The cAMP response element-binding protein (CREB) has been shown to mediate transcriptional activation of genes in response to both cAMP and calcium influx signal transduction pathways. The roles of two multifunctional calcium/calmodulin-dependent protein kinases, CaMKIV and CaMKII, were examined in transient transfection studies that utilized either the full-length or the constitutively active forms of these kinases. The results indicate that CaMKIV is much more potent than CaMKII in activating CREB in three different cell lines. It was also found in these studies that Ser133 of CREB is essential for its activation by CaMKIV. Because both CaMKII and CaMKIV can phosphorylate CREB, we pursued further the mechanism by which CaMKII and CaMKIV differentially regulate CREB activity. Mutagenesis studies and phosphopeptide mapping analysis demonstrated that in vitro, CaMKIV phosphorylates CREB at Ser133 only, whereas CaMKII phosphorylates CREB at Ser133 and a second site, Ser142. Transient transfection studies revealed that phosphorylation of Ser142 by CaMKII blocks the activation of CREB that would otherwise occur when Ser133 is phosphorylated. When Ser142 was mutated to alanine, CREB was activated by CaMKII, as well as by CaMKIV. Furthermore, mutation of Ser142 to alanine enhanced the ability of Ca2+ influx to activate CREB, suggesting a physiological role for the phosphorylation of Ser142 in modulation of CREB activity. These data provide evidence for a new mechanism for regulation of CREB activity involving phosphorylation of a negative regulatory site in the transcriptional activation domain. The studies also provide new insights into possible interactions between the cAMP and Ca2+ signaling pathways in the regulation of transcription. In particular, changes in intracellular Ca2+ have the potential to either inhibit or augment the ability of cAMP to stimulate transcription, depending on the presence of specific forms of Ca2+/calmodulin-dependent protein kinases.

Many drugs of abuse exert their addictive effects by increasing extracellular dopamine in the nucleus accumbens, where they likely alter the plasticity of corticostriatal glutamatergic transmission. This mechanism implies key molecular alterations in neurons in which both dopamine and glutamate inputs are activated. Extracellular signal-regulated kinase (ERK), an enzyme important for long-term synaptic plasticity, is a good candidate for playing such a role. Here, we show in mouse that d -amphetamine activates ERK in a subset of medium-size spiny neurons of the dorsal striatum and nucleus accumbens, through the combined action of glutamate NMDA and D1-dopamine receptors. Activation of ERK by d -amphetamine or by widely abused drugs, including cocaine, nicotine, morphine, and Δ ⁹ -tetrahydrocannabinol was absent in mice lacking dopamine- and cAMP-regulated phosphoprotein of M _r 32,000 (DARPP-32). The effects of d -amphetamine or cocaine on ERK activation in the striatum, but not in the prefrontal cortex, were prevented by point mutation of Thr-34, a DARPP-32 residue specifically involved in protein phosphatase-1 inhibition. Regulation by DARPP-32 occurred both upstream of ERK and at the level of striatal-enriched tyrosine phosphatase (STEP). Blockade of the ERK pathway or mutation of DARPP-32 altered locomotor sensitization induced by a single injection of psychostimulants, demonstrating the functional relevance of this regulation. Thus, activation of ERK, by a multilevel protein phosphatase-controlled mechanism, functions as a detector of coincidence of dopamine and glutamate signals converging on medium-size striatal neurons and is critical for long-lasting effects of drugs of abuse.

The cellular response to treatment with proinflammatory cytokines or exposure to environmental stress is mediated, in part, by the p38 group of mitogen-activated protein (MAP) kinases. We report the molecular cloning of a novel isoform of p38 MAP kinase, p38β2. This p38 MAP kinase, like p38α, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38α, p38β2, and p38γ MAP kinase isoforms, while MKK3 activates only p38α and p38γ MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases.

