n Interactive Fly, Drosophila



Table of contents

Pathways to CREB activation

Nerve growth factor (NGF) is a neurotrophic factor secreted by cells that are the targets of innervation of sympathetic and some sensory neurons. However, what remains unclear is the mechanism by which the NGF signal is propagated from the axon terminal to the cell body (which can be more than 1 meter away). This mechanism influences biochemical events critical for growth and survival of neurons. An NGF-mediated signal transmitted from the terminals and distal axons of cultured rat sympathetic neurons to their nuclei regulates phosphorylation of the transcription factor CREB (cyclic adenosine monophosphate response element-binding protein). Internalization of NGF and its receptor tyrosine kinase TrkA, and the transport of both to the cell body, are required for transmission of this signal. The tyrosine kinase activity of TrkA is required to maintain TrkA in an autophosphorylated state upon its arrival in the cell body and for propagation of the signal to CREB within neuronal nuclei. Thus, an NGF-TrkA complex is a messenger that delivers the NGF signal from axon terminals to cell bodies of sympathetic neurons (Riccio, 1997).

Nerve growth factor (NGF) activates multiple signaling pathways that mediate the phosphorylation of CREB at the critical regulatory site, serine 133 (Ser-133). NGF activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs), which in turn activate the pp90 ribosomal S6 kinase (RSK; see Drosophila RSK) family of Ser/Thr kinases, all three members of which were found to catalyze CREB Ser-133 phosphorylation in vitro and in vivo. In addition to the ERK/RSK pathway, NGF activates the p38 MAPK and its downstream effector, MAPK-activated protein kinase 2 (MAPKAP kinase 2), resulting in phosphorylation of CREB at Ser-133. Inhibition of either the ERK/RSK or the p38/MAPKAP kinase 2 pathway only partially blocks NGF-induced CREB Ser-133 phosphorylation, suggesting that either pathway alone is sufficient for coupling the NGF signal to CREB activation. However, inhibition of both the ERK/RSK and the p38/MAPKAP kinase 2 pathways completely abolish NGF-induced CREB Ser-133 phosphorylation. These findings indicate that NGF activates two distinct MAPK pathways, both of which contribute to the phosphorylation of the transcription factor CREB and the activation of immediate-early genes (Xing, 1998).

beta neuregulins (also called NDF, GGF, ARIA, and heregulins) are neuron-derived molecules that are likely to be responsible for Schwann cell precursor survival, proliferation, and maturation, both in vivo and in vitro. Although the receptors to which beta neuregulins bind have been defined, little is known about the transcription factors these important ligands activate. Using antibodies, quantitative imaging methods and Western blotting, it has been shown that beta neuregulin induces a high level of phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB) on Ser-133 in cultured rat Schwann cells and that the phosphorylation is prolonged over several hours. In contrast, neurotrophins, CNTF, FGF-2, EGF, and TGFbeta induce little or no phosphorylation of CREB despite the fact that receptors for these factors are present on Schwann cells. As expected, CREB phosphorylation is detected following cAMP elevation, and it is also induced by elevation of cytoplasmic Ca2+, endothelin 1, and PDGF-BB. The signal is lower than that seen in response to beta neuregulin, and transient, unlike the sustained CREB activation induced by beta neuregulin. These results suggest that the sustained phosphorylation of CREB on Ser-133 may contribute to the broad spectrum of effects that beta neuregulins have on cells of the Schwann cell lineage and that the CREB pathway may be important for transduction of neuregulin signals in Schwann cells (Tabernero, 1998).

The cAMP response element­binding protein (CREB) is a plasticity-associated transcription factor that can potentially integrate cAMP and calcium signals at the gene activation level. Convergence in Ser-133 phosphorylation of CREB via either dopamine D1/D5 receptors or L-type calcium channels has been tested in organotypic cultures of neonatal striatum. Such convergence occurs only transiently. Sustained CREB phosphorylation by dopamine D1/D5 receptor and L-type channel agonists is targeted to striosomes (striatal neurons) for D1/D5 agonists and to matrix (surrounding tissues) by L-type channel agonists. Subsequent expression of c-fos, the CRE-containing gene, matches the divergent patterns of sustained CREB phosphorylation, and both divergent patterns can be switched by inhibition of phosphatases, including calcineurin. Calcineurin is differentially concentrated in developing striosomes, and thus inhibition of Calcineurin results in a change in the pattern of sustained CREB phosphorylation induced by L-type Ca2+ channels. Control of the duration of CREB phosphorylation may be a critical regulator of CRE-mediated gene expression by dopamine and calcium (Liu, 1996).

