Deubiquitination of CXCR4 by USP14 is critical for both CXCL12-induced CXCR4 degradation and chemotaxis but not ERK ativation.
ABSTRACT The chemokine receptor CXCR4 plays important roles in the immune and nervous systems. Abnormal expression of CXCR4 contributes to cancer and inflammatory and neurodegenerative disorders. Although ligand-dependent CXCR4 ubiquitination is known to accelerate CXCR4 degradation, little is known about counter mechanisms for receptor deubiquitination. CXCL12, a CXCR4 agonist, induces a time-dependent association of USP14 with CXCR4, or its C terminus, that is not mimicked by USP2A, USP4, or USP7, other members of the deubiquitination catalytic family. Co-localization of CXCR4 and USP14 also is time-dependent following CXCL12 stimulation. The physical interaction of CXCR4 and USP14 is paralleled by USP14-catalyzed deubiquitination of the receptor; knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4 deubiquitination, whereas overexpression of USP14 promotes CXCR4 deubiquitination. We also observed that ubiquitination of CXCR4 facilitated receptor degradation, whereas overexpression of USP14 or RNAi-induced knockdown of USP14 blocked CXCL12-mediated CXCR4 degradation. Most interestingly, CXCR4-mediated chemotactic cell migration was blocked by either overexpression or RNAi-mediated knockdown of USP14, implying that a CXCR4-ubiquitin cycle on the receptor, rather than a particular ubiquitinated state of the receptor, is critical for the ligand gradient sensing and directed motility required for chemokine-mediated chemotaxis. Our observation that a mutant of CXCR4, HA-3K/R CXCR4, which cannot be ubiquitinated and does not mediate a chemotactic response to CXCL12, indicates the importance of this covalent modification not only in marking receptors for degradation but also for permitting CXCR4-mediated signaling. Finally, the indistinguishable activation of ERK by wild typeor 3K/R-CXCR4 suggests that chemotaxis in response to CXCL12 may be independent of the ERK cascade.
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ABSTRACT: Non-small cell lung cancer (NSCLC) accounts for most lung cancer. To develop new therapy required the elucidation of NSCLC pathogenesis. The deubiquitinating enzymes USP 28 has been identified and studied in colon and breast carcinomas. However, the role of USP28 in NSCLC is unknown. The level mRNA or protein level of USP28 were measured by qRT-PCR or immunohistochemistry (IHC). The role of USP28 in patient survival was revealed by Kaplan-Meier plot of overall survival in NSCLC patients. USP28 was up or down regulated by overexpression plasmid or siRNA transfection. Cell proliferation and apoptosis was assayed by MTT and FACS separately. Potential microRNAs, which targeted USP28, were predicated by bioinformatic algorithm and confirmed by Dual Luciferase reporter assay system. High mRNA and protein level of USP28 in NSCLC were both correlated with low patient survival rate. Overexpression of USP28 promoted NSCLC cells growth and vice versa. Down-regulation of USP28 induced cell apoptosis. USP28 was targeted by miR-4295. Overexpression of USP28 promoted NSCLC cells proliferation, and was associated with poor prognosis in NSCLC patients. The expression of USP28 may be regulated by miR-4295. Our data suggested that USP28 was a tumour-promoting factor and a promising therapeutic target for NSCLC. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.Journal of Cellular and Molecular Medicine 02/2015; 19(4). DOI:10.1111/jcmm.12426 · 3.70 Impact Factor
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ABSTRACT: Human and Simian Immunodeficiency virus (HIV-1, HIV-2, and SIV) encode an accessory protein, Nef, which is a pathogenesis and virulence factor. Nef is a multivalent adapter that dysregulates the trafficking of many immune cell receptors, including chemokine receptors (CKRs). Physiological endocytic itinerary of agonist occupied CXCR4 involves ubiquitinylation of the phosphorylated receptor at three critical lysine residues and dynamin-dependent trafficking through the ESCRT pathway into lysosomes for degradation. Likewise, Nef induced CXCR4 degradation was critically dependent on the three lysines in the C-terminal -SSLKILSKGK- motif. Nef directly recruits the HECT domain E3 ligases AIP4 or NEDD4 to CXCR4 in the resting state. This mechanism was confirmed by ternary interactions of Nef, CXCR4 and AIP4 or NEDD4; by reversal of Nef effect by expression of catalytically inactive AIP4-C830A mutant; and siRNA knockdown of AIP4, NEDD4 or some ESCRT-0 adapters. However, ubiquitinylation dependent lysosomal degradation was not the only mechanism by which Nef downregulated CKRs. Agonist and Nef mediated CXCR2 (and CXCR1) degradation was ubiquitinylation independent. Nef also profoundly downregulated the naturally truncated CXCR4 associated with WHIM syndrome and engineered variants of CXCR4 that resist CXCL12 induced internalization via an ubiquitinylation independent mechanism.PLoS ONE 01/2014; 9(1):e86998. DOI:10.1371/journal.pone.0086998 · 3.53 Impact Factor
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ABSTRACT: To understand adenosylmethionine decarboxylase 1 (AMD1)-mediated mRNA processing and cell adhesion activated & inhibited transition mechanisms between chimpanzee and human left hemisphere, AMD1-activated different complete (all no positive correlation, Pearson correlation coefficient < 0.25) and uncomplete (partly no positive correlation except AMD1, Pearson < 0.25) networks were identified in higher human compared with lower chimpanzee left hemisphere from the corresponding AMD1-stimulated (Pearson ≥ 0.25) or inhibited (Pearson ≤ -0.25) overlapping molecules of Pearson and GRNInfer, respectively. This result was verified by the corresponding scatter matrix. As visualized by GO, KEGG, GenMAPP, BioCarta, and disease database integration, we proposed mainly that AMD1-stimulated different complete network was involved in AMD1 activation with cytoplasm ubiquitin specific peptidase (tRNA-guanine transglycosylase) to nucleus paired box-induced mRNA processing, whereas the corresponding inhibited network participated in AMD1 repression with cytoplasm protocadherin gamma and adaptor-related protein complex 3-induced cell adhesion in lower chimpanzee left hemisphere. However, AMD1-stimulated network contained AMD1 activation with plakophilin and phosphodiesterase to SH3 binding glutamic acid-rich protein to dynein and zinc finger-induced cell adhesion, whereas the corresponding inhibited different complete network included AMD1 repression with mitochondrial denine nucleotide translocator, brain protein, and ADH dehydrogenase to ribonucleoprotein-induced mRNA processing in higher human left hemisphere. Our AMD1 different networks were verified by AMD1-activated or -inhibited complete and uncomplete networks within and between chimpanzee left hemisphere or (and) human left hemisphere.Cell biochemistry and biophysics 03/2014; 70(1). DOI:10.1007/s12013-014-9902-y · 2.38 Impact Factor