated HIF-1α expression and prolonged its protein half-life. Furthermore, Parkin influenced transforming growth factor β (TGF-β)/Smad signaling by targeting HIF-1α. Under hypoxic conditions, silencing Parkin enhanced KF proliferation and inhibited apoptosis through the TGF-β/Smad signaling pathway. Notably, metformin, an antidiabetic drug, could significantly induce Parkin expression and enhance the interaction between Parkin and HIF-1α. As a result, we revealed an important mechanism for Parkin in keloid development and suggested that targeting Parkin could be an alternative method for keloid treatment.     

The tight junction-specific protein occludin is a functional target of the E3 ubiquitin-protein ligase itch.

Tight junctions create a highly selective diffusion barrier between epithelial and endothelial cells by preventing the free passage of molecules and ions across the paracellular pathway. Although the regulation of this barrier is still enigmatic, there is evidence that junctional transmembrane proteins are critically involved. Recent evidence confirms the notion that occludin, a four-pass integral plasma-membrane protein, is a functional component of the paracellular barrier. The overall hydrophilicity of occludin predicts two extracellular loops bounded by NH(2)- and COOH-terminal cytoplasmic domains. To date, the binding of the COOH terminus of occludin to intracellular proteins is well documented, but information concerning the function of the cytoplasmic NH(2) terminus is still lacking. Using yeast two-hybrid screening we have identified a novel interaction between occludin and the E3 ubiquitin-protein ligase Itch, a member of the HECT domain-containing ubiquitin-protein ligases. We have found that the NH(2)-terminal portion of occludin binds specifically to a multidomain of Itch, consisting of four WW motifs. This interaction has been confirmed by our results from in vivo and in vitro co-immunoprecipitation experiments. In addition, we provide evidence that Itch is specifically involved in the ubiquitination of occludin in vivo, and that the degradation of occludin is sensitive to proteasome inhibition.     

Vitamin E protected cultured cortical neurons from oxidative stress-induced cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase

The role of vitamin E in the CNS has not been fully elucidated. In the present study, we found that pre-treatment with vitamin E analogs including alphaT (alpha-tocopherol), alphaT3 (alpha -tocotrienol), gammaT, and gammaT3 for 24 h prevented the cultured cortical neurons from cell death in oxidative stress stimulated by H2O2, while Trolox, a cell-permeable analog of alphaT, did not. The preventive effect of alphaT was dependent on de novo protein synthesis. Furthermore, we found that alphaT exposure induced the activation of both the MAP kinase (MAPK) and PI3 kinase (PI3K) pathways and that the alphaT-dependent survival effect was blocked by the inhibitors, U0126 (an MAPK pathway inhibitor) or LY294002 (a PI3K pathway inhibitor). Interestingly, the up-regulation of Bcl-2 (survival promoting molecule) was induced by alphaT application. The up-regulation of Bcl-2 did not occur in the presence of U0126 or LY294002, suggesting that alphaT-up-regulated Bcl-2 is mediated by these kinase pathways. These observations suggest that vitamin E analogs play an essential role in neuronal maintenance and survival in the CNS.     

Identification of determinants in E2 ubiquitin-conjugating enzymes required for hect E3 ubiquitin-protein ligase interaction

Members of the hect domain protein family are characterized by sequence similarity of their C-terminal regions to the C terminus of E6-AP, an E3 ubiquitin-protein ligase. An essential intermediate step in E6-AP-dependent ubiquitination is the formation of a thioester complex between E6-AP and ubiquitin in the presence of distinct E2 ubiquitin-conjugating enzymes including human UbcH5, a member of the UBC4/UBC5 subfamily of E2s. Similarly, several hect domain proteins, including Saccharomyces cerevisiae RSP5, form ubiquitin thioester complexes, indicating that hect domain proteins in general have E3 activity. We show here, by the use of chimeric E2s generated between UbcH5 and other E2s, that a region of UbcH5 encompassing the catalytic site cysteine residue is critical for its ability to interact with E6-AP and RSP5. Of particular importance is a phenylalanine residue at position 62 of UbcH5 that is conserved among the members of the UBC4/UBC5 subfamily but is not present in any of the other known E2s, whereas the N-terminal 60 amino acids do not contribute significantly to the specificity of these interactions. The conservation of this phenylalanine residue throughout evolution underlines the importance of the ability to interact with hect domain proteins for the cellular function of UBC4/UBC5 subfamily members.     

Autoregulation of Parkin activity through its ubiquitin-like domain

Parkin is an E3-ubiquitin ligase belonging to the RBR (RING-InBetweenRING-RING family), and is involved in the neurodegenerative disorder Parkinson's disease. Autosomal recessive juvenile Parkinsonism, which is one of the most common familial forms of the disease, is directly linked to mutations in the parkin gene. However, the molecular mechanisms of Parkin dysfunction in the disease state remain to be established. We now demonstrate that the ubiquitin-like domain of Parkin functions to inhibit its autoubiquitination. Moreover pathogenic Parkin mutations disrupt this autoinhibition, resulting in a constitutively active molecule. In addition, we show that the mechanism of autoregulation involves ubiquitin binding by a C-terminal region of Parkin. Our observations provide important molecular insights into the underlying basis of Parkinson's disease, and in the regulation of RBR E3-ligase activity.     

Staring,a novel E3 ubiquitin-protein ligase that targets syntaxin 1 for degradation

Syntaxin 1 is an essential component of the neurotransmitter release machinery, and regulation of syntaxin 1 expression levels is thought to contribute to the mechanism underlying learning and memory. However, the molecular events that control the degradation of syntaxin 1 remain undefined. Here we report the identification and characterization of a novel RING finger protein, Staring, that interacts with syntaxin 1. Staring is expressed throughout the brain, where it exists in both cytosolic and membrane-associated pools. Staring binds and recruits the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 to syntaxin 1 and facilitates the ubiquitination and proteasome-dependent degradation of syntaxin 1. These findings suggest that Staring is a novel E3 ubiquitin-protein ligase that targets syntaxin 1 for degradation by the ubiquitin-proteasome pathway.     

Enhancement of BRCA1 E3 ubiquitin ligase activity through direct interaction with the BARD1 protein

The breast and ovarian cancer-specific tumor suppressor RING finger protein BRCA1 has been identified as an E3 ubiquitin (Ub) ligase through in vitro studies, which demonstrated that its RING finger domain can autoubiquitylate and monoubiquitylate histone H2A when supplied with Ub, E1, and UBC4 (E2). Here we report that the E3 ligase activity of the N-terminal 110 amino acid residues of BRCA1, which encodes a stable domain containing the RING finger, as well as that of the full-length BRCA1, was significantly enhanced by the BARD1 protein (residues 8-142), whose RING finger domain itself lacked Ub ligase activity in vitro. The results of mutagenesis studies indicate that the enhancement of BRCA1 E3 ligase activity by BARD1 depends on direct interaction between the two proteins. Using K48A and K63A Ub mutants, we found that BARD1 stimulated the formation of both Lys(48)- and Lys(63)-linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys(63), which could potentially activate biological pathways other than BRCA1 degradation. We also found that co-expression of BRCA1 and BARD1 in living cells increased the abundance and stability of both proteins and that this depended on their ability to heterodimerize.