A mechanism by which the nerve growth factor (NGF) signal is transduced to the nucleus to induce gene expression has been characterized. An NGF-inducible, Ras-dependent protein kinase has been identified that catalyzes the phosphorylation of the cyclic AMP response element-binding protein (CREB) at Ser-133. Phosphorylation of Ser-133 stimulates the ability of CREB to activate transcription in NGF-treated cells. These findings suggest that CREB has a more widespread function than previously believed and functions in the nucleus as a general mediator of growth factor responses (Ginty, 1994).

Somatostatin (SRIF) was discovered as an inhibitor of GH secretion from pituitary somatotroph cells. SRIF analogs are very effective agents used to treat neuroendocrine tumors and are now being used with increasing frequency in clinical trials to treat more aggressive malignancies. However, the cellular components mediating SRIF signal transduction remain largely unknown. The SRIF type 2 receptor (SST2) was stabily expressed in GH4 rat somatomammotroph cells, establishing a physiologically relevant model system. In this model, a SRIF analog inhibits forskolin-induced cAMP accumulation, protein kinase A activation, cAMP response element-binding protein (CREB) phosphorylation, and Pit-1/GHF-1 promoter activation in an okadaic acid-insensitive manner. Pertussis toxin inhibits the effects of the NGF analog, documenting that SST2 signaling is coupled to Gi. The inhibitory effects of the NGF analog are reversed by overexpression of protein kinase A catalytic subunit, indicating that SRIF does not act via serine/threonine phosphatases, but rather, by lowering protein kinase A activity. These data define the components of the SRIF/SST2 receptor signaling pathway and provide important mechanistic insights into how SRIF controls neuroendocrine tumors (Tentler, 1997).

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. 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. The Ca2+-induced nuclear translocation of ERK and Rsk2 to the nucleus requires protein kinase A (PKA) activation. Interestingly, Ca2+-mediated CREB phosphorylation in wild-type PC12 cells is decreased by a selective PKA inhibitor. With a high efficiency transfection protocol, expression of dominant negative PKA also attenuates Ca2+-stimulated CREB phosphorylation. In addition, treatment with the PKA inhibitors also inhibits depolarization-mediated CREB phosphorylation in primary hippocampal neurons. These results suggest that in PC12 cells and hippocampal neurons, PKA activity is required for Ca2+-induced CREB phosphorylation (Impey, 1998a).

Because the nuclear translocation of ERK may be necessary for ERK-activated transcription, and PKA is required for Ca2+ stimulation of CREB phosphorylation, nuclear translocation of ERK was monitored when PKA was inhibited. To efficiently induce the nuclear translocation of ERK by Ca2+, PC12 cells were treated with KCl and a direct activator of L-type Ca2+ channels. Depolarization induces the phosphorylation of ERK and its translocation to the nucleus in both PC12 cells and hippocampal neurons. The specific PKA inhibitors inhibited the nuclear translocation of Erk in PC12 cells and hippocampal neurons. Western blotting of cytosolic fractions shows that the inhibition of Erk translocation by treatment with PKA inhibitors is not the result of an effect on Erk activation. To verify that PKA is required for the nuclear translocation of ERK, the cytosolic-to-nuclear ratio of phospho-ERK in KCl-stimulated hippocampal neurons was also quantitated. A specific PKA inhibitor significantly inhibited the translocation of ERK to the nucleus. The importance of PKA activity for ERK nuclear translocation was confirmed by transiently transfecting PC12 cells with a dominant negative PKA fused to green fluorescent protein. Only cells that express dominant negative PKA-GFP show impaired nuclear translocation of phospho-ERK. These results suggest that PKA is required for the phosphorylation and transactivation of CREB by Ca2+, because PKA is required for the nuclear translocation of ERK. However, since Rsk2 [a member of the pp90(RSK) family] is a major Ca2+-activated CREB kinase in PC12 cells, inhibition of Erk translocation should also block the activation of nuclear but not cytosolic Rsk2. Accordingly, inhibition of PKA blocks the activation of Rsk2 in the nuclear fraction but not in the cytosolic fraction. Treatment with PKA inhibitors attenuates the nuclear translocation of Rsk2. This is not surprising, because it is known that both ERK and Rsk2 are tightly associated in vivo and that they cotranslocate to the nucleus. Collectively, these data indicate that PKA may be necessary for the phosphorylation and transactivation of CREB by Ca2+, because PKA is required for the nuclear translocation of ERK and subsequent nuclear activation of the CREB kinase Rsk2. Inhibition of PKA also significantly impairs the translocation of ERK to the nucleus in response to NGF. Interestingly, coexpression of dominant negative PKA attenuates NGF-stimulated Elk1 transcriptional activation. Evidently, the modulation of ERK translocation by PKA activity plays a general role in the activation of transcription by mitogens and neurotrophic factors. NGF does not detectably elevate intracellular cAMP, suggesting that basal PKA activity is sufficient for neurotrophic factors and other strong ERK activators to induce nuclear translocation of ERK. Nevertheless, in the case of depolarization, which activates ERK to a lesser degree, the concomitant depolarization-mediated increase in cAMP levels enhances ERK translocation. These results 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 (Impey, 1998a).

