A Novel Cyclic AMP-Dependent Epac-Rit Signaling Pathway Contributes to PACAP38-Mediated Neuronal Differentiation (original) (raw)
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Molecular Biology of the Cell, 2010
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent neuropeptide that possesses both neurotrophic and neurodevelopmental effects. Recently, the Rit GTPase was found to be activated by a novel G␣/cAMP/exchange protein activated by cyclic AMP (Epac)-dependent signaling pathway and required for PACAP-dependent cAMP response element-binding protein activation and neuronal differentiation. However, Epac did not function as a Rit guanine nucleotide exchange factor (GEF), and the nature of the PACAP regulatory cascade remained unclear. Here, we show that PACAP-mediated Rit activation involves Src family kinase-dependent TrkA receptor transactivation. PACAP receptor (PACR1) stimulation triggered both G i ␣ and G s ␣/cAMP/Epac regulatory cascades resulting in Src kinase activity, which in turn induced TrkA kinase tyrosine phosphorylation. Importantly, Src inhibition, or the lack of functional Trk receptors, was found to inhibit PACAP-mediated Rit activation, whereas constitutively active Src alone was sufficient to stimulate Rit-guanosine triphosphate levels. A single tyrosine (Y 499 ) phosphorylation event was identified as critical to both PACAP-mediated transactivation and TrkA-dependent Rit activation. Accordingly, PACAP stimulation resulted in TrkA-dependent phosphorylation of both the Shc adaptor and son of sevenless (SOS)1/2 GEFs, and Rit activation was inhibited by RNA interference silencing of SOS1/2, implicating a TrkA/Shc/SOS signaling complex in Rit regulation. Together, these observations expand upon the nature of PACR1-mediated transactivation and identify TrkA-Rit signaling as a key contributor to PACAP-dependent neuronal differentiation.
The FASEB Journal, 2012
Both cAMP and ERK are necessary for neuroendocrine cell neuritogenesis, and pituitary adenylate cyclase-activating polypeptide (PACAP) activates each. It is important to know whether cAMP and ERK are arranged in a novel, linear pathway or in two parallel pathways using known signaling mechanisms. Native cellular responses [cAMP elevation, ERK phosphorylation, cAMP responsive element binding (CREB) phosphorylation, and neuritogenesis] and promoter-reporter gene activation after treatment with forskolin, cAMP analogs, and PACAP were measured in Neuroscreen-1 (NS-1) cells, a PC12 variant enabling simultaneous morphological, molecular biological, and biochemical analysis. Forskolin (25 M) and cAMP analogs (8-bromo-cAMP, dibutyryl-cAMP, and 8-chlorophenylthio-cAMP) stimulated ERK phosphorylation and neuritogenesis in NS-1 cells. Both ERK phosphorylation and neuritogenesis were MEK dependent (blocked by 10 M U0126) and PKA independent (insensitive to 30 M H-89 or 100 nM myristoylated protein kinase A inhibitor). CREB phosphorylation induced by PACAP was blocked by H-89. The exchange protein activated by cAMP (Epac)-selective 8-(4-chlorophenylthio)-2=-O-Me-cAMP (100-500 M) activated Rap1 without affecting the other cAMP-dependent processes. Thus, PACAP-38 potently stimulated two distinct and independent cAMP pathways leading to CREB or ERK activation in NS-1 cells. Drug concentrations for appropriate effect were derived from control data for all compounds. In summary, a novel PKA-and Epac-independent signaling pathway: PACAP ¡ adenylate cyclase ¡ cAMP ¡ ERK ¡ neuritogenesis has been identified.-Emery, A. C., Eiden, L. E. Signaling through the neuropeptide GPCR PAC 1 induces neuritogenesis via a single linear cAMP-and ERK-dependent pathway using a novel cAMP sensor.
A cAMP-activated pathway, including PKA and PI3K, regulates neuronal differentiation
Neurochemistry International, 2004
Neuronal differentiation is a complex process in which many different signalling pathways may be involved. An increase in the intracellular levels of cyclic AMP (cAMP) has been shown to induce neuronal differentiation and also to cooperate with NGF to induce PC12 neurite outgrowth in a Ras-dependent manner. However, the neuritogenic activities associated with cAMP are still not well understood.
Journal of Neurochemistry, 2003
Nuclear calcium signals associated with electrical activation of neurons can control the activity of the transcription factor cAMP-response element binding protein (CREB). Yet, cAMP is thought to be the key messenger that links synaptic activity to the regulation of CREB-mediated transcription. It is generally assumed that synaptic activity increases the intracellular levels of cAMP; this causes activation of the cAMP-dependent protein kinase (PKA) that regulates CREB-mediated transcription either directly or through controlling nuclear signalling of the MAP kinases/extracellular signal-regulated kinases (ERK1/2) pathway. Here we show that, in hippocampal neurons, synaptic activity failed to increase global levels of cAMP that would be required for the cAMP-PKA system to induce nuclear events. Even near-continuous bursting of action potentials, giving rise to large nuclear calcium signals and robust CREB-dependent transcription, left global intracellular levels of cAMP unchanged. These results suggest that the cAMP-PKA system does not function as the transducer of synaptic signals to the nucleus. They indicate that the known inhibitory effects of blockers of PKA on gene expression and longlasting plasticity triggered by calcium entry reflect a gating function of basal activity of PKA that renders neurons permissive for nuclear calcium-regulated, CREB/CBP-dependent gene expression.
