The deubiquitinating enzyme USP17 blocks N-Ras membrane trafficking and activation but leaves K-Ras unaffected.
Ontology highlight
ABSTRACT:
SUBMITTER: de la Vega M
PROVIDER: S-EPMC2852940 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Deubiquitinating enzymes are now emerging as potential therapeutic targets that control many cellular processes, but few have been demonstrated to control cell motility. Here, we show that ubiquitin-specific protease 17 (USP17) is rapidly and transiently induced in response to chemokines SDF-1/CXCL12 and IL-8/CXCL8 in both primary cells and cell lines, and that its depletion completely blocks chemokine-induced cell migration and cytoskeletal rearrangements. Using live cell imaging, we demonstrate that USP17 is required for both elongated and amoeboid motility, in addition to chemotaxis. USP17 has previously been reported to disrupt Ras localization and we now find that USP17 depletion blocks chemokine-induced subcellular relocalization of GTPases Cdc42, Rac and RhoA, which are GTPases essential for cell motility. Collectively, these results demonstrate that USP17 has a critical role in cell migration and may be a useful drug target for both inflammatory and metastatic disease.
| S-EPMC3072070 | biostudies-literature
Project description:Interaction of platelet-derived growth factor (PDGF) isoforms with their receptors results in activation and internalization of receptors, with a concomitant activation of downstream signalling pathways. Ubiquitination of PDGFRs serves as a mark to direct the internalization and sorting of the receptors. By overexpressing a panel of deubiquitinating enzymes (DUBs), we found that USP17 and USP4 efficiently deubiquitinate PDGF receptor β (PDGFRβ) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFRβ did not affect its stability, but regulated the timing of its trafficking, whereby USP17 prolonged the presence of the receptor at the cell surface, while USP4 affected the speed of trafficking towards early endosomes. Induction of each of the DUBs in BJhTERT fibroblasts and U2OS osteosarcoma cells led to prolonged and/or shifted activation of STAT3 in response to PDGF-BB stimulation, which in turn led to increased transcriptional activity of STAT3. Induction of USP17 promoted acute upregulation of the mRNA expression of STAT3-inducible genes STAT3, CSF1, junB and c-myc, while causing long-term changes in the expression of myc and CDKN1A. Deletion of USP17 was lethal to fibroblasts, while deletion of USP4 led to a decreased proliferative response to stimulation by PDGF-BB. Thus, USP17- and USP4-mediated changes in ubiquitination of PDFGRβ lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.
| S-EPMC8782881 | biostudies-literature
Project description:The proto-oncogene Ras undergoes a series of post-translational modifications at its carboxyl-terminal CAAX motif that are essential for its proper membrane localization and function. One step in this process is the cleavage of the CAAX motif by the enzyme Ras-converting enzyme 1 (RCE1). Here we show that the deubiquitinating enzyme USP17 negatively regulates the activity of RCE1. We demonstrate that USP17 expression blocks Ras membrane localization and activation, thereby inhibiting phosphorylation of the downstream kinases MEK and ERK. Furthermore, we show that this effect is caused by the loss of RCE1 catalytic activity as a result of its deubiquitination by USP17. We also show that USP17 and RCE1 co-localize at the endoplasmic reticulum and that USP17 cannot block proliferation or Ras membrane localization in RCE1 null cells. These studies demonstrate that USP17 modulates Ras processing and activation, at least in part, by regulating RCE1 activity.
| S-EPMC2666611 | biostudies-literature
Project description:SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.