Exposure of Syrian hamsters to light 1 h after lights-off rapidly (10 min) induces nuclear immunoreactivity (-ir) to the phospho-Ser133 form of the Ca2+/cAMP response element (CRE) binding protein (pCREB) in the retinorecipient zone of the suprachiasmatic nuclei (SCN). Light also induces nuclear Fos-ir in the same region of the SCN after 1 h. The glutamatergic N-methyl-D-aspartate (NMDA) receptor blocker MK801 attenuates the photic induction of both factors. To investigate glutamatergic regulation of pCREB and Fos further, tissue blocks and primary cultures of neonatal hamster SCN were examined by Western blotting and immunocytochemistry in vitro. The pCREB-ir signal at 45 kDa is enhanced by glutamate or a mixture of glutamatergic agonists [NMDA, amino-methyl proprionic acid (AMPA), and Kainate (KA)], whereas total CREB does not change. Glutamate or the mixture of agonists also induces a 56 kDa band identified as Fos protein in SCN tissue. In dissociated cultures of SCN, glutamate causes a rapid (15 min) induction of nuclear pCREB-ir and Fos-ir (after 60 min) exclusively in neurons, both GABA-ir and others. Treatment with NMDA alone has no effect on pCREB-ir. AMPA alone causes a slight increase in pCREB-ir. However, kainate alone or in combination with NMDA and AMPA induces nuclear pCREB-ir equal to that induced by glutamate. The effects of glutamate on pCREB-ir and Fos-ir are blocked by antagonists of both NMDA (MK801) and AMPA/KA (NBQX) receptors. In the absence of extracellular Mg2+, MK801 blocks glutamatergic induction of Fos-ir. However, the AMPA/KA receptor antagonist is no longer effective at blocking glutamatergic induction of either Fos-ir or pCREB-ir, consistent with the model that glutamate regulates gene expression in the SCN by a co-ordinate action through both NMDA and AMPA/KA receptors. Glutamatergic induction of nuclear pCREB-ir in GABA-ir neurons is blocked by an inhibitor of Ca2+/Calmodulin (CaM)-dependent kinases, implicating Ca2+-dependent signaling pathways in the glutamatergic regulation of gene expression in the SCN (Schurov, 1999).

Activation of the mitogen-activated protein kinase (MAPK) cascade plays an important role in synaptic plasticity in area CA1 of rat hippocampus. However, the upstream mechanisms regulating MAPK activity and the downstream effectors of MAPK in the hippocampus are uncharacterized. Hippocampal MAPK activation is regulated by both the PKA and PKC systems; moreover, a wide variety of neuromodulatory neurotransmitter receptors (metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, and beta-adrenergic receptors) couple to MAPK activation via these two cascades. PKC is a powerful regulator of CREB phosphorylation in area CA1. MAPK plays a critical role in transcriptional regulation by PKC, because MAPK activation is a necessary component for increased CREB phosphorylation in response to the activation of this kinase. Surprisingly, MAPK activation is necessary for PKA coupling to CREB phosphorylation in area CA1. Overall, these studies indicate an unexpected richness of diversity in the regulation of MAPK in the hippocampus and suggest the possibility of a broad role for the MAPK cascade in regulating gene expression in long-term forms of hippocampal synaptic plasticity (Roberson, 1999).

Although the circadian time-keeping properties of the suprachiasmatic nuclei (SCN) require gene expression, little is known about the signal transduction pathways that initiate transcription. A brief exposure to light during the subjective night, but not during the subjective day, activates the p44/42 mitogen-activated protein kinase (MAPK) signaling cascade in the SCN. In addition, MAPK stimulation activates CREB (cAMP response element binding protein), indicating that potential downstream transcription factors are stimulated by the MAPK pathway in the SCN. Striking circadian variations are observed in MAPK activity within the SCN, suggesting that the MAPK cascade is involved in clock rhythmicity (Obrietan, 1999).

The deposition of amyloid beta protein (A beta) in the cerebral cortex is the pathological characteristic of Alzheimer's disease (AD); patients with AD suffer from progressive memory loss. Transgenic experiments have revealed that long-term memory is dependent on cyclic AMP-response element binding protein, CREB. CREB phosphorylation at serine-133 is essential for its transcriptional activity. At a concentration more than 1 microM, A beta(1-40) induces CREB phosphorylation at serine-133 in rat pheochromocytoma PC12 cells, while PD98059, a MEK1 inhibitor, inhibits A beta(1-40)-induced CREB phosphorylation at serine-133. A beta(1-40) induces phosphorylation of p44 and p42 MAP kinases (Erk1 and Erk2) at tyrosine-204. It is concluded that elevated A beta(1-40) level induces CREB phosphorylation at serine-133 via p44/42 MAP kinase-dependent pathway (Sato, 1997).

