Transcriptional Program of Apoptosis Induction following Interleukin 2 Deprivation: Identification of RC3, a Calcium/Calmodulin Binding Protein, as a Novel Proapoptotic Factor

Abstract

Apoptosis of mature T lymphocytes preserves immune system homeostasis by counteracting transient increases in T-cell number. This process is regulated, at least in part, by the cytokine interleukin 2 (IL-2): T cells deprived of IL-2 undergo apoptosis. The mechanism of apoptosis induction by IL-2 deprivation remains to be determined but is known to require RNA synthesis, implying the existence of transcriptionally activated genes whose products induce cell death. To identify such genes, we have performed expression profiling in IL-2-dependent T cells following cytokine deprivation. Our results reveal an intricate transcriptional program entailing the induction of known proapoptotic factors and the simultaneous repression of known antiapoptotic factors. Surprisingly, one gene whose transcription substantially increased was RC3 (also called neurogranin), which encodes a calmodulin binding protein thought to be a neural-specific factor involved in learning and memory. We show that ectopic expression of RC3 in IL-2-dependent T cells increases the intracellular Ca²⁺ concentration and induces apoptosis even in the presence of cytokine. Buffering the Ca²⁺ increase with the cytoplasmic Ca²⁺ chelator BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N1,N-tetraacetic acid] blocks RC3-induced apoptosis, indicating that the rise in intracellular Ca²⁺ is required for apoptotic death. RC3 mutants unable to bind calmodulin fail to increase intracellular Ca²⁺ levels and to induce apoptosis. Based upon these results, we propose that IL-2 deprivation raises the level of RC3 and other apoptotic factors, which induce apoptosis by increasing the intracellular Ca²⁺ concentration.

ACKNOWLEDGMENTS

We thank J. G. Sutcliffe for the RC3 cDNA and antibody, D. Storm for RC3 mutants, C. Sagerstorm for help with the microscopy, A. Earhardt and D. Lambright for help with the spectrofluorometry, the University of Massachusetts Medical School Center for AIDS Research (CFAR) for human peripheral blood lymphocytes, P. Furcinitti for help with the Ca²⁺ single-cell imaging, members of our laboratory for helpful discussions and comments on the manuscript, and S. Evans for editorial assistance.

This work was supported in part by an NIH grant (to M.R.G.) and by the Leukemia and Lymphoma Society (to L.R.D.). M.R.G. is an investigator of the Howard Hughes Medical Institute.