| S-EPMC5016256 | biostudies-literature
Project description:Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPARs) are the primary mediators for inter-neuronal communication and play a crucial role in higher brain functions including learning and memory. Our previous work demonstrated that AMPARs are subject to ubiquitination by the E3 ligase Nedd4, resulting in EPS15-mediated receptor internalization and Ubiquitin (Ub)-proteasome pathway (UPP)-dependent degradation. Protein ubiquitination is a highly dynamic and reversible process, achieved via the balance between ubiquitination and deubiquitination. However, deubiquitination of mammalian AMPARs and the responsible deubiquitinating enzymes remain elusive. In this study, we identify USP46 as the deubiquitinating enzyme for AMPARs. We find that AMPARs are subject to K63 type ubiquitination, and USP46 is able to deubiquitinate AMPARs in vivo and in vitro. In heterologous cells and neurons, expression of USP46 results in a significant reduction in AMPAR ubiquitination, accompanied by a reduced rate in AMPAR degradation and an increase in surface AMPAR accumulation. By contrast, knockdown of USP46 by RNAi leads to elevated AMPAR ubiquitination and a reduction in surface AMPARs at synapses in neurons. Consistently, miniature excitatory postsynaptic currents recordings show reduced synaptic strength in neurons expressing USP46-selective RNAi. These results demonstrate USP46-mediated regulation of AMPAR ubiquitination and turnover, which may play an important role in synaptic plasticity and brain function. Protein ubiquitination is a highly dynamic and reversible process, achieved via the balance between ubiquitination and deubiquitination. The glutamatergic AMPARs, which mediate most of the excitatory synaptic transmission in the brain, are known to be subjected to Nedd4-mediated ubiquitination; however, the deubiquitination process and the responsible deubiquitinating enzymes (DUBs) for mammalian AMPARs remain elusive. We find that AMPARs are subject to K63-type ubiquitination, and identify USP46 as the DUB for AMPARs. USP46 deubiquitinates AMPARs in vitro and in vivo. Up- or down-regulation of USP46 leads to changes in AMPAR ubiquitination, surface expression, and trafficking, as well as the strength of synaptic transmission. USP46-mediated regulation of AMPAR ubiquitination and turnover may play an important role in synaptic plasticity and brain function.
| S-EPMC4668950 | biostudies-literature
Project description:USP17 is a deubiquitinating enzyme that is upregulated in numerous cancers and therefore a drug target. We developed a robust expression, purification, and assay system for USP17 enabling its enzymatic and structural characterization. USP17 was expressed in E. coli as inclusion bodies and then solubilized, refolded, and purified using affinity and size-exclusion chromatography. Milligram quantities of pure USP17 can be produced that is catalytically more efficient (kcat/Km = 1500 (x103) M-1sec-1) than other human USPs studied to date. Analytical size-exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering studies suggest that the quaternary structure of USP17 is a monomer. Steady-state kinetic studies show that USP17 efficiently hydrolyzes both ubiquitin-AMC (kcat = 1.5 sec-1 and Km = 1.0 ?M) and ubiquitin-rhodamine110 (kcat = 1.8 sec-1 and Km = 2.0 ?M) substrates. Ubiquitin chain cleavage assays reveal that USP17 efficiently cleaves di-ubiquitin chains with Lys11, Lys33, Lys48 and Lys63 linkages and tetra-ubiquitin chains with Lys11, Lys48 and Lys63 linkages but is inefficient in cleaving di-ubiquitin chains with Lys6, Lys27, or Lys29 linkages or linear ubiquitin chains. The substrate specificity of USP17 is most similar to that of USP1, where both USPs display higher specificity than other characterized members of the USP family.
| S-EPMC5868344 | biostudies-literature
Project description:Checkpoint kinase 1 (CHK1), a Ser/Thr protein kinase, is modified by the K63-linked ubiquitin chain in response to genotoxic stress, which promotes its nuclear localization, chromatin association, and activation. Interestingly, this bulky modification is linked to a critical residue, K132, at the kinase active site. It is unclear how this modification affects the kinase activity and how it is removed to enable the release of CHK1 from chromatin. Herein, we show that the K63-linked ubiquitin chain at CHK1's K132 residue has an inhibitory effect on the kinase activity. Furthermore, we demonstrate that this modification can be removed by ubiquitin-specific protease 3 (USP3), a deubiquitinating enzyme that targets K63-linked ubiquitin chains. Wild-type USP3, but not the catalytically defective or nuclear localization sequence-deficient mutants, reduced CHK1 K63-linked ubiquitination. Conversely, USP3 knockdown elevated K63-linked ubiquitination of the kinase, leading to prolonged CHK1 chromatin association and phosphorylation. Paradoxically, by removing the bulky ubiquitin chain at the active site, USP3 also increased the accessibility of CHK1 to its substrates. Thus, our findings on the dual roles of USP3 (namely, one to release CHK1 from the chromatin and the other to open up the active site) provide further insights into the regulation of CHK1 following DNA damage.