Cyclic AMP-Induced Neuronal Differentiation via Activation of p38 Mitogen-Activated Protein Kinase
Journal of Neurochemistry, 2002
The p38 mitogen-activated protein kinase (MAPK) pathway mediates cellular responses to inflammatory cytokines and environmental stress, but recent studies have indicated that p38 MAPK may be involved in a more widespread set of cellular functions. Here we show that activation of the cyclic AMP (cAMP) pathway induces a rapid, dose-dependent phosphorylation and activation of p38 MAPK and that combined stimulation with forskolin and growth factors results in additive stimulation of p38 MAPK. Forskolin-stimulated neurite outgrowth in rat pheochromocytoma PC12 cells was inhibited by the p38 MAPK inhibitor SB203580. With the combination of forskolin and nerve growth factor, neurite outgrowth was additively increased, and this effect was also inhibited by SB203580. Finally, transfection of p38AGF, which exhibits a mutated activation loop, inhibited cAMP-mediated neuronal differentiation. The results indicate that p38 MAPK is a downstream target of the cAMP signaling pathway and that p38 MAPK plays a key role in neuronal differentiation induced by cAMP and growth factors by integration of signals from both pathways. Key Words: p38 mitogen-activated protein kinase -Cyclic AMP-Protein kinase A-Neuronal differentiation.
Molecular and Cellular Biology, 2005
Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.
Journal of Biological Chemistry, 2005
In neuronal precursor cells, the magnitude and longevity of mitogen-activated protein (MAP) kinase cascade activation contribute to the nature of the cellular response, differentiation, or proliferation. However, the mechanisms by which neurotrophins promote prolonged MAP kinase signaling are not well understood. Here we defined the Rin GTPase as a novel component of the regulatory machinery contributing to the selective integration of MAP kinase signaling and neuronal development. Rin is expressed exclusively in neurons and is activated by neurotrophin signaling, and loss-offunction analysis demonstrates that Rin makes an essential contribution to nerve growth factor (NGF)-mediated neuronal differentiation. Most surprisingly, although Rin was unable to stimulate MAP kinase activity in NIH 3T3 cells, it potently activated isoform-specific p38␣ MAP kinase signaling and weakly stimulated ERK signaling in pheochromocytoma (PC6) cells. This cell-type specificity is explained in part by the finding that Rin binds and stimulates b-Raf but does not activate c-Raf. Accordingly, selective down-regulation of Rin in PC6 cells suppressed neurotrophin-elicited activation of b-Raf and p38, without obvious effects on NGF-induced ERK activation. Moreover, the ability of NGF to promote neurite outgrowth was inhibited by Rin knockdown. Together, these observations establish Rin as a neuronal specific regulator of neurotrophin signaling, required to couple NGF stimulation to sustain activation of p38 MAP kinase and b-Raf signaling cascades required for neuronal development.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1997
To understand how extracellular signals may produce long-term effects in neural cells, we have analyzed the mechanism by which neurotransmitters and growth factors induce phosphorylation of the transcription factor cAMP response element binding protein (CREB) in cortical oligodendrocyte progenitor (OP) cells. Activation of glutamate receptor channels by kainate, as well as stimulation of G-protein-coupled cholinergic receptors by carbachol and tyrosine kinase receptors by basic fibroblast growth factor (bFGF), rapidly leads to mitogen-activated protein kinase (MAPK) phosphorylation and ribosomal S6 kinase (RSK) activation. Kainate and carbachol activation of the MAPK pathway requires extracellular calcium influx and is accompanied by protein kinase C (PKC) induction, with no significant increase in GTP binding to Ras. Conversely, growth factor-stimulated MAPK phosphorylation is independent of extracellular calcium and is accompanied by Ras activation. Both basal and stimulated MAPK ...
Peptides, 2011
Pituitary adenylate cyclase-activating polypeptide (PACAP)-mediated activation of its G proteincoupled receptor PAC1 results in activation of the two G proteins Gs and Gq to alter second messenger generation and gene transcription in the nervous system, important for homeostatic responses to stress and injury. Heterologous expression of the three major splice variants of the rat PAC1 receptor, PAC1hop, null and hip, in neural NG108-15 cells conferred PACAP-mediated intracellular cAMP generation, while elevation of [Ca 2+ ] i occurred only in PAC1hop-, and to a lesser extent in PAC1null-expressing cells. Induction of vasoactive intestinal polypeptide (VIP) and stanniocalcin 1 (STC1), two genes potentially involved in PACAP's homeostatic responses, was examined as a function of the expressed PAC1 variant. VIP induction was greatest in PAC1hop-expressing cells, suggesting that a maximal transcriptional response requires combinatorial signaling through both cAMP and Ca 2+. STC1 induction was similar for all three receptor splice variants and was mimicked by the adenylate cyclase activator forskolin, indicating that cAMP elevation is sufficient to induce STC1. The degree of activation of two different second messenger pathways appears to determine the transcriptional response, suggesting that cellular responses to stressors are fine-tuned through differential receptor isoform expression. Signaling to the VIP gene proceeded through cAMP and protein kinase A (PKA) in these cells, independently of the MAP kinase ERK1/2. STC1 gene induction by PACAP was dependent on cAMP and ERK1/2, independently of PKA. Differential gene induction via different cAMP dependent signaling pathways potentially provides further targets for the design of treatments for stressassociated disorders.