The mammalian hypothalamic suprachiasmatic nucleus (SCN) is an endogenous pacemaker generating circadian rhythms. SCN activity is synchronized with environmental light/dark cycles by photic information primarily transmitted via the retinohypothalamic tract (RHT). The SCN controls synthesis and release of melatonin, the hormone of the pineal gland. Melatonin itself feeds back to the SCN. Using brain slice technique and immunocytochemistry it has been demonstrated that (1) pituitary adenylate cyclase-activating polypeptide (PACAP) induces the phosphorylation of the transcription factor cAMP response element binding protein (CREB) in the SCN during late subjective day and (2) melatonin inhibits this PACAP-induced phosphorylation. These data suggest that PACAP is a neurotransmitter that affects gene expression in the SCN, probably via the cAMP signaling pathway, and that the antagonistic effect of melatonin mirrors a feed-back loop within the circadian system (Kopp, 1997).

What signaling pathways control the phosphorylation state of CREB in mammals? Evidence linking PKA to memory is lacking in mammals. In fact, PKA-deficient mice appear normal with regard to learning and long-term memory, although long-term potentiation and long-term depression are perturbed (Huang, 1995). There are two important Ca++/calmodulin (CaM)-regulated mechanisms in hippocampal neurons that control CREB phosphorylation: a CaM kinase cascade involving nuclear CaMKIV and a calcineurin-dependent regulation of nuclear protein phosphatase 1 activity. Kinase inhibitors were used to identify CaMKIV as a regulator of CREB. CaMKII (alpha and beta isoforms) are either undetectable or low in concentration in the nucleus, while only CaMKIV has a predominant nuclear location. Suppression of nuclear CaMKIV with antisense mRNA significantly decreases the number of neurons immunopositive for both CaMKIV and CREB. Even a short depolarization of neurons (1 min) induces a rapid and maximal phosphorylation of CREB and both rapidly and maximimally activates CaMKIV (Bito, 1996).

In cell culture systems, the TCF Elk-1 represents a convergence point for extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) subclasses of mitogen-activated protein kinase (MAPK) cascades. Its phosphorylation strongly potentiates its ability to activate transcription of the c-fos promoter through a ternary complex assembled on the c-fos serum response element. In rat brain postmitotic neurons, Elk-1 is strongly expressed. However, its physiological role in these postmitotic neurons remains to be established. To investigate biochemically the signaling pathways targeting Elk-1 and c-fos in mature neurons, a semi-in vivo system was used, composed of brain slices stimulated with the excitatory neurotransmitter glutamate. Glutamate treatment leads to a robust, progressive activation of the ERK and JNK/SAPK MAPK cascades. This corresponds kinetically to a significant increase in Ser383-phosphorylated Elk-1 and the appearance of c-fos mRNA. Glutamate also causes increased levels of Ser133-phosphorylated cyclic AMP-responsive element-binding protein (CREB) but only transiently relative to Elk-1 and c-fos. ERK and Elk-1 phosphorylation are blocked by the MAPK kinase inhibitor PD98059, indicating the primary role of the ERK cascade in mediating glutamate signaling to Elk-1 in the rat striatum in vivo. Glutamate-mediated CREB phosphorylation is also inhibited by PD98059 treatment. Interestingly, KN62, which interferes with calcium-calmodulin kinase (CaM-K) activity, leads to a reduction of glutamate-induced ERK activation and of CREB phosphorylation. These data indicate that ERK functions as a common component in two signaling pathways (ERK/Elk-1 and ERK/?/CREB) converging on the c-fos promoter in postmitotic neuronal cells and that CaM-Ks act as positive regulators of these pathways (Vanhoutte, 1999)

The c-jun proto-oncogene encodes a transcription factor that is activated by mitogens both transcriptionally and as a result of phosphorylation by Jun N-terminal kinase (JNK). The cellular signaling pathways involved in epidermal growth factor (EGF) induction of the c-jun promoter have been investigated. Two sequence elements that bind ATF1 (a leucine zipper DNA binding protein) and MEF2D transcription factors are required in HeLa cells, although these elements are not sufficient for maximal induction. Activated forms of Ras, RacI, Cdc42Hs, and MEKK increase expression of the c-jun promoter, while dominant negative forms of Ras, RacI, and MEK kinase (MEKK) inhibit EGF induction. These results suggest that EGF activates the c-jun promoter by a Ras-to-Rac-to-MEKK pathway. No change is found in protein binding to the jun ATF1 site in EGF-treated cells. A potential mechanism for regulation of ATF1 and CREB is phosphorylation (Clarke, 1997).

