The Ca2+-activated protein phosphatase calcineurin induces apoptosis, but the mechanism is unknown. Calcineurin was found to dephosphorylate BAD, a pro-apoptotic member of the Bcl-2 family, thus enhancing BAD heterodimerization with Bcl-xL and promoting apoptosis. The Ca2+-induced dephosphorylation of BAD correlated with its dissociation from 14-3-3 in the cytosol and translocation to mitochondria where Bcl-xL resides. In hippocampal neurons, L-glutamate, an inducer of Ca2+ influx and calcineurin activation, triggered mitochondrial targeting of BAD and apoptosis, which were both suppressible by coexpression of a dominant-inhibitory mutant of calcineurin or pharmacological inhibitors of this phosphatase. Thus, a Ca2+-inducible mechanism for apoptosis induction operates by regulating BAD phosphorylation and localization in cells.

A new facet of calcium signalling involves the nuclear import of the NF-AT transcription factors from their dormant position in the cytoplasm. The protein phosphatase calcineurin appears to play an essential role in activating NF-AT nuclear import, as the calcineurin inhibitors cyclosporin A and FK506 block dephosphorylation and nuclear import of NF-AT (refs 4-7). Here we show that calcium signalling induces an association between NF-AT4 and calcineurin, and that these molecules are transported, as a complex, to the nucleus, where calcineurin continues to dephosphorylate NF-AT4. We propose that a nuclear complex of NF-AT4 and calcineurin maintains calcium signalling by counteracting a vigorous nuclear NF-AT kinase.

T cell activation requires the import of NF-AT transcription factors to the nucleus, a process promoted by calcineurin-dependent dephosphorylation and inhibited by poorly understood protein kinases. Here, we report the identification of two protein kinases that oppose NF-AT4 nuclear import. Casein kinase Ialpha directly binds and phosphorylates NF-AT4, resulting in the inhibiton of NF-AT4 nuclear translocation. MEKK1 indirectly suppresses NF-AT4 nuclear import by stabilizing the interaction between NF-AT4 and CKIalpha. CKIalpha thus acts to establish an intramolecular masking of the nuclear location signal on NF-AT4, while MEKK1 augments this mechanism, and may further provide a link to signal transduction pathways regulating NF-AT4.

It is not known how the protein Bcl-2 inhibits cell death induced by calcium signalling and growth-factor withdrawal. Here we report that Bcl-2 forms a tight complex with calcineurin, resulting in the targeting of calcineurin to Bcl-2 sites on cytoplasmic membranes, and show that this interaction is dependent on the BH4 domain of Bcl-2. Calcineurin bound to Bcl-2 is an active phosphatase but is unable to promote the nuclear translocation of NF-AT, a transcription-factor required for induction of interleukin-2 expression, suggesting a mechanism by which Bcl-2 suppresses NF-AT activity. We also show that Bax, a pro-apoptotic member of the Bcl-2 family, interferes with interactions between calcineurin and Bcl-2. We propose that the ability of Bcl-2 to block NF-AT signalling is due to the sequestering of active calcineurin to the same domain of Bcl-2 which associates with Rad-1 (ref. 5), and that calcineurin may act in Bcl-2-regulated functions.

Cofilin, an essential regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 and reactivated by dephosphorylation. Although cofilin undergoes dephosphorylation in response to extracellular stimuli that elevate intracellular Ca 2؉ concentrations, signaling mechanisms mediating Ca 2؉ -induced cofilin dephosphorylation have remained unknown. We investigated the role of Slingshot (SSH) 1L, a member of a SSH family of protein phosphatases, in mediating Ca 2؉ -induced cofilin dephosphorylation. The Ca 2؉ ionophore A23187 and Ca 2؉ -mobilizing agonists, ATP and histamine, induced SSH1L activation and cofilin dephosphorylation in cultured cells. A23187-or histamine-induced SSH1L activation and cofilin dephosphorylation were blocked by calcineurin inhibitors or a dominant-negative form of calcineurin, indicating that calcineurin mediates Ca 2؉ -induced SSH1L activation and cofilin dephosphorylation. Importantly, knockdown of SSH1L expression by RNA interference abolished A23187-or calcineurin-induced cofilin dephosphorylation. Furthermore, calcineurin dephosphorylated SSH1L and increased the cofilin-phosphatase activity of SSH1L in cell-free assays. Based on these findings, we suggest that Ca 2؉ -induced cofilin dephosphorylation is mediated by calcineurin-dependent activation of SSH1L.

Long recognized as an important regulatory mechanism in biosignal processes, modulation of the phosphorylation state of proteins has emerged as the most important mechanism for understanding signal transduction. In contrast to the multitude of protein kinases and the clear signal transduction pathways, relatively few protein phosphatases are known and their regulation is unclear. Among them, calcineurin, a calcium/calmodulin-dependent phosphatase (PP2B), is the best enzyme to unveil the phosphatase function, because it was shown to be the direct target for immunosuppressants CsA and FK506, which are powerful tools for understanding this function in diseases as well as in several tissues and organs. Although calcineurin has been found in the highest concentrations in brain, it has also been detected in many other mammalian tissues. Well characterized in T cell activation by analysing the transcription factor NFAT, the function of calcineurin, however, was less well understood in other tissues and organs. Since the mid-1990s, several novel functions of this phosphatase have been reported, revealing that it plays important roles as a multifunctional regulator under the direct regulation of calcium signaling.

Abstract. Calcineurin is a calcium-dependent protein phosphatase that functions in T cell activation. We present evidence that calcineurin functions more generally in calcium-triggered apoptosis in mammalian cells deprived of growth factors. Specifically, expression of epitope-tagged calcineurin A induces rapid cell death upon calcium signaling in the absence of growth factors. We show that this apoptosis does not require new protein synthesis and therefore calcineurin must operate through existing substrates. Co-expression of the Bcl-2 protooncogene efficiently blocks calcineurin-induced cell death. Significantly, we demonstrate that a calciumindependent calcineurin mutant induces apoptosis in the absence of calcium, and that this apoptotic response is a direct consequence of calcineurin's phosphatase activity. These data suggest that calcineurin plays an important role in mediating the upstream events in calcium-activated cell death.

Hypoxia-inducible factor (HIF)-