Fluxes in amounts of intracellular calcium ions are important determinants of gene expression. So far, Ca2+-regulated kinases and phosphatases have been implicated in changing the phosphorylation status of key transcription factors and thereby modulating their function. In addition, direct effectors of Ca2+-induced gene expression have been suggested to exist in the nucleus, although no such effectors have been identified yet. Expression of the human prodynorphin gene, which is involved in memory acquisition and pain, is regulated through its downstream regulatory element (DRE) sequence, which acts as a location-dependent gene silencer. Here we isolate a new transcriptional repressor, DRE-antagonist modulator (DREAM), which specifically binds to the DRE. DREAM contains four Ca2+-binding domains of the EF-hand type. Upon stimulation by Ca2+, DREAM's ability to bind to the DRE and its repressor function are prevented. Mutation of the EF-hands abolishes the response of DREAM to Ca2+. In addition to the prodynorphin promoter, DREAM represses transcription from the early response gene c-fos. Thus, DREAM represents the first known Ca2+-binding protein to function as a DNA-binding transcriptional regulator.

Trans-synaptic activation of gene expression is linked to long-term plastic adaptations in the nervous system. To examine the molecular program induced by synaptic activity, we have employed molecular cloning techniques to identify an immediate early gene that is rapidly induced in the brain. We here report the entire nucleotide sequence of the cDNA, which encodes an open reading frame of 396 amino acids. Within the hippocampus, constitutive expression was low. Basal levels of expression in the cortex were high but can be markedly reduced by blockade of N-methyl-D-aspartate receptors. By contrast, synaptic activity induced by convulsive seizures increased mRNA levels in neurons of the cortex and hippocampus. High-frequency stimulation of the perforant path resulted in long-term potentiation and a spatially confined dramatic increase in the level of mRNA in the granule cells of the ipsilateral dentate gyrus. Transcripts were localized to the soma and to the dendrites of the granule cells. The dendritic localization of the transcripts offers the potential for local synthesis of the protein at activated postsynaptic sites and may underlie synapse-specific modifications during longterm plastic events.Activity-dependent alterations of neuronal connectivity are characteristic of plastic events in the nervous system. Plasticity is associated with physiological processes such as learning and memory as well as neuropathological states, including epilepsy. Seizure episodes set in motion a cascade of events that include gene expression, sprouting of fibers, and the establishment of new synaptic contacts (1). These long-lasting alterations are reminiscent of changes that occur during long-term potentiation (LTP) of synaptic transmission in the mammalian brain. LTP is an activity-dependent and persistent enhancement of synaptic efficacy that may underlie certain forms of long-term memory (2, 3). In both invertebrates and vertebrates, longterm memory differs from short-term memory in that it requires RNA and protein synthesis (4, 5). As is the case for memory in the intact animal, LTP is blocked by inhibitors of RNA and protein synthesis (6-9). Attention therefore has been focused on identifying activity-induced genes. In invertebrates, behavioral training elicits changes in the level of specific mRNAs in cells involved in learning (10, 11). In mammalian brain and spinal chord neurons, a variety of physiological and pathological stimuli induce rapid and transient activation of immediate early genes (IEGs) (12). Many of the IEGs encode transcription factors that may control the expression of downstream effector genes (13-15). More recent studies have identified genes that may themselves have effector function with the potential to quickly promote long-term alterations in neuronal phenotype during plastic processes, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fac...

PTEN/PI3K/AKT constitutes an important pathway regulating the signaling of multiple biological processes such as apoptosis, metabolism, cell proliferation and cell growth. PTEN is a dual protein/lipid phosphatase which main substrate is the phosphatidyl-inositol,3,4,5 triphosphate (PIP3), the product of PI3K. Increase in PIP3 recruits AKT to the membrane where it is activated by other kinases also dependent on PIP3. Many components of this pathway have been described as causal forces in cancer. PTEN activity is lost by mutations, deletions or promoter methylation silencing at high frequency in many primary and metastatic human cancers. Germ line mutations of PTEN are found in several familial cancer predisposition syndromes. Activating mutations which have been reported for PI3K and AKT, in tumours are able to confer tumourigenic properties in several cellular systems. Additionally, the binding of PI3K to oncogenic ras is essential for the transforming properties of ras. In summary, the data strongly support the view of the PTEN/PI3K/AKT pathway as an important target for drug discovery.

SummaryAging constitutes the key risk factor for age‐related diseases such as cancer and cardiovascular and neurodegenerative disorders. Human longevity and healthy aging are complex phenotypes influenced by both environmental and genetic factors. The fact that genetic contribution to lifespan strongly increases with greater age provides basis for research on which “protective genes” are carried by long‐lived individuals. Studies have consistently revealed FOXO (Forkhead box O) transcription factors as important determinants in aging and longevity. FOXO proteins represent a subfamily of transcription factors conserved from Caenorhabditis elegans to mammals that act as key regulators of longevity downstream of insulin and insulin‐like growth factor signaling. Invertebrate genomes have one FOXO gene, while mammals have four FOXO genes: FOXO1, FOXO3, FOXO4, and FOXO6. In mammals, this subfamily is involved in a wide range of crucial cellular processes regulating stress resistance, metabolism, cell cycle arrest, and apoptosis. Their role in longevity determination is complex and remains to be fully elucidated. Throughout this review, the mechanisms by which FOXO factors contribute to longevity will be discussed in diverse animal models, from Hydra to mammals. Moreover, compelling evidence of FOXOs as contributors for extreme longevity and health span in humans will be addressed.

