Caspase-Mediated Calcineurin Activation Contributes to IL-2 Release during T Cell Activation Neeta Mukerjee,* Kim M. McGinnis,* , † ,1 Margaret E. Gnegy,† and Kevin K. W. Wang* , † ,2 *Laboratory of Neuro-biochemistry, Department of CNS Molecular Sciences, Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105; and †Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109 Received June 21, 2001 Calcineurin, a Ca 2 /calmodulin-dependent Ser/Thr phosphatase (protein phosphatase 2B), plays a critical role in IL-2 production during T cell activation. It has been previously reported that IL-2 release in activated Jurkat T requires caspase-like activity (Posmantur et al. (1998) Exp. Cell. Res. 244, 302–309). We report here that the 60-kDa catalytic subunit of calcineurin A (Cn A) was partially cleaved to a 45-kDa form in phytohe- magglutinin A (PHA) or phorbol ester  ionomycin (P  I)-activated Jurkat cells. In parallel, proteolytic ac- tivation of upstream caspases (caspase-8 and -9) as well as effector caspase-3 was also observed. Cn A cleavage was caspase mediated, since it was inhibit- able by pan-caspase inhibitor Cbz-Asp-CH 2 OC(O)-2,6- dichlorobenzene (Z-D-DCB). Cn A cleavage was also observed when purified calcineurin was digested in vitro with caspase-3. Truncated Cn A was associated with enhanced phosphatase activity and reduced cal- modulin sensitivity. Furthermore, in PHA or P  I-activated Jurkat cells, dephosphorylation of cal- cineurin substrate NFATc (a transcription factor known to be involved in transactivation of the IL-2 gene), was also suppressed by Z-D-DCB. Taken to- gether, our results suggest that caspase-mediated cleavage of Cn A contributes to IL-2 production during T cell activation. © 2001 Academic Press Key Words: calcineurin; protein phosphatase; cas- pase; NFAT; IL-2; T cell activation. Antigen presentation in context with MHC mole- cules results in the physiological activation of T cells (1, 2). During T cell activation, signaling events are triggered that lead to production of hematopoietic growth factors such as IL-2, IL-3, and GM-CSF. IL-2 is necessary for T cell growth and clonal expansion. Among the early T cell signaling events are activation of protein tyrosine kinases (fyn, ZAP-70, lck), phos- phatidyl inositol metabolism and increases in the lev- els of intracellular calcium. These events lead to acti- vation of PKC/MAPK pathways as well as activation of protein phosphatase 2B (calcineurin). Activation of cal- cineurin causes dephosphorylation and nuclear trans- location of NFAT, a transcription factor critical for IL-2 production (3). NFAT, once inside the nucleus, forms DNA binding complexes by cooperatively interacting with AP-1 family members (Fos/Jun) and transacti- vates cytokine genes such as IL-2. Thus calcineurin plays a key role in T cell activation. Calcineurin is a highly conserved Ca 2+ /calmodulin (CaM) dependent Ser/Thr phosphatase (4). The het- erodimeric molecule of calcineurin consists of a 60-kDa catalytic subunit (Cn A) and a 19-kDa Ca +2 binding regulatory subunit (Cn B). Calcineurin is the molecu- lar target of immunosuppressive agents, cyclosporine A (CsA) and FK506 (4, 5). NFAT, a substrate of cal- cineurin, was originally described to be a transcription factor critical for IL-2 expression. NFAT binding sites have since been reported to be present within the pro- moter regions of other cytokines including IL-3, GM- CSF and TNF as well as cell surface receptors such as CD40L and Fas L (3). NFAT protein family consists of several isoforms; NFATp (NFAT1 or NFATc2), NFATc (NFAT2 or NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx or NFATc3) and a very recently identified isoform NFAT5 (6). NFATp and NFATc have been shown to strongly activate IL-2 and IL-4 promoters (3). All known isoforms of NFAT are activated by cal- cineurin, except NFAT5, which is constitutively present inside the nucleus. Studies on inactivation of Abbreviations used: PKC, protein kinase C; MAPK, mitogen- activated protein kinase; PHA, phytohemagglutinin A; PMA, phorbol 12-myristate 13-acetate; I, ionomycin; CaM, calmodulin; Cn A, cal- cineurin A; CsA, cyclosporine A; PARP; poly(ADP-ribose) polymer- ase; Z-D-DCB, Cbz-Asp-CH 2 OC(O)-2,6-dichlorobenzene); NFAT, nu- clear factor of activated T cells and DTT, dithiothreitol. 1 Current address: Molecular Neurogenetics Unit, Massachusetts General Hospital 13th Street, Building 149, Charlestown, MA 02129. 2 To whom correspondence should be addressed at Laboratory of Neuro-biochemistry, Department of CNS Molecular Sciences, Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105. Fax: 734-622-7178. E-mail: kevin.wang@pfizer.com. Biochemical and Biophysical Research Communications 285, 1192–1199 (2001) doi:10.1006/bbrc.2001.5278, available online at http://www.idealibrary.com on 1192 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.