The synaptic basal lamina protein agrin is essential for the formation of neuromuscular junctions. Agrin mediates the postsynaptic clustering of acetylcholine receptors and regulates transcription in muscles. Agrin expression is not restricted to motor neurons but can be demonstrated throughout the CNS. The functional significance of agrin expression in neurons other than motor neurons is unknown. To test whether agrin triggers responses in neurons that lead to the activation of transcription factors, phosphorylation of the transcriptional regulatory site serine 133 of the transcription factor CREB (cAMP response element binding protein) was analyzed in primary hippocampal neurons. The neuronal (Ag4,8), but not the non-neuronal (Ag0,0), isoform of agrin induces CREB phosphorylation in hippocampal neurons. The kinetics of agrin- and BDNF-induced CREB phosphorylation are similar: peak levels are reached in minutes and are strongly reduced 2 hr later. Neuronal responses to agrin require extracellular calcium, and, in contrast to tyrosine kinase inhibitors, the specific inhibition of protein kinase A (PKA) does not affect agrin-evoked CREB phosphorylation. These results show that hippocampal neurons respond specifically to neuronal agrin in a Ca2+-dependent manner and via the activation of tyrosine kinases (Ji, 1998).

One of the early events in AChR clustering is the binding of agrin to MuSK, which becomes phosphorylated at tyrosine residues within minutes of agrin addition. Other phosphorylation events downstream of the initial activation of MuSK also occur. Tyrosine phosphorylation of the beta-subunit of the AChR is concomitant with AChR clustering, but it does not seem to be necessary for this process. As in myotubes, the activation of tyrosine kinases is an essential step in the signaling cascade activated by agrin in neurons. There is a similar requirement for extracellular Ca2+ for agrin function in neurons and muscle. Extracellular Ca2+ is required for both agrin-induced CREB phosphorylation in hippocampal neurons and for the agrin-induced aggregation of AChR on myotubes. There are two possible explanations for the Ca2+ dependence in muscle. Ca2+ may influence the binding of agrin to its receptor; it has been reported that in the absence of Ca2+ agrin does not bind to the myotube surface. Alternatively, engagement of the agrin receptor may trigger a signaling cascade that leads to Ca2+ influx. Because the addition of neither the AChR antagonist alpha-bungarotoxin nor the Na+ channel blocker tetrodotoxin affects agrin-induced AChR aggregation, neither AChR activation nor Na+ channel-dependent depolarization must be required for Ca2+ influx into muscle fibers. A role for a transient rise of intracellular Ca2+ concentration has been proposed for agrin-induced AChR clustering. However, the precise source responsible for these intracellular calcium transients remains undetermined. There are other possible roles for Ca2+ in the neural response to agrin. Again, a Ca2+ requirement for agrin binding to its receptor(s) or the activation of voltage-gated Ca2+ channels may be involved. In neurons, however, the addition of TTX or glutamate receptor antagonists reduces the magnitude of the agrin response even before the establishment of functional synapses. This suggests that a component of the neuronal response to agrin is attributable to acute release of glutamate and/or increased glutamate responsivity. Both cases would result in Na+ channel-dependent depolarization, which would activate voltage-gated Ca2+ channels. How could agrin stimulate neurotransmitter release? It has been reported that agrin promotes the differentiation of presynaptic specializations in motor neurons. It is possible that an acute exposure to agrin may affect the proteins involved in synaptic vesicle release, thus favoring the fusion of vesicles that are docked either along, or in proximity to, the plasma membrane of growth cones. Alternatively, agrin may change the cellular responses to glutamate. This may occur via sensitization of glutamate receptors, possibly via post-translational modifications, or via stimulation of receptor insertion in the plasma membrane, as proposed for silent synapses (Ji, 1998 and references).

A mechanism by which the Ras-mitogen-activated protein kinase (MAPK) signaling pathway mediates growth factor-dependent cell survival has been characterized. The neurotrophin BDNF (brain-derived neurotrophic factor) and its receptor TrkB regulate the survival of newly generated granule neurons within the developing cerebellum. BDNF promotes the survival of cultured rat cerebellar granule neurons; upon BDNF withdrawal, these neurons die by apoptosis. BDNF induces phosphorylation of MAPK. Inhibition of MAPK activity by PD098059, a pharmacological agent that blocks MEK activity, diminishes the effect of BDNF on the survival of cerebellar granule cells. Likewise, the introduction of a dominant interfering form of MEK (MEK-KA97) blocks BDNF-enhancement of neuronal survival. These results indicate that activation of MAPK is required for BDNF-induced survival of cerebellar granule neurons (Bonni, 1999).

