Mitogen-activated protein kinase kinase (MEK) is a dual-specificity protein kinase that is located primarily in the cellular cytosol, both prior to and upon mitogenic stimulation. The existence of a nuclear export signal in the N-terminal domain of MEK [Fukuda, M., Gotoh, I., Gotoh, Y. & Nishida, E. (1996) J. Biol. Chem. 271, [20024][20025][20026][20027][20028] suggests that there are circumstances under which MEK enters the nucleus and must be exported. Using mutants of MEK, we show that the deletion of the nuclear export signal sequence from constitutively active MEK caused constitutive localization of MEK in the nucleus of COS7 and HEK-293T cells. However, when the same region was deleted from a catalytically inactive MEK, cytoplasmic localization was observed in resting cells, which turned nuclear upon stimulation. Confocal microscopy of COS7 cells expressing the above mutants showed localization of the active MEK in the nuclear envelope and also in the cell periphery. The differences in cellular localization between the wild-type and mutant MEKs are not due to severe changes in specificity because the recombinant, constitutively active MEK that lacked its Nterminal region exhibited the same substrate specificity as the wild-type MEK, both in vitro and in intact cells. Taken together, our results indicate that upon mitogenic stimulation, MEK, like extracellular signal responsive kinase and p90 RSK , is massively translocated to the nucleus. Rapid export from the nucleus, which is mediated by the nuclear export signal, is probably the cause for the cytoplasmic distribution observed with wild-type MEK.The transmission of extracellular signals from the cell surface into the nucleus involves several groups of protein kinases, which are collectively known as the mitogen-activated protein kinase (MAPK) signaling cascades. One of these cascades, the extracellular signal responsive kinase (ERK) signaling cascade, involves a sequential phosphorylation and activation of Raf1, MAPK kinase (MAPKK, also known as MEK), ERK, p90 RSK , and under some conditions also GSK3 (reviewed in ref. 1). Other MAPK signaling cascades are the JNK (stress-activated protein kinases) cascade, the p38RK (HOG, reviewed in ref.2), and other, less-characterized cascades. A key step in the signaling mechanism of the ERK cascade is the translocation of both ERK1 and 2 and p90 RSK into the nucleus (3, 4). This translocation, which occurs in response to mitogenic stimulation, is rapid (occurs within 5-30 min), and might be a prerequisite for activation of nuclear processes such as transcription (5, 6). The mechanism by which protein kinases are translocated to the nucleus upon stimulation is not yet known. The sequences of the ERKs and p90 RSK do not contain a nuclear localization signal (NLS), and although the size of ERK may permit a simple diffusion via nuclear pores, such diffusion would probably be much slower than the rapid movement observed upon activation. However, because kinase-deficient mutants of ERK can translocate to the nucl...

Gene expression is necessary for the formation and consolidation of long term memory in both invertebrates and vertebrates. Here, we describe the expression and characterization of candidate plasticity gene 16 (cpg16), a protein serine/threonine kinase that was previously isolated from rat hippocampus as a plasticity-related gene. CPG16, when expressed in and purified from bacteria and COS7 cells, was only capable of autophosphorylation and phosphorylation of myelin basic protein but failed to phosphorylate many other peptides and proteins in in vitro phosphorylation assays. Recombinant CPG16, when overexpressed and purified from COS7 cells, had a relatively low level of autophosphorylation activity. This activity was significantly stimulated when cAMP-elevating agents (forskolin, 8-bromo-cAMP) were added to the cells but not by any other extracellular stimuli tested, e.g. serum, phorbol esters, and a calcium ionophore. Although the stimulation of CPG16 activity was inhibited by the cAMP-dependent protein kinase inhibitor H-89, it did not serve as a direct substrate for this kinase. This suggests that CPG16 may be activated by a cAMP-stimulated protein kinase cascade. Immunolocalization studies in COS7 and NIH-3T3 cells showed mostly cytoplasmic localization of CPG16 that turned partially nuclear upon stimulation with 8-bromo-cAMP. Moreover, overexpression of CPG16 seems to partially inhibit cAMP-stimulated activity of the transcription factor CREB (cAMP response element-binding protein), suggesting its involvement in the down-regulation of cAMP-induced transcription. Thus, CPG16 is a protein serine/threonine kinase that may be involved in a novel signaling pathway downstream of cAMP-dependent protein kinase.Learning and memory processes in the brain are characterized by plasticity changes in central nervous system neurons. In a comprehensive search for candidate plasticity-related genes (CPGs 1 (1, 2)), a novel cDNA encoding for a putative protein kinase, termed cpg16, has been isolated from kainatetreated rat hippocampus. Here we show that CPG16 is a protein serine/threonine kinase that, when expressed in COS7 cells, is activated by cAMP via a cAMP-dependent protein kinase (PKA)-induced mechanism. Studies on the effect of CPG16 on transcription have revealed that it may be involved in the down-regulation of cAMP response element-binding protein (CREB) activity. Thus, it is possible that CPG16 participates in the regulation of neuronal plasticity by down-regulating PKA-stimulated transcription. EXPERIMENTAL PROCEDURESNorthern Blot Analysis-Northern blotting was performed as described previously (3) with 5 g of RNA/lane. The probe used for detection was the 1600-base pair cpg16 cDNA (2), which was labeled by the random priming technique (U. S. Biochemical Corp.) using [␣-32 P]dCTP according to the manufacturer's instructions. Construction of Expression Vectors for CPG16 -The polymerase chain reaction method was used to clone the cDNA encoding the open reading frame for cpg16 (2) in-frame with glutathione S-trans...