Signal transduction via MAP kinase pathways plays a key role in a variety of cellular responses, including growth factor‐induced proliferation, differentiation and cell death. In mammalian cells, p38 MAP kinase can be activated by multiple stimuli, such as pro‐inflammatory cytokines and environmental stress. Although p38 MAP kinase is implicated in the control of inflammatory responses, the molecular mechanisms remain unclear. Upon activation, CD4+ T cells differentiate into Th2 cells, which potentiate the humoral immune response or pro‐inflammatory Th1 cells. Here, we show that pyridinyl imidazole compounds (specific inhibitors of p38 MAP kinase) block the production of interferon‐γ (IFNγ) by Th1 cells without affecting IL‐4 production by Th2 cells. These drugs also inhibit transcription driven by the IFNγ promoter. In transgenic mice, inhibition of the p38 MAP kinase pathway by the expression of dominant‐negative p38 MAP kinase results in selective impairment of Th1 responses. In contrast, activation of the p38 MAP kinase pathway by the expression of constitutivelyactivated MAP kinase kinase 6 in transgenic mice caused increased production of IFNγ during the differentiation and activation of Th1 cells. Together, these data demonstrate that the p38 MAP kinase is relevant for Th1 cells, not Th2 cells, and that inhibition of p38 MAP kinase represents a possible site of therapeutic intervention in diseases where a predominant Th1 immune response leads to a pathological outcome. Moreover, our study provides an additional mechanism by which the p38 MAP kinase pathway controls inflammatory responses.

Endocannabinoids form a novel class of intercellular messengers, the functions of which include retrograde signaling in the brain and mediation or modulation of several types of synaptic plasticity. Yet, the signaling mechanisms and long-term effects of the stimulation of CB1 cannabinoid receptors (CB1-R) are poorly understood. We show that anandamide, 2-arachidonoyl-glycerol, and Δ9-tetrahydrocannabinol (THC) activated extracellular signal-regulated kinase (ERK) in hippocampal slices. In living mice, THC activated ERK in hippocampal neurons and induced its accumulation in the nuclei of pyramidal cells in CA1 and CA3. Both effects were attributable to stimulation of CB1-R and activation of MAP kinase/ERK kinase (MEK). In hippocampal slices, the stimulation of ERK was independent of phosphatidyl-inositol-3-kinase but was regulated by cAMP. The endocannabinoid-induced stimulation of ERK was lost in Fyn knock-out mice, in slices andin vivo, although it was insensitive to inhibitors of Src-family tyrosine kinasesin vitro, suggesting a noncatalytic role of Fyn. Finally, the effects of cannabinoids on ERK activation were dependent on the activity of glutamate NMDA receptorsin vivo, but not in hippocampal slices, indicating the existence of several pathways linking CB1-R to the ERK cascade.In vivoTHC induced the expression of immediate-early genes products (c-Fos protein, Zif268, and BDNF mRNAs), and this induction was prevented by an inhibitor of MEK. The strong potential of cannabinoids for inducing long-term alterations in hippocampal neurons through the activation of the ERK pathway may be important for the physiological control of synaptic plasticity and for the general effects of THC in the context of drug abuse.

We have characterized Ca2+/calmodulin-dependent protein kinase IV (CaM kinase IV), expressed using the baculovirus/Sf9 cell system, to assess its potential role in Ca2+-dependent transcriptional regulation. CaM kinase IV was strongly inhibited in vitro by KN-62, a specific CaM kinase inhibitor which suppresses Ca2+-dependent transcription of several genes, so we tested whether CaM kinase IV could stimulate transcription. Co-transfection of COS-1 cells by cDNA for CaM kinase IV gave 3-fold stimulation of a reporter gene expression, whereas co-transfection with CaM kinase II gave no transcriptional stimulation. Since this transcriptional response was mediated by phosphorylation of cAMP responsive element-binding protein (CREB), we determined the kinetics and site specificities of CaM kinases IV and II for phosphorylating CREB in vitro. CaM kinases IV and II and cAMP kinase (protein kinase A) all had similar Km values for CREB (1-5 microns), but the Vmax of CaM kinase IV was 40-fold lower than those of CaM kinase II and protein kinase A. Although all three kinases phosphorylated Ser133 in CREB, CaM kinase II also gave equal phosphorylation of a second site which was not Ser98. The two CREB phosphorylation sites were separately 32P-labeled, and the abilities of protein phosphatases 1, 2A, and 2B (calcineurin) to dephosphorylate them were tested. Our results show that all three phosphatases could dephosphorylate both sites, and calcineurin was a stronger catalyst for dephosphorylating site 1 (Ser133) than for site 2. These results indicate that CaM kinase IV may be important in Ca2+-dependent transcriptional regulation through phosphorylation of Ser133 in CREB. The fact that CaM kinase II phosphorylates another site in addition to Ser133 in CREB raises the possibility that this second phosphorylation site may account for the suppressed phosphorylation site may account for the suppressed ability of CaM kinase II to enhance transcription through the CRE/CREB system. In addition multiple protein phosphatases, including calcineurin, may exert a modulatory effect on transcription depending on which site they dephosphorylate.