Like BDNF, insulin-like growth factor 1 (IGF-1) (or a high concentration of insulin that stimulates the IGF-1 receptor) promotes the survival of cerebellar granule neurons. Both BDNF and IGF-1 activate phosphatidylinositol 3-kinase (PI-3K) and the protein kinase Akt (PKB) cascade in cerebellar granule neurons. Although the PI-3K-Akt signaling pathway mediates the survival-promoting effects of BDNF and IGF-1, inhibition of MAPK in cerebellar neurons has no effect on IGF-1 receptor-mediated cell survival. These results suggest that BDNF and IGF-1 promote cell survival at least in part by distinct mechanisms (Bonni, 1999).

The MAPK-activated kinases, the Rsks, catalyze the phosphorylation of the pro-apoptotic protein BAD at serine 112 both in vitro and in vivo. The Rsk-induced phosphorylation of BAD at serine 112 suppresses BAD-mediated apoptosis in neurons. Rsks also are known to phosphorylate the transcription factor CREB (cAMP response element-binding protein) at serine 133. Activated CREB promotes cell survival, and inhibition of CREB phosphorylation at serine 133 triggers apoptosis. These findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism comprising the posttranslational modification and inactivation of a component of the cell death machinery and the increased transcription of pro-survival genes (Bonni, 1999).

To determine whether CREB contributes to BDNF's ability to enhance cerebellar granule cell survival, the effects of two distinct dominant interfering forms of CREB on the BDNF survival response were tested. K-CREB, in which Arg287 is converted to Leu, forms dimers with endogenous CREB proteins via its leucine zipper domain. K-CREB inhibits the binding of endogenous CREB to the promoters of CREB-responsive genes. M1-CREB, in which Ser133 is converted to Ala, competes with endogenous CREB proteins for binding to the promoters of CREB-responsive genes. However, once bound to DNA, M1-CREB does not activate transcription. When transfected into cerebellar granule neurons, either K-CREB or M1-CREB inhibits the effect of BDNF on cell survival. However, the dominant interfering forms of CREB do not inhibit IGF-1-mediated cerebellar granule cell survival; this finding suggests that these proteins act specifically to block the BDNF response. In addition, M1-CREB does not lead to inhibition of Rsk function because its expression in 293T cells does not inhibit the MEK-induced phosphorylation of BAD at Ser112 (Bonni, 1999).

CREB has been implicated in mediating adaptive responses of neurons to trans-synaptic stimuli. These findings indicate that CREB may also have a function in the regulation of neuronal survival in the developing central nervous system. Mice in which the CREB gene has been disrupted die perinatally before the majority of cerebellar granule neurons are generated However, analysis of the CREB-/- mouse embryos has revealed a number of abnormalities in brain development that may reflect the contribution of CREB to the regulation of the survival of neurons. These findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism that modulates the cell death machinery directly by phosphorylating and thereby inhibiting the pro-apoptotic protein BAD, and by inducing the expression of pro-survival genes in a CREB-dependent manner. Suppression of BAD-mediated cell death by Rsk occurs relatively early after the removal of extracellular survival factors, whereas the contribution of CREB-mediated cell survival is detected significantly later. Therefore, the two arms of the MAPK-Rsk-regulated mechanism might act with different kinetics or at different times in developing neurons (Bonni, 1999).

During development of the cerebellum, Sonic hedgehog is expressed in migrating and settled Purkinje neurons and is directly responsible for proliferation of granule cell precursors in the external germinal layer. SHH interacts with vitronectin in the differentiation of spinal motor neurons. Whether similar interactions between SHH and extracellular matrix glycoproteins regulate subsequent steps of granule cell development has been examined. Laminins and their integrin receptor subunit alpha6 accumulate in the outer most external germinal layer where proliferation of granule cell precursors is maximal. Consistent with this expression pattern, laminin significantly increases SHH-induced proliferation in primary cultures of cerebellar granule cells. Vitronectin and its integrin receptor subunits alphav are expressed in the inner part of the external germinal layer where granule cell precursors exit the cell cycle and commence differentiation. In cultures, vitronectin is able to overcome SHH-induced proliferation, thus allowing granule cell differentiation. The pathway in granule cell precursors responsible for the conversion of a proliferative SHH-mediated response to a differentiation signal depends on CREB. Vitronectin stimulates phosphorylation of cyclic-AMP responsive element-binding protein (CREB), and over-expression of CREB is sufficient to induce granule cell differentiation in the presence of SHH. Although at the present time the different components mediating VN stimulation of CREB phosphorylation are not known, it has been reported that integrins stimulate Ca2+ influx through Calreticulin family members. Ca2+ is a pleiotrophic second messenger that activates a wide variety of kinases. Taken together, these data suggest that granule neuron differentiation is regulated by the vitronectin-induced phosphorylation of CREB, a critical event that terminates SHH-mediated proliferation and permits the differentiation program to proceed in these cells (Pons, 2001).