| S-EPMC6003459 | biostudies-literature
Project description:A new ubiquitin-processing protease (Ubp-M) has been identified in mammalian cells that is phosphorylated at the onset of mitosis and dephosphorylated during the metaphase/anaphase transition. The carboxyl-terminal domain of this 823-aa protein can be phosphorylated in vitro with either extracts of mitotic cells or purified cdc-2/cyclin B complexes. Recombinant Ubp-M is able to deubiquitinate histone H2A in vitro, and the phosphorylated form is also enzymatically active. Wild-type Ubp-M, transiently expressed as green fluorescent protein-fusion proteins, localizes in the cytoplasm of cultured cells, but mutant forms, lacking an active-site cysteine, associate closely with mitotic chromosomes during all stages of cell division and remain within the nucleus during the postmitotic period. Cells transfected with plasmids containing mutant Ubp-M genes stop dividing and eventually undergo apoptosis. Ubp-M may deubiquitinate one or more critical proteins that are involved in the condensation of mitotic chromosomes, possibly acting selectively on histones H2A and H2B, the major ubiquitinated proteins of chromatin.
| S-EPMC15854 | biostudies-literature
Project description:BackgroundProtein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. Deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway.ResultsWhile identifying putative ubiquitin specific protease (USP) enzymes that contain a conserved Asp (I) domain in humans, 4 USP17 subfamily members, highly homologous to DUB-3, have been found (USP17K, USP17L, USP17M, and USP17N), from human chorionic villi. Expression analysis showed that USP17 transcripts are highly expressed in the heart, liver, and pancreas and are expressed moderately in various human cancerous cell lines. Amino acid sequence analysis revealed that they contain the highly conserved Cys, His, and Asp domains which are responsible for the deubiquitinating activity. Biochemical enzyme assays indicated that they have deubiquitinating activity. Interestingly, the sequence analysis showed that these proteins, with exception of USP17N, contain the putative hyaluronan/RNA binding motifs, and cetylpyridinium chloride (CPC)-precipitation analysis confirmed the association between these proteins and intracellular hyaluronan and RNA.ConclusionHere, we report that the overexpression of these proteins, with exception of USP17N, leads to apoptosis, suggesting that the hyaluronan and RNA binding motifs in these enzymes play an important role in regulating signal transduction involved in cell death.
| S-EPMC1665497 | biostudies-literature
Project description:The mechano-electrical transduction (MET) channel of the inner ear receptor cells, termed hair cells, is a protein complex that enables our senses of hearing and balance. Hair cell MET requires an elaborate interplay of multiple proteins that form the MET channel. One of the MET complex components is the transmembrane protein LHFPL5, which is required for hair cell MET and hearing. LHFPL5 is thought to form a multi-protein complex with other MET channel proteins, such as PCDH15, TMIE, and TMC1. Despite localizing to the plasma membrane of stereocilia, the mechanosensing organelles of hair cells, LHFPL5 requires its binding partner within the MET complex, PCDH15, to localize to the stereocilia tips in hair cells and to the plasma membrane in heterologous cells. Using the Aquaporin 3-tGFP reporter (AGR) for plasma membrane localization, we found that a region within extracellular loop 1, which interacts with PCDH15, precludes the trafficking of AGR reporter to the plasma membrane in heterologous cell lines. Our results suggest that the presence of protein partners may mask endoplasmic reticulum retention regions or enable the proper folding and trafficking of the MET complex components, to facilitate expression of the MET complex at the stereocilia membrane.
| S-EPMC9925724 | biostudies-literature