Western blot analysis of neuronal tissues taken from fear-conditioned rats showed a selective activation of phosphatidylinositol 3-kinase (PI-3 kinase) in the amygdala. PI-3 kinase was also activated in response to long-term potentiation (LTP)-inducing tetanic stimulation. PI-3 kinase inhibitors blocked tetanus-induced LTP as well as PI-3 kinase activation. In parallel, these inhibitors interfered with long-term fear memory while leaving short-term memory intact. Tetanus and forskolin-induced activation of mitogen-activated protein kinase (MAPK) was blocked by PI-3 kinase inhibitors, which also inhibited cAMP response element binding protein (CREB) phosphorylation. These results provide novel evidence of a requirement of PI-3 kinase activation in the amygdala for synaptic plasticity and memory consolidation, and this activation may occur at a point upstream of MAPK activation.

Memory consolidation is mediated by new protein synthesis. However, the transcriptional pathways induced in neurons by behavioral training that activate gene responses have yet to be fully delineated. We have previously shown that nuclear factor B (NF-B) is activated in the amygdala after fear conditioning. Here we report that fear conditioning resulted in an increase in histone acetyl-transferase activity, the association between NF-B p65 and CBP, and the increase in acetylated p65. Pretreating animals with histone deacetylase (HDAC) inhibitors prolonged the nuclear expression of acetyl-p65 and increased its DNA binding activity. Consistent with these results, HDAC inhibitors enhanced long-term but not short-term fear memory, and this effect was attenuated by B decoy DNA, whereas scrambled DNA was without effect. This study provides evidence that HDAC-mediated deacetylation functions as an intranuclear molecular switch culminating in the termination of NF-B transcriptional response that is involved in the formation of fear memory.

Memory extinction refers to a gradual decrease of the previously acquired response when exposed to conditional stimulus without pairing with unconditional stimulus. Here we show for the first time that fear training-induced phosphorylation of specific substrates in the rat amygdala is reduced after extinction trials and is accompanied by an increase in the protein level and enzymatic activity of calcineurin. In parallel, calcineurin inhibitors prevented extinction-induced protein dephosphorylation as well as extinction of fear memory. Thus, extinction training increased phosphatase activity likely via an expression of calcineurin. Calcineurin then created a negative-feedback loop and directly or indirectly dephosphorylated specific substrates, which, in their phosphorylated state, were required for memory consolidation. Accordingly, in our experimental condition, extinction could be ascribed at least in part to a weakening of the original signaling.

The transcription factor Sp1 is ubiquitously expressed in different cells and thereby regulates the expression of genes involved in many cellular processes. This study reveals that Sp1 was phosphorylated during the mitotic stage in three epithelial tumor cell lines and one glioma cell line. By using different kinase inhibitors, we found that during mitosis in HeLa cells, the c-Jun NH 2 -terminal kinase (JNK) 1 was activated that was then required for the phosphorylation of Sp1. In addition, blockade of the Sp1 phosphorylation via inhibition JNK1 activity in mitosis resulted in the ubiquitination and degradation of Sp1. JNK1 phosphorylated Sp1 at Thr278/739. The Sp1 mutated at Thr278/739 was unstable during mitosis, possessing less transcriptional activity for the 12(S)-lipoxygenase expression and exhibiting a decreased cell growth rate compared with wild-type Sp1 in HeLa cells. In N-methyl-N-nitrosourea-induced mammary tumors, JNK1 activation provided a potential relevance with the accumulation of Sp1. Together, our results indicate that JNK1 activation is necessary to phosphorylate Sp1 and to shield Sp1 from the ubiquitin-dependent degradation pathway during mitosis in tumor cell lines. INTRODUCTIONThe transcription factor Sp1 is ubiquitously expressed in mammalian cells, and it is important in a variety of physiological processes, including cell cycle regulation, apoptosis, and differentiation (Firestone and Bjeldanes, 2003;Chu and Ferro, 2005;Wong et al., 2005;Deniaud et al., 2006). Sp1 binds specifically to the GC-rich promoter elements, via three C 2 H 2 -type zinc finger regions at the C terminus of Sp1, and it regulates the transcriptional activity of the target genes by using two major glutamine-rich transactivation domains localized, respectively, at the N terminus and the medial region (Suske, 1999;Bouwman and Philipsen, 2002). In addition, in Sp1, there are serine/threonine-rich sequences between the two transactivation domains that may be a target for posttranslational modification (Bouwman and Philipsen, 2002).The transcriptional activity of a transcription factor is determined at least by three factors: transactivational activity, DNA binding affinity, and protein level. Previous studies on the regulation of Sp1 activities focused mostly on transactivational activity, thereby allowing study of its interaction with other proteins and its DNA binding affinity. However, one of the apparent key elements regulating the activity of Sp1 is via its stability, which certainly needs to be explored and established. Recent studies revealed that the DNA binding ability, transactivational activity, and protein stability of Sp1 might be influenced by its posttranslational modifications such as sumoylation, glycosylation, ubiquitination, acetylation, and phosphorylation (Han and Kudlow, 1997;Mortensen et al., 1997;Wells et al., 2001;Ryu et al., 2003;Abdelrahim and Safe, 2005;Chu and Ferro, 2005;Hung et al., 2006;Spengler and Brattain, 2006). For example, Sp1 is sumoylated at Lys16, which might repress the transact...

