Activity-dependent regulation of neuronal events such as cell survival and synaptic plasticity is controlled by increases in neuronal calcium levels. These actions often involve stimulation of intracellular kinase signaling pathways. For example, the mitogen-activated protein kinase, or extracellular signal-regulated kinase (ERK), signaling cascade has increasingly been shown to be important for the induction of gene expression and long term potentiation. However, the mechanisms leading to ERK activation by neuronal calcium are still unclear. In the present study, we describe a protein kinase A (PKA)-dependent signaling pathway that may link neuronal calcium influx to ERKs via the small G-protein, Rap1, and the neuronal Raf isoform, B-Raf. Thus, in PC12 cells, depolarization-mediated calcium influx led to the activation of B-Raf, but not Raf-1, via PKA. Furthermore, depolarization also induced the PKA-dependent stimulation of Rap1 and led to the formation of a Rap1/B-Raf signaling complex. In contrast, depolarization did not lead to the association of Ras with B-Raf. The major action of PKA-dependent Rap1/B-Raf signaling in neuronal cells is the activation of ERKs. Thus, we further show that, in both PC12 cells and hippocampal neurons, depolarization-induced calcium influx stimulates ERK activity in a PKA-dependent manner. Given the fact that both Rap1 and B-Raf are highly expressed in the central nervous system, we suggest that this signaling pathway may regulate a number of activity-dependent neuronal functions.

In cerebellar granule cells, the mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) cascade mediates multiple functions, including proliferation, differentiation, and survival. In these cells, ERKs are activated by diverse stimuli, including cyclic adenosine monophosphate (cAMP), pituitary adenylate cyclase activating protein (PACAP), depolarization induced by elevated extracellular potassium (KCl), and the neurotrophin brain-derived neurotrophic factor. Extensive studies in neuronal cell lines have implicated the small G proteins Ras and Rap1 in the activation of ERKs by cAMP, PACAP, and KCl. However, the requirement of Ras and Rap1 in these pathways in cerebellar granule cells has not been addressed. In this study, we utilize multiple biochemical assays to determine the mechanisms of action and requirement of Ras and Rap1 in cultured cerebellar granule cells. We show that both Ras and Rap1 can be activated by cAMP or PACAP via protein kinase (PKA)-dependent mechanisms. KCl activation of Ras also required PKA. Using both adenoviral and transgenic approaches, we show that Ras plays a major role in ERK activation by cAMP, PACAP, and KCl, while Rap1 also mediates activation of a selective membrane-associated pool of ERKs. Furthermore, Rap1, but not Ras, activation by PKA appears to require the action of Src family kinases.

The heterotrimeric G proteins are an extended family of guanyl nucleotide-binding proteins that serve essential functions in the mature nervous system but whose contributions to neuronal development remain poorly understood. We have investigated the potential role of one specific G protein, Go(alpha), in the control of neuronal migration. During embryogenesis of the moth, Manduca sexta, an identified population of undifferentiated neurons (the EP cells) migrate along sets of visceral muscle bands to form part of the enteric nervous system. Previously, immunohistochemical studies indicated the presence of Go(alpha)-related proteins in the EP cells during migration. We have now verified this result, using probes derived from the Go(alpha) gene in Manduca. A clone containing the full-length coding domain for Go(alpha) was sequenced from a Manduca cDNA library; digoxigenin-labeled probes were then made from this clone and used to examine the developmental expression of the Go(alpha) gene during embryogenesis. Go(alpha)-specific transcripts could first be detected in the EP cells several hours before the onset of their migration. The level of Go(alpha) expression in all of the EP cells continued to increase during migration, but subsequently was down-regulated in a subset of the postmigratory neurons at the time of their terminal differentiation. This pattern of regulated expression is consistent with the distribution of Go(alpha)-related protein in the EP cells. We also used a semi-intact culture preparation of staged embryos to investigate the effects of G protein-specific toxins on the migratory process. Intracellular injections of the wasp toxin mastoparan, a specific activator of Go(alpha)-and Gi(alpha)-related proteins, inhibited the migration of individual EP cells. Injections of pertussis toxin (an inhibitor of Go(alpha) and Gi(alpha)) or cholera toxin (a selective activator of Gs(alpha)) had no effect on migration, although pertussis toxin treatments did cause a measurable increase in the subsequent outgrowth of axonal processes. However, co-injection of mastoparan with pertussis toxin blocked the inhibitory effects of mastoparan alone. These results suggest that Go(alpha)-coupled signaling events within the EP cells may down-regulate their migratory behavior, possibly in response to inhibitory cues that normally guide migration in the developing embryo.

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