SUMMARY A20 is a potent anti-inflammatory protein that inhibits NF-κB, and A20 dysfunction is associated with autoimmunity and B-cell lymphoma. A20 harbors a deubiquitination enzyme domain and can employ multiple mechanisms to antagonize ubiquitination upstream of NEMO, a regulatory subunit of the IκB kinase complex (IKK). However, direct evidence of IKK inhibition by A20 is lacking, and the inhibitory mechanism remains poorly understood. Here we show that A20 can directly impair IKK activation without deubiquitination or impairment of ubiquitination enzymes. We find that polyubiquitin binding by A20, which is largely dependent on A20’s seventh zinc finger motif (ZnF7), induces specific binding to NEMO. Remarkably, this ubiquitin-induced recruitment of A20 to NEMO is sufficient to block IKK phosphorylation by its upstream kinase TAK1. Our results suggest a non-catalytic mechanism of IKK inhibition by A20 and a means by which polyubiquitin chains can specify a signaling outcome.
Photodynamic therapy (PDT) is an efficient inducer of apoptosis, an active form of cell death that can be inhibited by the BCL-2 oncoprotein. The ability of BCL-2 to modulate PDT-induced apoptosis and overall cell killing has been studied in a pair of Chinese hamster ovary cell lines that differ from one another by a transfected human BCL-2 gene in one of them (Bissonnette et al, Nature 359, 552-554, 1992). Cells were exposed to the phthalocyanine photosensitizer Pc 4 and various fluences of red light. Pc 4 uptake was identical in the two cell lines. The parental cells displayed a high incidence of apoptosis after PDT, whereas at each fluence there was a much lower incidence of apoptosis in the BCL-2-expressing cells. Apoptosis was monitored by (a) observation of 50 kbp and oligonucleosome-size DNA fragments by gel electrophoresis, (b) flow cytometry of cells labeled with fluorescently tagged dUTP by terminal deoxynucleotidyl transferase and (c) fluorescence microscopy of acridine orange-stained cells. The time course of apoptosis varied with the PDT dose, suggesting that only after moderately high doses (> 99% loss of clonogenicity) was there a relatively synchronous and rapid entry of many cells into apoptosis. At PDT doses reducing cell survival by 90 or 99%, significant increases in apoptotic cells were found in the population after 6-12 h. Clonogenic assays showed that BCL-2 protein inhibited not only apoptosis but overall cell killing as well, effecting a two-fold resistance at the 10% survival level. Thus, BCL-2-expressing cells may be relatively resistant to PDT.
One of the primary neurodegenerative events occurring in amyotrophic lateral sclerosis (ALS) is the selective loss of spinal cord alpha motor neurons. To study the potential role of apoptosis in the degeneration of these motor neurons, in situ hybridization was used to measure the expression of two apoptotic cell death genes, bcl-2 and bax, in control and ALS lumbar spinal cord sections. The strongest hybridization signal for bcl-2 mRNA in neurological and nonneurological control spinal cords was found primarily in lamina IX alpha motor neurons, while a weaker hybridization signal was found in neurons of Clarke's nucleus and the proper sensory nucleus of the dorsal horn. Surviving lamina IX motor neurons in ALS spinal cord sections also expressed bcl-2 mRNA, but at levels that were significantly and selectively decreased (4.7-fold) compared with controls. bax mRNA hybridization signal was detected in several cells throughout the gray matter in control and ALS lumbar spinal cord, but was significantly and selectively increased (2.8-fold) in ALS motor neurons. Given the proposed interactive roles of these genes in apoptosis, the present findings favor a scenario in which this mode of cell death would contribute to spinal cord motor neuron degeneration in ALS.
Prostate carcinoma (PCA) is the most frequently diagnosed malignancy in American men. PCA at advanced stages can both proliferate abnormally and resist apoptosis. Among the many known signal transduction pathways, phosphatidylinositide-3'OH kinase (PI3-kinase) has been shown to play an important role in cell survival and resistance to apoptosis. In this study, we investigate the involvement of Etk/Bmx, a newly discovered tyrosine kinase that is a substrate of PI3-kinase, in protection of prostate cancer cells from apoptosis. Parental LNCaP cells and two derivative cell lines, one overexpressing wild type Etk (Etkwt) and the other expressing a dominant negative Etk (EtkDN), were used to study the function of Etk. The cells were treated with photodynamic therapy (PDT), a newly approved cancer treatment which employs a photosensitizer and visible light to produce an oxidative stress in cells, often leading to apoptosis. Our results indicate that PDT induces apoptosis in LNCaP cells, as measured by DNA fragmentation and by cleavage of poly(ADP-ribose) polymerase (PARP), and moreover, the extent of apoptosis was much reduced in Etkwt cells as compared to LNCaP or EtkDN cells. Assay of overall cell viability con®rmed that Etkwt cells were considerably less sensitive to PDT than were the parental LNCaP or EtkDN cells. Similar results were found in response to thapsigargin (TG). A speci®c inhibitor of PI3-kinase, LY294002, abolished Etk activity and markedly increased TG-induced PARP cleavage. The results suggest that Etk/Bmx is an e cient e ector of PI3-kinase and that the newly described PI3-kinase/Etk pathway is involved in the protection of prostate carcinoma cells from apoptosis in response to PDT or TG.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.