A mechanism that triggers neuronal apoptosis has been characterized. We report that the cell cycle-regulated protein kinase Cdc2 is expressed in postmitotic granule neurons of the developing rat cerebellum and that Cdc2 mediates apoptosis of cerebellar granule neurons upon the suppression of neuronal activity. Cdc2 catalyzes the phosphorylation of the BH3-only protein BAD at a distinct site, serine 128, and thereby induces BAD-mediated apoptosis in primary neurons by opposing growth factor inhibition of the apoptotic effect of BAD. The phosphorylation of BAD serine 128 inhibits the interaction of growth factor-induced serine 136-phosphorylated BAD with 14-3-3 proteins. Our results suggest that a critical component of the cell cycle couples an apoptotic signal to the cell death machinery via a phosphorylation-dependent mechanism that may generally modulate protein-protein interactions.

The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in mediating apoptosis in the developing and mature organism. The JNK signaling pathway is thought to induce apoptosis via transcription-dependent and transcription-independent mechanisms that remain to be elucidated. In this study, we report a novel mechanism by which the JNK signaling pathway directly activates a component of the cell death machinery. We have found that JNK catalyzes the phosphorylation of the BH3-only protein BAD at the distinct site of serine 128 in vitro. Activation of the JNK signaling pathway induces the BAD serine 128 phosphorylation in vivo, including in primary granule neurons of the developing rat cerebellum. The JNK-induced BAD serine 128 phosphorylation promotes the apoptotic effect of BAD in primary neurons by antagonizing the ability of growth factors to inhibit BAD-mediated apoptosis. These findings indicate that BAD is a novel substrate of JNK that links the stress-activated signaling pathway to the cell death machinery.Regulation of cell death is critical to the normal development and homeostasis of multicellular organisms. In the developing nervous system, neurons are produced in excess, and naturally occurring neuronal cell death is thus critical in ensuring that neurons form the appropriate connections (1). In the mature nervous system, abnormally occurring neuronal cell death contributes to the pathogenesis of a variety of diseases including stroke, epilepsy, and neurodegenerative diseases (2-4). Not surprisingly, the mechanisms that underlie neuronal cell death have been the subject of intense interest.Studies of cell death in a variety of organisms have revealed that members of the Bcl-2 family of proteins act as gatekeepers of the cell death machinery (5-7). Under conditions in which cells are destined to undergo programmed cell death (apoptosis), the proapoptotic multidomain Bcl-2 proteins, including Bax, induce the release of cytochrome c from mitochondria leading to the activation of a caspase cascade that executes the cell death program (8, 9). The prosurvival multidomain Bcl-2 proteins, including Bcl-2 and Bcl-xl, interact with and inhibit the proapoptotic multidomain Bcl-2 proteins (10).The BH3-only subfamily of Bcl-2 proteins appears to play an important role in regulating the function of the multidomain Bcl-2 protein in response to signals from the cell surface or the cytoskeleton (11). The BH3-only protein BAD promotes cell death by interacting with and inhibiting the prosurvival multidomain Bcl-2 proteins (10). Survival factors suppress BADmediated apoptosis by inducing the phosphorylation of BAD at serine 136, serine 112, or both, culminating in the interaction and sequestration of phosphorylated BAD by members of the 14-3-3 family of proteins (12). The protein kinases Akt, p21-activated kinase 1, and p70S6 kinase appear to mediate survival factor-induced phosphorylation of BAD serine 136 (13-17). On the other hand, the protein kinases Rsk, protein kinase A, and p21-activated kinase 1 ar...

The temporally specific nature of neurotrophic factor-induced responses is a general feature of mammalian nervous system development, the mechanisms of which remain to be elucidated. We characterized a mechanism underlying the temporal specificity by which BDNF selectively promotes the survival of newly generated, but not mature, granule neurons of the mammalian cerebellum. We found that BDNF specifically induces the extracellular signal-regulated kinase 5 (ERK5)-myocyte enhancer factor (MEF2) signaling pathway in newly generated granule neurons and thereby induces transcription of neurotrophin-3 (NT-3), a novel gene target of MEF2. Inhibition of endogenous ERK5, MEF2, or NT-3 in neurons by several approaches including disruption of the NT-3 gene in mice revealed a requirement for the ERK5-MEF2-NT-3 signaling pathway in BDNF-induced survival of newly generated granule neurons. These findings define a novel mechanism that underlies the antiapoptotic effect of neurotrophins in a temporally defined pattern in the developing mammalian brain.