Although Ca2+-stimulated cAMP response element binding protein- (CREB-) dependent transcription has been implicated in growth, differentiation, and neuroplasticity, mechanisms for Ca2+-activated transcription have not been defined. Here, we report that extracellular signal-related protein kinase (ERK) signaling is obligatory for Ca2+-stimulated transcription in PC12 cells and hippocampal neurons. The sequential activation of ERK and Rsk2 by Ca2+ leads to the phosphorylation and transactivation of CREB. Interestingly, the Ca2+-induced nuclear translocation of ERK and Rsk2 to the nucleus requires protein kinase A (PKA) activation. This may explain why PKA activity is required for Ca2+-stimulated CREB-dependent transcription. Furthermore, the full expression of the late phase of long-term potentiation (L-LTP) and L-LTP-associated CRE-mediated transcription requires ERK activation, suggesting that the activation of CREB by ERK plays a critical role in the formation of long lasting neuronal plasticity.

The protection against apoptosis provided by growth factors in several cell lines is due to stimulation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, which results in activation of protein kinase B (PKB; also known as c-Akt and Rac) and phosphorylation and sequestration to protein 14-3-3 of the proapoptotic Bcl-2-family member BAD. A modest increase in intracellular Ca2+ concentration also promotes survival of some cultured neurons through a pathway that requires calmodulin but is independent of PI(3)K and the MAP kinases. Here we report that Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) activates PKB directly, resulting in phosphorylation of BAD on serine residue 136 and the interaction of BAD with protein 14-3-3. Serum withdrawal induced a three- to fourfold increase in cell death of NG108 neuroblastoma cells, and this apoptosis was largely blocked by increasing the intracellular Ca2+ concentration with NMDA (N-methyl-D-aspartate) or KCl or by transfection with constitutively active CaM-KK. The effect of NMDA on cell survival was blocked by transfection with dominant-negative forms of CaM-KK or PKB. These results identify a Ca2+-triggered signalling cascade in which CaM-KK activates PKB, which in turn phosphorylates BAD and protects cells from apoptosis.

Brain ischemia induces apoptosis in neuronal cells, but the mechanism is not well understood. When wild-type mice were subjected to bilateral common carotid arteries occlusion (BCCAO) for 15 min, apoptosis-associated morphological changes and appearance of TUNEL-positive cells were observed in the striatum and in the hippocampus at 48 h after occlusion. RT-PCR analysis revealed that mRNAs for ER stress-associated proapoptotic factor CHOP and an ER chaperone BiP are markedly induced at 12 h after BCCAO. Immunohistochemical analysis showed that CHOP protein is induced in nuclei of damaged neurons at 24 h after occlusion. In contrast, ischemia-associated apoptotic loss of neurons was decreased in CHOP À/À mice. Primary hippocampal neurons from CHOP À/À mice were more resistant to hypoxiareoxygenation-induced apoptosis than those from wild-type animals. These results indicate that ischemia-induced neuronal cell death is mediated by the ER stress pathway involving CHOP induction.