A new signaling pathway leading to the activation of cAMP-responsive element-binding protein (CREB) has been characterized in several cell lines affected by mitochondrial dysfunction. In vitro kinase assays, inhibitors of several kinase pathways and overexpression of a dominant-negative mutant for calcium/calmodulin kinase IV (CaMKIV), which blocks the activation of CREB, shows that CaMKIV is activated by a mitochondrial activity impairment. A high calcium concentration leading to the disruption of the protein interaction with protein phosphatase 2A explains CaMKIV activation in these conditions. Transcriptionally active phosphorylated CREB was also found in a rho0 143B human osteosarcoma cell line and in a MERRF hybrid cell line mutated for tRNALys (A8344G). Phosphorylated CREB is involved in the proliferation defect induced by a mitochondrial dysfunction. Indeed, cell proliferation inhibition can be prevented by CaMKIV inhibition and CREB dominant-negative mutants. Finally, the data suggest that phosphorylated CREB recruits p53 tumor suppressor protein, modifies its transcriptional activity and increases the expression of p21Waf1/Cip1, a p53-regulated cyclin-dependent kinase inhibitor. These results suggest that the activation of a 'retrograde communication' signaling pathway initiated by a mitochondrial dysfunction could be an important modulator of the cell cycle in cells submitted to energetic stress conditions (Arnould, 2002).

Adenylyl cyclase types 1 (AC1) and 8 (AC8), the two major calmodulin-stimulated adenylyl cyclases in the brain, couple NMDA receptor activation to cAMP signaling pathways. Cyclic AMP signaling pathways are important for many brain functions, such as learning and memory, drug addiction, and development. Wild-type, AC1, AC8, or AC1&8 double knockout (DKO) mice are indistinguishable in tests of acute pain, whereas behavioral responses to peripheral injection of two inflammatory stimuli, formalin and complete Freund's adjuvant, are reduced or abolished in AC1&8 DKO mice. AC1 and AC8 are highly expressed in the anterior cingulate cortex (ACC), and contribute to inflammation-induced activation of CREB. Allodynia is the inflammation-related behavioral sensitization to a non-noxious stimulus. Intra-ACC administration of forskolin rescues behavioral allodynia defective in the AC1&8 DKO mice. These studies suggest that AC1 and AC8 in the ACC selectively contribute to behavioral allodynia (Wei, 2002).

Evidence is presented that AC1 and AC8 are important for CREB activation following tissue injury and inflammation in the ACC and insular cortex. Deletion of AC1 or AC8 causes significant reduction of CREB activated by inflammation. Interestingly, no further reduction was found in the AC1&8 DKO mice. Furthermore, injury-triggered CREB activation is not completely blocked in any mice, suggesting that other signaling pathways also contribute to CREB activation in the forebrain. These findings are slightly different from those in the spinal cord. In spinal cord dorsal horn, injury-induced activation of CREB is completely blocked in AC1&8 DKO mice. Future studies are needed to identify other signaling molecules for injury-related CREB activation in the ACC and insular cortex. In both the spinal cord and ACC, signaling molecules downstream of activated CREB remain to be identified (Wei, 2002).

Developmentally regulated role for Ras-GRFs in coupling NMDA glutamate receptors to Ras, Erk and CREB

p140 Ras-GRF1 and p130 Ras-GRF2 constitute a family of calcium/calmodulin-regulated guanine-nucleotide exchange factors that activate the Ras GTPases. Studies on mice lacking these exchange factors revealed that both p140 Ras-GRF1 and p130 Ras-GRF2 couple NMDA glutamate receptors (NMDARs) to the activation of the Ras/Erk signaling cascade and to the maintenance of CREB transcription factor activity in cortical neurons of adult mice. Consistent with this function for Ras-GRFs and the known neuroprotective effect of CREB activity, ischemia-induced CREB activation is reduced in the brains of adult Ras-GRF knockout mice and neuronal damage is enhanced. Interestingly, in cortical neurons of neonatal animals NMDARs signal through Sos rather than Ras-GRF exchange factors, implying that Ras-GRFs endow NMDARs with functions unique to mature neurons (Tian, 2004).

Ca2+-stimulated adenylyl cyclases regulate ERK-dependent activation of MSK1 during fear conditioning

The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation has not been elucidated. This study reports that PKA, MAPK, and MSK1, a CREB kinase, are coactivated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca2+-stimulated adenylyl cyclase activity. It is concluded that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory (Sindreu, 2007).

One of the major objectives of this study was to identify which MAPK-activated CREB kinase is stimulated during memory formation. Furthermore, it was important to define the relationship between MAPK and cAMP signaling following training for contextual fear conditioning, and to determine why Ca2+-stimulated adenylyl cyclase activity is required for contextual memory. There are several mechanisms by which cAMP could contribute to memory, including regulation of AMPA receptor trafficking and MAPK activation. No increased PKA phosphorylation of AMPA receptors was detected following contextual fear conditioning. Consequently, focus was placed on the role of cAMP signaling in MAPK activation because of the central role played by MAPK during memory formation. Confocal imaging was used to identify individual hippocampal cells in which PKA, MAPK, and CREB kinases are activated after contextual fear conditioning. It has not been previously shown that contextual fear conditioning activates PKA, nor was it known that PKA and MAPK are activated in the same neurons in the hippocampus. Furthermore, there was no evidence for activation of specific CREB kinases following fear conditioning (Sindreu, 2007).

