Deubiquitinating enzyme USP36 contains the PEST motif and is polyubiquitinated

The ubiquitin-mediated protein degradation pathway has been emphasized for the regulation of numerous cellular mechanisms and the significance of deubiquitination, mediated by deubiquitinating (DUB) enzymes, has been emerging as an essential regulatory step to control these cellular mechanisms. Previously, we demonstrated a human DUB enzyme, HeLa DUB-1, expressed in human ovarian cancer cells. Here, we report human USP36, which has the extension of the C-terminal region of HeLa DUB-1 and has conserved amino acid domains as previously shown in other DUBs. Human USP36, encoding a DUB enzyme, was isolated from ovarian cancer cells using RT-PCR and characterized. We identified DUB enzyme activity of USP36 by analyzing its capability to cleave the ubiquitin. Interestingly, structural and immunoprecipitation analyses revealed for the first time that USP36 contains the PEST motif and is polyubiquitinated.     

Ubiquitin-specific cysteine protease 2a (USP2a) regulates the stability of Aurora-A

The ubiquitin/proteasome pathway plays critical roles in virtually all aspects of cell biology. Enzymes of the ubiquitin pathway add (ligases) or remove (deubiquitinases) ubiquitin tags to or from their target proteins in a selective fashion. USP2a is a member of a subfamily of deubiquitinases, called ubiquitin-specific cysteine proteases (USPs). Although USP2a has been reported to be a bona fide oncogene that regulates the stability of MDM2, MDMX, and FAS, it is likely that there are other unidentified substrates for USP2a. In this study, we show that USP2a mediates mitotic progression by regulating the stability of Aurora-A. Through cell-based screening of a USP siRNA library, we discovered that knockdown of USP2a reduced the protein levels of Aurora-A. USP2a interacts with Aurora-A directly in vitro and in vivo. In addition, Aurora-A is a substrate for USP2a in vitro and in vivo. Our study provides a novel mechanism for the role of USP2a in mediating the stability of Aurora-A.     

Deubiquitination of Chfr, a checkpoint protein, by USP7/HAUSP regulates its stability and activity

Chfr, a mitotic stress checkpoint, plays an important role in cell cycle progression, tumor suppression and the processes that require the E3 ubiquitin ligase activity mediated by the RING finger domain. Chfr stimulates the formation of polyubiquitin chains by ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins including Plk1 and Aurora A. In this study, we identified USP7 (also known as HAUSP), which is a member of a family of proteins that cleave polyubiquitin chains and/or ubiquitin precursors, as an interacting protein with Chfr by immunoaffinity purification and mass spectrometry, and their interaction greatly increases the stability of Chfr. In fact, USP7 can remove ubiquitin moiety from the autoubiquitinated Chfr both in vivo and in vitro, which results in the accumulation of Chfr in the cell. Thus, our finding suggests that USP7-mediated deubiquitination of Chfr leads to its accumulation, which might be a key regulatory step for Chfr activation and that USP7 may play an important role in the regulation of Chfr-mediated cellular processes including cell cycle progression and tumor suppression.     

The Histone H2A Deubiquitinase USP16 Interacts with HERC2 and Fine-tunes Cellular Response to DNA Damage

Histone ubiquitination at DNA double strand breaks facilitates the recruitment of downstream repair proteins; however, how the ubiquitination is dynamically regulated during repair and terminated after repair is not well understood. Here we report that the histone H2A deubiquitinase USP16 interacts with HERC2, fine-tunes the ubiquitin signal during repair, and importantly, is required for terminating the ubiquitination signal after repair. HERC2 interacts with the coiled-coil domain of USP16 through its C-terminal HECT domain. HERC2 knockdown affects the levels of ubiquitinated H2A through the action of USP16. In response to DNA damage, USP16 levels increase, and this increase is dependent on HERC2. Increased USP16 serves as a negative regulator for DNA damage-induced ubiquitin foci formation and affects downstream factor recruitment and DNA damage response. The functional significance of USP16 is further manifested in human Down syndrome patient cells, which contain three copies of USP16 genes and have altered cellular response to DNA damage. Finally, we demonstrated that USP16 could deubiquitinate both H2A Lys-119 and H2A Lys-15 ubiquitination in vitro. Therefore, this study identifies USP16 as a critical regulator of DNA damage response and H2A Lys-15 ubiquitination as a potential target of USP16.     

The Deubiquitinating Enzyme USP8 Promotes Trafficking and Degradation of the Chemokine Receptor 4 at the Sorting Endosome

Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the early-to-late endosome transition.     

