Bim and Bcl-2 Mutually Affect the Expression of the Other in T Cells Trine N. Jorgensen, Amy McKee, Michael Wang, Ella Kushnir, Janice White, Yosef Refaeli, John W. Kappler and This information is current as Philippa Marrack of September 25, 2021. J Immunol 2007; 179:3417-3424; ; doi: 10.4049/jimmunol.179.6.3417 http://www.jimmunol.org/content/179/6/3417 Downloaded from References This article cites 54 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/179/6/3417.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Bim and Bcl-2 Mutually Affect the Expression of the Other in T Cells1 Trine N. Jorgensen,2* Amy McKee,2*ʈ Michael Wang,2† Ella Kushnir,*ʈ Janice White,*ʈ Yosef Refaeli,** John W. Kappler,*‡§ʈ and Philippa Marrack3*§¶ʈ The life and death of T cells is controlled to a large extent by the relative amounts of Bcl-2-related proteins they contain. The antiapoptotic protein Bcl-2 and the proapoptotic protein Bim are particularly important in this process with the amount of Bcl-2 per cell dropping by about one-half when T cells prepare to die. In this study we show that Bcl-2 and Bim each control the expression of the other. Absence of Bim leads to a drop in the amount of intracellular Bcl-2 protein, while having no effect on the amounts of mRNA for Bcl-2. Conversely, high amounts of Bcl-2 per cell allow high amounts of Bim, although in this case the effect involves increases in Bim mRNA. These mutual effects occur even if Bcl-2 is induced acutely. Thus these two proteins control the expression of the other, at either the protein or mRNA level. The Journal of Immunology, 2007, 179: 3417–3424. Downloaded from t is well known that members of the Bcl-2 family of proteins via PI3K and AKT (18). Expression of the gene for the proapop- interact with each other to control the fate of the cell (1–3). totic protein, Bim, has likewise been studied in detail. The bim I In many types of cells, overexpression of antiapoptotic pro- transcription is controlled by transcription factors in the FOXO teins, such as Bcl-2, inhibits cell death and, conversely, underex- family (19), and the activity of these proteins, in turn, is inhibited pression of these same proteins promotes death (4–10). Not un- by phosphorylation via PI3K and AKT and also perhaps by MAPK http://www.jimmunol.org/ expectedly, opposite effects result from over or underexpression of pathways (20, 21). the proapoptotic members of the same family. Moreover, experi- As is the case for many proteins, the activities of Bcl-2-related ments have shown that Bcl-2-related proteins bind to each other, proteins are controlled not only by transcription of their genes but with different pairs present in different cells under different cir- also by posttranslational modifications, their location in the cell, cumstances (11, 12). and their rates of synthesis and degradation. The activity of Bcl-2 Because the Bcl-2 family controls the life and death of many is no exception, being affected by phosphorylation, nitrosylation, kinds of cells, including cancer cells, a lot of attention has been and degradation (22–25). Likewise the effectiveness of Bim is con- paid to the mechanisms that control transcription of their genes and trolled in several ways. In some cell types, Bim is sequestered much is now known about the processes. For example, bcl-2 tran- away from target organelles such as the mitochondrion, and is only by guest on September 25, 2021 scription is induced by CREB and other factors (13–16) and may released to bind Bcl-2 on mitochondria and precipitate death under be inhibited by factors binding to negative regulatory elements particular circumstances (26, 27). In T cells, however, Bim is al- (17). The transcription factors that increase bcl-2 transcription are ways mitochondrially located bound to Bcl-2 (11). themselves in turn induced by extracellular signals acting through Our laboratory has been studying the relative contributions of well-known intracellular pathways. In normal T cells, for example, Bcl-2 and Bim to the death of Ag-activated T cells. In resting T bcl-2 is induced by members of the IL-2 family of cytokines acting cells much of the Bim is bound to Bcl-2 on mitochondria (11). The level and location of Bim does not change when activated T cells die. However, levels of Bcl-2 fall to ϳ50% of normal just before † ‡ *Integrated Department of Immunology, and Department of Pediatrics, and Depart- activated T cells die (28). Inhibition of this fall in Bcl-2, by over- ment of Pharmacology, §Department of Medicine, and ¶Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO expression of the protein or by its induction via antioxidants such 80262; ʈHoward Hughes Medical Institute, and **Department of Pediatrics, National as MnTBAP, prevents activated T cells from dying (8, 29, 30). Jewish Medical and Research Center, Denver, CO 80206 Thus it seems that this quite subtle change to the ratio between Received for publication June 1, 2007. Accepted for publication June 11, 2007. Bcl-2 and one of its binding partners, Bim, has dramatic effects on The costs of publication of this article were defrayed in part by the payment of page T cell life expectancy. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Because of these ideas we became interested in the relative 1 This work was supported in part by a National Institute of Child Health and Human amounts of Bcl-2 and Bim in T cells. A preliminary experiment led Development Award K12-HD00850 (to M.W.). This work was also supported by us to measure the amounts of Bcl-2 mRNA and protein in T cells Grants AI-17134, AI-18785, and AI-22295 from the U.S. Public Health Service, by that contained or lacked Bim. Absence of Bim had no statistically Grant AI-002 from the Autoimmunity Center of Excellence, and by Grant CA-046934 from the Cancer Center Core Funding (to P.M. and J.W.K.). This work is supported significant effect on the amounts of Bcl-2 mRNA. However, T by Grant CA-117802 from the U.S. Public Health Service and by a Translational cells lacking Bim halved their amounts of Bcl-2 protein, by com- Research Award from the Leukemia and Lymphoma Society (to Y.R.). M.W. is a parison with wild-type cells. This effect was also observed with National Institute of Child Health and Human Development Fellow of the Pediatric Scientist Development Program. Bcl-2 expressed via a transgene; T cells expressing a Bcl-2 trans- 2 T.N.J., A.M., and M.W. contributed equally to the work. gene had about one-half the amount of the transgenic Bcl-2 protein 3 Address correspondence and reprint requests to Dr. Philippa Marrack, Integrated per cell if they lacked, rather than contained, Bim. The effect Department of Immunology, Howard Hughes Medical Institute, National Jewish proved to be reciprocal, that is, overexpression, via a transgene, of Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail ad- Bcl-2 raised intracellular amounts of Bim and simultaneously re- dress: [email protected] duced the amounts of endogenous Bcl-2 protein. These phenomena Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 were not caused solely by long time effects, due to development in www.jimmunol.org 3418 Bim AND Bcl-2 AFFECT EXPRESSION IN EACH OTHER the absence of Bim or in the presence of high levels of Bcl-2. Thus without reduction on SDS-PAGE 10–20% gradient Criterion gels and blot- Bcl-2 and Bim mutually control each other at the protein level. ted onto nitrocellulose. The mBcl-2 was analyzed with hamster anti-mouse Bcl-2, 3F11 (34), Bim with BD Biosciences rabbit anti-Bim, Bax, with Santa Cruz Biotechnology rabbit anti-Bax (N-20) and actin with Santa Materials and Methods Cruz Biotechnology goat anti-actin. Secondary Abs were HRP goat anti- Mice hamster Ig or HRP donkey anti-rabbit Ig from The Jackson Laboratory or HRP anti-goat Ig from Santa Cruz Biotechnology. Western blots were de- Mice lacking Bim and backcrossed at least 10 times to C57BL/6 animals veloped with the ECL reagents. were the gifts of Drs. P. Bouillet and A. Strasser (The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia) (4). Mice expressing human Bcl-2 (hBcl-2)4 under the control of the ␮-chain promoter, but only Cell culture in T cells, were the gifts from Drs.