Fear conditioning has been ascribed to presynaptic mechanisms, particularly presynaptic facilitation of transmission at thalamo-and cortico-amygdala synapses. Here, by labeling surface receptors with biotin or using membrane fractionation approaches, we report that fear conditioning resulted in an increase in surface expression of GluR1 subunit of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the amygdala, whereas total GluR1 mRNA and protein levels were unchanged. The control group that received conditioned stimulus (CS) and unconditioned stimulus in an unpaired fashion did not present any increase, indicating that GluR1 increase was specific to the learning component of the task. Conditioninginduced increase in surface expression of GluR1 depended on the activation of N-methyl-D-aspartate receptors and protein kinases and required the synthesis of new proteins. CS-alone trials applied 24 h before training attenuated fear-potentiated startle and prevented conditioning-induced increase in surface expression of GluR1. Increase in GluR1 was also observed in the amygdala slices after delivery of tetanic stimulation that elicited long-term potentiation of synaptic transmission. Proteasome inhibitor increased surface expression of GluR1 in a time-and dose-dependent manner. Furthermore, pretraining administration of proteasome inhibitor into the amygdala facilitated the fear-potentiated startle. These results suggest that long-term memory formation is correlated with the change in synaptic expression of GluR1, and trafficking of GluR1 to the synaptic sites contributes at least in part to the expression of fear memory.Fear conditioning, an animal model of emotional learning and post-traumatic stress disorder, is initiated by a cue (conditioned stimulus, CS) that is previously paired with an aversive stimulus (unconditioned stimulus, US) such as footshock. Neuronal changes mediating the association between the CS and US occur in the lateral (LA) and basolateral (BLA) amygdala (McKernan and Shinnick-Gallagher, 1997;Rogan et al., 1997;Pare, 2003). It is generally recognized that consolidation of long-term memory in vertebrate and invertebrate brains requires transcription and translation of new proteins (Dudai, 1996;McGaugh, 2000). Newly synthesized proteins are believed to deposit at the synapses that encode the persistent changes in synaptic strength. Despite the signal cascades that have been identified to be responsible for the consolidation of fear memory, little is known about the downstream effectors leading to the expression of memory. Previous study has showed that conditioned fear is associated with a reduction in paired-pulse facilitation and an enhancement in the probability of transmitter release in the thalamo-amygdala pathway (McKernan and Shinnick-Gallagher, 1997). Quantal analysis of unitary synaptic responses in cortico-amygdala pathway after induction of long-term potentiation (LTP) revealed a marked decrease in the fraction of failures, with no change in potency of CV of the...