Training for contextual memory caused a 5- to 6-fold increase in MAPK activation in approximately 10% of CA1 pyramidal neurons in two distinct intracellular pools: a nuclear pool and a postsynaptic pool. Furthermore, PKA was activated in the same subset of neurons as MAPK, and both showed increased nuclear activities after training. MAPK activation strongly correlated with activation of MSK1, a CREB kinase. Most importantly, the training-induced increases in MAPK, PKA, and MSK1 activities were ablated in mice lacking Ca2+-stimulated adenylyl cyclase activity. It is concluded that one of the major roles of cAMP signaling in memory is to support the activation and nuclear translocation of MAPK in CA1 pyramidal neurons (Sindreu, 2007).

Signal transduction pathways are usually implicated in memory formation because they are activated in specific areas of the brain by training and inhibition of the pathway blocks memory. For example, MAPK activity is stimulated in area CA1 following training for hippocampus-dependent memory, and administration of MEK inhibitors blocks both training-induced increases in MAPK and memory formation. Ca2+-stimulated adenylyl cyclase and PKA activities are required for memory formation, suggesting that either basal PKA activity is necessary or that an increment in PKA activity contributes to memory. Using an antibody that recognizes phosphorylated PKA substrates (pPKA-s), it was discovered that PKA is not only activated in area CA1 following contextual fear conditioning, but there is also a strong correlation between neurons showing MAPK activation and those in which PKA is activated. In keeping with this, increased nuclear levels of the PKA catalytic α subunit was observed in pERK+ neurons after training. The increase in pPKA-s was readily blocked by inhibitors of PKA and lost in mice lacking Ca2+-stimulated adenylyl cyclase activity, thus validating the use of the pPKA-s antibody to monitor PKA activation (Sindreu, 2007).

The observation that fear conditioning activates MAPK selectively in area CA1 agrees with other evidence that stimulation of transcription in this area of the hippocampus is particularly important for contextual memory formation. Much less was known, however, about the identity and size of the cellular population activated during training for contextual memory, and the intracellular compartments in which MAPK is stimulated. Although this analysis focused on the role of MAPK in the nucleus because of its importance for CREB-mediated transcription, MAPK was simultaneously activated in dendrites and at distal synapses following fear conditioning. It is noteworthy that MAPK regulates a number of other proteins, including dendritic K+ channels and glutamate receptors, and it may also control dendritic protein synthesis. Thus, the parallel activation of synaptodendritic and somatonuclear pools of MAPK supports the general hypothesis that memory formation depends on several MAPK-regulated events, including synaptic activity, dendritic protein synthesis, and transcription (Sindreu, 2007).

Although CREB-mediated transcription is necessary for memory formation and depends on MAPK signaling, the CREB kinase activated by MAPK following training for contextual memory was not certain. It was particularly interesting to determine if training for contextual fear activates RSK2 or MSK1 because studies using cultured neurons have implicated both kinases in CREB-mediated transcription through the phosphorylation of transcription factors and histones. This study discovered that fear conditioning activates MSK1, but not RSK2, in CA1 neurons, and that activation of MAPK and MSK1 is tightly correlated on a cell-by-cell basis. Furthermore, the activation of MSK1 induced by training is abrogated in mice lacking Ca2+-stimulated adenylyl cyclases or by post-training inhibition of MEK1/2. This strongly implicates MSK1 in MAPK-dependent CREB phosphorylation during formation of contextual memory. The identification of MSK1, and not RSK2, as the activated CREB kinase emphasizes that signaling mechanisms inferred from cultured neuron studies do not necessarily apply in vivo. Definitive evidence as to the relative importance of both CREB kinases during memory formation may come from the use of conditional mutant mice or novel MSK1 antagonists (Sindreu, 2007).

In summary, the data indicates that stimulation of MAPK in dendrites and the nucleus following training for contextual memory depends on Ca2+-stimulated adenylyl cyclase activity and leads to the activation of the CREB kinase MSK1. Furthermore, signaling elements for CREB-mediated transcription, starting with the initial cAMP signal, and including PKA, MAPK, MSK1, and CREB, are all activated in the same subset of neurons after training. It is concluded that one of the major roles of adenylyl cyclase activity in memory is to support the activation of MAPK, MSK1, and CREB in hippocampal neurons (Sindreu, 2007).

Table of contents

CrebB-17A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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