Ataxin-3 deubiquitination is coupled to Parkin ubiquitination via E2 ubiquitin-conjugating enzyme

We reported previously that parkin, a Parkinson disease-associated E3 ubiquitin-ligase interacts with ataxin-3, a deubiquitinating enzyme associated with Machado-Joseph disease. Ataxin-3 was found to counteract parkin self-ubiquitination both in vitro and in cells. Moreover, ataxin-3-dependent deubiquitination of parkin required the catalytic cysteine 14 in ataxin-3, although the precise mechanism remained unclear. We report here that ataxin-3 interferes with the attachment of ubiquitin (Ub) onto parkin in real-time during conjugation but is unable to hydrolyze previously assembled parkin-Ub conjugates. The mechanism involves an ataxin-3-dependent stabilization of the complex between parkin and the E2 Ub-conjugating enzyme, which impedes the efficient charging of the E2 with Ub. Moreover, within this complex, the transfer of Ub from the E2 is diverted away from parkin and onto ataxin-3, further explaining how ataxin-3 deubiquitination is coupled to parkin ubiquitination. Taken together, our findings reveal an unexpected convergence upon the E2 Ub-conjugating enzyme in the regulation of an E3/deubiquitinating enzyme pair, with important implications for the function of parkin and ataxin-3, two proteins responsible for closely related neurodegenerative diseases.     

USP8 and PARK2/parkin-mediated mitophagy

The Parkinson disease (PD)-associated E3-ubiquitin (Ub) ligase PARK2/parkin plays a central role in many stress response pathways, and in particular, in mitochondrial quality control. Within this pathway, PARK2 activation is accompanied by a robust increase in its autoubiquitination, followed by clearance of the damaged mitochondria by selective autophagy (mitophagy). Yet, little is known about how this auto-ubiquitination is regulated during mitophagy. In our study, we demonstrate that PARK2 forms predominantly K6-linked Ub conjugates on itself. Moreover, PARK2 interacts with the deubiquitinating enzyme USP8 that preferentially removes these K6-linked conjugates, thereby regulating the activity and function of PARK2 in the pathway. When USP8 is silenced, a persistence of K6-linked Ub conjugates on PARK2 delays both its translocation to damaged mitochondria and successful completion of mitophagy. Taken together, these findings implicate a novel role for K6-linked Ub conjugates and USP8-mediated deubiquitination in the regulation of PARK2 in mitochondrial quality control.     

Epidermal Growth Factor Receptor is a Common Element in the Signaling Pathways Activated by Cell Volume Changes in Isosmotic,Hyposmotic or Hyperosmotic Conditions

Changes in external osmolarity, including both hyper- or hyposmotic conditions, elicit the tyrosine phosphorylation of a number of tyrosine kinase receptors (TKR). We show here that the epidermal growth factor receptor (EGFR) is activated by both cell swelling (hyposmolarity, isosmotic urea, hyperosmotic sorbitol) or shrinkage (hyperosmotic NaCl or raffinose) and discuss the mechanisms by which these apparently opposed conditions come to the same effect, i.e., EGFR activation. Evidence suggests that this results from early activation of integrins, p38 and tyrosine kinases of the Src family, which are all activated in the two anisosmotic conditions. TKR transactivation by integrins and p38 is likely occurring via an effect on the metalloproteinases. Information discussed in this review, points to TKR as elements in osmotransduction as a useful mechanism to amplify and diversify the initial response to anisosmolarity and cell volume changes, due to their privileged situation as convergence point for numerous intracellular signaling pathways. The variety of effector pathways connected to TKR is advantageous for the cell to cope with the changes in cell volume including adaptation to stress, cytoskeleton remodeling, adhesion reactions, cell survival and the adaptive mechanisms to ultimately restore the original cell volume. Special Issue dedicated to Dr. Simo S. Oja     

Caspase-8-mediated cleavage inhibits IRF-3 protein by facilitating its proteasome-mediated degradation

Interferon regulatory factor 3 (IRF-3) plays a central role in inducing the expression of cellular antiviral genes, including the interferon-β gene, in response to Pattern Recognition Receptors. IRF-3 is targeted for proteasome-mediated degradation, which modulates the strength and duration of the innate immune responses that depend on it. We have found that caspase-8, which is activated by cytosolic RIG-I-dependent signaling, catalyzes an essential intermediate step in the ubiquitination and proteasome-mediated degradation of IRF-3. Mutation of a consensus cleavage site within IRF-3 generates a form that is not cleaved by caspase-8 and that is protected from ubiquitination and degradation. An in vitro assay confirms the direct action of caspase-8 cleavage on IRF-3. We also show that caspase-8-mediated cleavage of IRF-3 helps to modulate dsRNA-dependent gene induction.     

Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme found in all eukaryotes that catalyzes the removal of ubiquitin from specific target proteins. Here, we report that UbE2E1, an E2 ubiquitin conjugation enzyme with a unique N-terminal extension, is a novel USP7-interacting protein. USP7 forms a complex with UbE2E1 in vitro and in vivo through the ASTS USP7 binding motif within its N-terminal extension in an identical manner with other known USP7 binding proteins. We show that USP7 attenuates UbE2E1-mediated ubiquitination, an effect that requires the N-terminal ASTS sequence of UbE2E1 as well as the catalytic activity of USP7. Additionally, USP7 is critical in maintaining the steady state levels of UbE2E1 in cells. This study reveals a new cellular mechanism that couples the opposing activities of the ubiquitination machinery and a deubiquitinating enzyme to maintain and modulate the dynamic balance of the ubiquitin-proteasome system.     

CD2-associated Protein (CD2AP) Enhances Casitas B Lineage Lymphoma-3/c (Cbl-3/c)-mediated Ret Isoform-specific Ubiquitination and Degradation via Its Amino-terminal Src Homology 3 Domains

Ret is the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor (GDNF) family of neuronal growth factors. Upon activation by GDNF, Ret is rapidly polyubiquitinated and degraded. This degradation process is isoform-selective, with the longer Ret51 isoform exhibiting different degradation kinetics than the shorter isoform, Ret9. In sympathetic neurons, Ret degradation is induced, at least in part, by a complex consisting of the adaptor protein CD2AP and the E3-ligase Cbl-3/c. Knockdown of Cbl-3/c using siRNA reduced the GDNF-induced ubiquitination and degradation of Ret51 in neurons and podocytes, suggesting that Cbl-3/c was a predominant E3 ligase for Ret. Coexpression of CD2AP with Cbl-3/c augmented the ubiquitination of Ret51 as compared with the expression of Cbl-3/c alone. Ret51 ubiquitination by the CD2AP·Cbl-3/c complex required a functional ring finger and TKB domain in Cbl-3/c. The SH3 domains of CD2AP were sufficient to drive the Cbl-3/c-dependent ubiquitination of Ret51, whereas the carboxyl-terminal coiled-coil domain of CD2AP was dispensable. Interestingly, activated Ret induced the degradation of CD2AP, but not Cbl-3/c, suggesting a potential inhibitory feedback mechanism. There were only two major ubiquitination sites in Ret51, Lys¹⁰⁶⁰ and Lys¹¹⁰⁷, and the combined mutation of these lysines almost completely eliminated both the ubiquitination and degradation of Ret51. Ret9 was not ubiquitinated by the CD2AP·Cbl-3/c complex, suggesting that Ret9 was down-regulated by a fundamentally different mechanism. Taken together, these results suggest that only the SH3 domains of CD2AP were necessary to enhance the E3 ligase activity of Cbl-3/c toward Ret51.     

Evolutionary conserved N-terminal domain of Nrf2 is essential for the Keap1-mediated degradation of the protein by proteasome

Under homeostatic conditions, Nrf2 activity is constitutively repressed. This process is dependent on Keap1, to which Nrf2 binds through the Neh2 domain. Since the N-terminal subdomain of Neh2 (Neh2-NT) contains evolutionarily conserved motifs, we examined the roles they play in the degradation of Nrf2. In Neh2-NT, we defined a novel motif that is distinct from the previously characterized DIDLID motif and designated it DLG motif. Deletion of Neh2-NT or mutation of the DLG motif largely abolished the Keap1-mediated degradation of Nrf2. These mutations were found to enfeeble the binding affinity of Nrf2 to Keap1. The Neh2-NT subdomain directed DLG-dependent, Keap1-independent, degradation of a reporter protein in the nucleus. By contrast, mutation of DLG did not affect the half-life of native Nrf2 protein in the nucleus under oxidative stress conditions. These results thus demonstrate that DLG motif plays essential roles in the Keap1-mediated proteasomal degradation of Nrf2 in the cytoplasm.     

