The cAMP-responsive element binding protein (CREB), a key regulator of gene expression, is activated by phosphorylation on Ser-133. Several different protein kinases possess the capability of driving this phosphorylation, making it a point of potential convergence for multiple intracellular signaling cascades. Previous work in neurons has indicated that physiologic synaptic stimulation recruits a fast calmodulin kinase IV (CaMKIV)-dependent pathway that dominates early signaling to CREB. Here we show in hippocampal neurons that the fast, CaMK-dependent pathway can be followed by a slower pathway that depends on Ras/mitogen-activated protein kinase (MAPK), along with CaMK. This pathway was blocked by dominant-negative Ras and was specifically recruited by depolarizations that produced strong intracellular Ca 2+ transients. When both pathways were recruited, phosphorylated CREB (pCREB) formation was overwhelmingly dominated by the CaMK pathway between 0 and 10 min, and by the MAPK pathway at 60 min, whereas the two pathways acted in concert at 30 min. The Ca 2+ signals that produced only rapid CaMK signaling to pCREB or both rapid CaMK and slow MAPK signaling deviated significantly for only ≈1 min, yet their differential impact on pCREB extended over a much longer period, between 20 and 60 min and beyond, which is of likely significance for gene expression. The CaMK-dependent MAPK pathway may inform the nucleus about stimulus amplitude. In contrast, the CaMKIV pathway may be well suited to conveying information on the precise timing of localized synaptic stimuli, befitting its greater speed and sensitivity, whereas the previously described calcineurin pathway may carry information about stimulus duration.
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The dynamic complexity of information processing within neuronal networks is greatly increased by activity-dependent changes in gene expression within individual neurons. Mammalian neurons express many tens of thousands of genes, several times more than any other known cell type. Alterations in the expression of these genes and in the state of the individual protein molecules they encode provide enormous capabilities for intracellular computation. Although little is known as yet about the extent to which such computation is actually used in the brain, understanding how various forms of neuronal activity control gene transcription is an obvious first step.
A leading paradigm of such regulation is the activation of the nuclear transcription factor CREB, the Ca 2+- and cAMP-responsive element binding protein. CREB becomes activated when phosphorylated on Ser-133 and, through interaction with its nuclear partner CREB-binding protein, drives the transcription of a large number of genes. Although other residues can also be phosphorylated, with possible functional consequences, Ser-133 has been the predominant focus of studies of transcriptional regulation. Ser-133 phosphorylation of CREB can be caused by electrical activity, neurotransmitter or hormone action on G-protein-coupled receptors, or neurotrophin effects on receptor tyrosine kinases. CREB has been strongly implicated in memory formation in a wide range of species. The richness of CREB signaling is greatly increased by its responsiveness to multiple intracellular signal transduction cascades. For example, direct activation of cAMP signaling (e.g., with forskolin, dopamine D1/D5 receptor stimulation, or cAMP analogs) gives rise to robust CREB phosphorylation. On the other hand, behavioral, synaptic or membrane-depolarizing stimuli chiefly recruit a calmodulin (CaM)/CaM kinase IV (CaMKIV)-mediated pathway. In some cases, a Ras/mitogen-activated protein kinase (MAPK) or extracellular signal-regulated protein kinase (ERK)-mediated pathway, modulated by protein kinase A (PKA) and protein kinase C, is also recruited. The functional importance of these signaling cascades is not entirely clear. For example, it has been claimed that the CaMKIV pathway can be dispensable in hippocampal neurons, and that the Ras-ERK-Rsk2 pathway can be a predominant pathway to CREB phosphorylation. On the other hand, genetic deletion of CaMKIV greatly attenuated both basal and activity-dependent CREB phosphorylation in a wide variety of central neurons at time points after stimulation ranging from 2 min to 45 min or more in addition to blocking the activation of CREB-dependent gene expression. These studies raised fresh questions about the possible role of the MAPK pathway.