SummaryThe eukaryotic cell is organized into membrane-covered compartments that are characterized by specific sets of proteins and biochemically distinct cellular processes. The appropriate subcellular localization of proteins is crucial because it provides the physiological context for their function. In this Commentary, we give a brief overview of the different mechanisms that are involved in protein trafficking and describe how aberrant localization of proteins contributes to the pathogenesis of many human diseases, such as metabolic, cardiovascular and neurodegenerative diseases, as well as cancer. Accordingly, modifying the disease-related subcellular mislocalization of proteins might be an attractive means of therapeutic intervention. In particular, cellular processes that link protein folding and cell signaling, as well as nuclear import and export, to the subcellular localization of proteins have been proposed as targets for therapeutic intervention. We discuss the concepts involved in the therapeutic restoration of disrupted physiological protein localization and therapeutic mislocalization as a strategy to inactivate disease-causing proteins. Journal of Cell Sciencecompartment has been associated with human diseases, and in the following sections we will discuss some of the mechanisms that can lead to such changes in protein localization. Mislocalization through alterations of the protein trafficking machineryDysregulation of the protein trafficking machinery can have dramatic effects on general protein transport processes, modifying cell morphology and physiology. Along these lines, changes in the nuclear pore complex (NPC) have been linked to several genetic disorders (Chahine and Pierce, 2009). For example, in patients with familial atrial fibrillation, the homozygous mutation R391H in the nucleoporin NUP155 has been shown to reduce nuclear envelope permeability and affect the export of Hsp70 mRNA and import of HSP70 protein (Zhang et al., 2008). That study was the first to link a nucleoporin defect to cardiovascular disease.Mutations in other components of the NPC, such as the nucleoporin p62 protein and ALADIN (alacrima achalasia adrenal insufficiency neurologic disorder, officially known as AAAS) are thought to cause the neurodegenerative diseases infantile bilateral striatal necrosis and triple A syndrome, respectively (Basel-Vanagaite et al., 2006; Kiriyama et al., 2008). Mutant ALADIN prevents nuclear entry of the DNA repair proteins aprataxin and DNA ligase I and, therefore, results in increased DNA damage and subsequent cell death caused by oxidative stress (Kiriyama et al., 2008). In a similar fashion, protein import into other organelles can be affected by mutations in the trafficking machinery. For instance, mutations in the peroxin gene PEX7, which encodes a peroxisomal import receptor that is responsible for the transport of several essential peroxisomal enzymes, have been found to cause the peroxisome biogenesis disorder rhizomelic chondrodysplasia punctata type 1 (RCDP1) (Braverman e...

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The spatial separation of DNA replication and gene transcription in the nucleus and protein translation in the cytoplasm is a uniform principle of eukaryotic cells. This compartmentalization imposes a requirement for a transport network of macromolecules to shuttle these components in and out of the nucleus. This nucleo-cytoplasmic transport of macromolecules is critical for both cell physiology and pathology. Consequently, investigating its regulation and disease-associated alterations can reveal novel therapeutic approaches to fight human diseases, such as cancer or viral infection. The characterization of the nuclear pore complex, the identification of transport signals and transport receptors, as well as the characterization of the Ran system (providing the energy source for efficient cargo transport) has greatly facilitated our understanding of the components, mechanisms and regulation of the nucleo-cytoplasmic transport of proteins in our cells. Here we review this knowledge with a specific emphasis on the selection of disease-relevant molecular targets for potential therapeutic intervention.

Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, ␣CREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of ␣CREM is not required for the interaction, but phospho-␣CREM shows higher affinity for DREAM. The interaction with ␣CREM is independent of the Ca 2؉ -binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both ␣CREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca 2؉ .Transcriptional derepression is an important mechanism for the accurate control of gene expression. Transcriptional repressors can bind directly to DNA or act indirectly by interacting with other DNA-associated proteins (23,32). DREAM, a calcium-binding protein, represses basal expression of target genes through specific interaction with downstream regulatory elements (DREs) in the DNA (5, 6). Release of binding of DREAM from the DRE results in derepression, a process that is regulated by Ca 2ϩ and protein kinase A (PKA) activation (5, 6). Other central players in the nuclear response to cyclic AMP (cAMP) and Ca 2ϩ are activator and repressor basic regionleucine zipper (LZ) transcription factors that bind to cAMPresponsive promoter elements (CREs) (10,15,25). They include proteins encoded by the CREB and CREM genes whose function is tightly regulated via phosphorylation by several kinases, including PKA and Ca 2ϩ -calmodulin-dependent kinases (8,12,13). As such, they represent the convergence point for various signaling cascades.The transcriptional repressor DREAM contains four EF hands, of which three (II, III, and IV) are responsible for the binding of calcium ions. In the absence of stimulated levels of nuclear calcium, DREAM binds with high affinity to the DRE sequence as a tetramer. Upon stimulation and increase in intracellular calcium, DREAM detaches from DNA without disruption of the tetrameric structure (6). The regulation by intracellular Ca 2ϩ of DREAM binding to DRE sites is a general mechanism that depends primarily on the EF-hand domains of DREAM. Mutation of two key amino acids within any of the functional EF hands results in mutated DREAM forms that stay bound to DNA also after calcium stimulation. Since DREAM binds to DRE sites as a tetramer, DREAM mutants insensitive to Ca 2ϩ behave as dominant negative mutants in a background of wild-type DREAM (unpublished observation). Similarly, PKA activation also results in loss of DREAM binding to the DRE and derepression of the target gene prodynorphin in human neuroblastoma cells (5). The molecular mechanism or the domains in DREAM that mediate this derepression by cAMP are unknown, and ...