Negative regulation of EGFR-Vav2 signaling axis by Cbl ubiquitin ligase controls EGF receptor-mediated epithelial cell adherens junction dynamics and cell migration

The E3 ubiquitin ligase Casitas B lymphoma protein (Cbl) controls the ubiquitin-dependent degradation of EGF receptor (EGFR), but its role in regulating downstream signaling elements with which it associates and its impact on biological outcomes of EGFR signaling are less clear. Here, we demonstrate that stimulation of EGFR on human mammary epithelial cells disrupts adherens junctions (AJs) through Vav2 and Rac1/Cdc42 activation. In EGF-stimulated cells, Cbl regulates the levels of phosphorylated Vav2 thereby attenuating Rac1/Cdc42 activity. Knockdown of Cbl and Cbl-b enhanced the EGF-induced disruption of AJs and cell motility. Overexpression of constitutively active Vav2 activated Rac1/Cdc42 and reorganized junctional actin cytoskeleton; these effects were suppressed by WT Cbl and enhanced by a ubiquitin ligase-deficient Cbl mutant. Cbl forms a complex with phospho-EGFR and phospho-Vav2 and facilitates phospho-Vav2 ubiquitinylation. Cbl can also interact with Vav2 directly in a Cbl Tyr-700-dependent manner. A ubiquitin ligase-deficient Cbl mutant enhanced the morphological transformation of mammary epithelial cells induced by constitutively active Vav2; this effect requires an intact Cbl Tyr-700. These results indicate that Cbl ubiquitin ligase plays a critical role in the maintenance of AJs and suppression of cell migration through down-regulation of EGFR-Vav2 signaling.     

Partial adenosine A1 receptor agonists for cardiovascular therapies

Extracellular purines and pyrimidines have emerged as key regulators of a wide range of physiological and pathophysiological cellular processes acting through P1 and P2 cell surface receptors. Increasing evidence suggests that purinergic receptors can interact with and/or modulate the activity of other classes of receptors and ion channels. This review will focus on the interactions of purinergic receptors with other GPCRs, ion channels, receptor tyrosine kinases, and steroid hormone receptors. Also, the signal transduction pathways regulated by these complexes and their new functional properties are discussed.     

Interaction Between Syntaxin 8 and HECTd3, a HECT Domain Ligase

Ubiquitination of proteins and their degradation within the proteasome has emerged as the major proteolytic mechanism used by mammalian cells to regulate cytosolic and nuclear protein levels. Substrate ubiquitylation is mediated by ubiquitin (Ub) ligases, also called E3 Ub ligases. HECT-E3 Ub ligases are characterized by the presence of a C-terminal HECT domain that contains the active site for Ub transfer onto substrates. Among the many E3 Ub ligases, the family homologous to E6-Ap C-terminus (HECT) E3 Ub ligases, which includes the yeast protein Rsp5p and the mammalian homolog NEDD4, AIP4/Itch, and Smurf, has been shown to ubiquitylate membrane proteins and, in some instances, to induce their degradation. In this report, we have identified Syntaxin 8 as a binding protein to a novel HECT domain protein, HECT domain containing 3 (HECTd3), by yeast two-hybrid screen. Besides HECT domain, HECTd3 contains an anaphase-promoting complex, subunit 10 (APC10) domain. Our co-immunoprecipitation experiments show that Syntaxin 8 directly interacts with HECTd3 and that the overexpression of HECTd3 promotes the ubiquitination of Syntaxin 8. Immunofluorescence results show that Syntaxin 8 and HECTd3 have similar subcellular localization.     

Fyn is a downstream target of the pleiotrophin/receptor protein tyrosine phosphatase beta/zeta-signaling pathway: regulation of tyrosine phosphorylation of Fyn by pleiotrophin

Pleiotrophin (PTN the protein, Ptn the gene) signals downstream targets through inactivation of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)beta/zeta, disrupting the balanced activity of RPTPbeta/zeta and the activity of a constitutively active tyrosine kinase. As a consequence of the inactivation of RPTPbeta/zeta, PTN stimulates a sharp increase in the levels of tyrosine phosphorylation of the substrates of RPTPbeta/zeta in PTN-stimulated cells. We now report that the Src family member Fyn interacts with the intracellular domain of RPTPbeta/zeta in a yeast two-hybrid system. We further demonstrate that Fyn is a substrate of RPTPbeta/zeta, and that tyrosine phosphorylation of Fyn is sharply increased in PTN-stimulated cells. In previous studies, we demonstrated that beta-catenin and beta-adducin are targets of the PTN/RPTPbeta/zeta-signaling pathway and defined the mechanisms through which tyrosine phosphorylation of beta-catenin and beta-adducin disrupts cytoskeletal protein complexes. We conclude that Fyn is a downstream target of the PTN/RPTPbeta/zeta-signaling pathway and suggest that PTN coordinately regulates tyrosine phosphorylation of beta-catenin, beta-adducin, and Fyn through the PTN/RPTPbeta/zeta-signaling pathway and that together Fyn, beta-adducin, and beta-catenin may be effectors of the previously described PTN-stimulated disruption of cytoskeletal stability, increased cell plasticity, and loss of cell-cell adhesion that are characteristic of PTN-stimulated cells and a feature of many human malignant cells in which mutations have established constitutive expression of the Ptn gene.