This paper describes experiments that focus on the kinetic contributions of various signaling pathways to the overall time course of CREB phosphorylation. The results provide information about how individual signaling pathways to CREB can be activated selectively and the possible functional significance of multiple converging pathways in carrying distinct information to the nucleus.
Calcium phosphate transfections, immunocytochemistry, and culture of dissociated hippocampal CA3/CA1 pyramidal neurons were carried out as previously described. Before stimulation, neurons at 8–9 days in vitro were preincubated in 1 μM tetrodotoxin (TTX) for 1–2 h to block spontaneous neuronal activity; TTX was also present during the poststimulus phase. The usual stimulus (90 mM KCl in isotonic Tyrode's solution) clamps the membrane potential of the neurons close to 0 mV and, in light of known current–voltage relations, provides maximal activation of voltage-gated sources of calcium influx. TTX was typically not present during the 90 mM K+ stimulus; as would be expected, inclusion of TTX during this type of stimulus has no effect (data not shown). However, for a 20 mM K+ stimulus TTX was included, as this is a submaximal stimulus. Stimuli were applied by rapidly changing the perfusion solution over the coverslip substrate bearing the cultured neurons from control to high-K Tyrode's solution. Pharmacological agents were added 15–20 min before stimulation unless otherwise indicated and were present in the recovery solutions. Drugs were obtained from Calbiochem [TTX, KN-93, KN-92, KT 5720, R P isomer of adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS), and forskolin] and Promega (U0126 and PD98059). The enhanced green fluorescent protein (EGFP) expression plasmid was obtained from CLONTECH; the expression plasmid for RasN17 was purchased from Upstate Biotechnology (Lake Placid, NY).
Antibody to Ser-133-phosphorylated CREB (pCREB) (Upstate Biotechnology) was used at 1:500. Antibody to dually phosphorylated ERK1/2 was obtained from Promega or Upstate Biotechnology and used at 1:1000. Anti-MAP2 monoclonal antibody (Roche Molecular Biochemicals) was used at 1:500. Secondary antibodies were from Jackson ImmunoResearch. To image and quantify immunofluorescence, confocal microscopy was performed as previously described. For purposes of quantification of dually phosphorylated MAPK (pMAPK), apical dendrites from all clearly identifiable individual neurons within randomly selected fields were included. Intracellular calcium imaging with fura-2 was conducted as previously described.
To distinguish the kinetics of different activity-dependent signaling pathways to CREB, we followed the time course of depolarization-induced CREB phosphorylation in the presence of different pharmacological inhibitors and under different stimulus conditions. First, PD98059, a MAPK kinase (MEK) inhibitor, was used to block the MAPK signaling pathway to CREB, and KN-93, an inhibitor of CaM-dependent kinases, was used to block the CaMK signaling pathway.
We provided a maximal depolarizing stimulus to the neurons (3 min of 90 mM K+) and tracked the resulting dynamics of pCREB. When neurons were fixed immediately after depolarization in the absence of pharmacological inhibition, robust formation of nuclear pCREB was detected with a phospho-specific antibody (shown in green). When instead fixation was delayed by a 60-min recovery period in control solution after stimulation, the majority of neurons still showed prominent nuclear pCREB, whereas approximately one-third of the cells had returned to their baseline state. This result is shown quantitatively in the intensity histograms below the images. During this recovery phase, nuclear pCREB intensity in individual neurons displayed an all-or-none character, as previously observed, suggesting that when CREB dephosphorylation occurs, it takes place rather abruptly and completely.
A very different pattern was observed when the MAPK pathway was inhibited with PD98059. Whereas the formation of pCREB immediately after the K+-depolarization was virtually unchanged by the MEK inhibitor, pCREB at the late time point was strongly reduced. Similar results were obtained with the distinct MEK inhibitor U0126 (data not shown). These data suggested that MAPK signaling played a selective role in the late phase of CREB phosphorylation. Yet another pattern of results was found in the presence of the potent CaMK inhibitor KN-93. In this case, the immediate increase in pCREB was abolished, consistent with the known role of CaM kinases in the fast phase of depolarization-induced pCREB formation.