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Rabies virus glycoprotein (RVG) is a trimeric and surface-exposed viral coat protein that has been shown to interact with the murine p75 neurotrophin receptor. We have investigated binding of RVG to p75 and describe several features that distinguish the p75-RVG interaction from conventional neurotrophin binding to p75. RVG binds mammalian but not avian p75 and does not bind to any of the Trk neurotrophin receptors. The mammalian p75 specificity of RVG binding may partly explain the phyletic specificity of rabies infection. Radioiodinated nerve growth factor (NGF) and RVG both bind to rat p75 but do not compete with each other's binding site. Although neurotrophins bind to the second and third cysteine-rich domains (CRD) of p75, RVG specifically interacts with high affinity (K d 30 -35 pM) with the first CRD (CRD1). Substitution of Gln 33 in p75-CRD1 by Glu completely abolishes RVG binding. Our data therefore firmly establish RVG as a trimeric high affinity ligand for a non-neurotrophin binding site on p75. Interestingly, the CRD1 in another TNF/NGF family receptor was recently shown to be involved in the binding of the herpes virus glycoprotein gD, suggesting that the CRD1 of TNF/NGF family members may be a widely used binding domain for viral glycoproteins. Rabies virus (RV)1 is a nonsegmented, negative strand RNA virus belonging to the genus Lyssavirus from the Rhabdoviridae (1). It is an enveloped virus with a single type I glycoprotein G (RVG) of 65 kDa inserted in its membrane. RVG is organized as a trimer and is responsible for the binding of the virus to the target cells and for the fusion between viral and cell membranes during the endocytosis of the virus. RV and other lyssaviruses are highly neurotropic and can cause fatal encephalomyelitis in mammals (2). Infection of both wild type and laboratory strains of RV is mainly targeted to neurons in vivo. In contrast in vitro infections with laboratory strains can target a range of cell types, and only wild type RV still shows specific neurotropism under these conditions. Specific interactions between RVG and neuronal cell surface molecules have been demonstrated (3), suggesting the existence of specific neuronal receptor(s). An expression cloning approach using soluble RVG to screen a neuroblastoma cell library led us to the identification of the murine p75 neurotrophin receptor as a candidate RVG receptor (4). Subsequent examination of six other lyssavirus genotypes revealed one additional viral glycoprotein (from European Bat lyssavirus 2) that can also bind p75 (5).The p75 neurotrophin receptor is highly conserved in vertebrates, and its expression is developmentally regulated (6, 7). During embryogenesis, p75 is present in neuronal as well as in non-neuronal cells, but in the adult its expression is essentially restricted to the peripheral and the central nervous system (8). p75 contains four cysteine-rich domains (CRD) in the N-terminal ectodomain and a type II death domain in its cytoplasmic C-terminal segment (8), both features characteristic of the...

The mechanisms underlying evolution of complex nervous systems are not well understood. In recent years there have been a number of attempts to correlate specific gene families or evolutionary processes with increased brain complexity in the vertebrate lineage. Candidates for evocation of complexity include genes involved in regulating brain size, such as neurotrophic factors or microcephaly-related genes; or wider evolutionary processes, such as accelerated evolution of brain-expressed genes or enhanced RNA splicing or editing events in primates. An inherent weakness of these studies is that they are correlative by nature, and almost exclusively focused on the mammalian and specifically the primate lineage. Another problem with genomic analyses is that it is difficult to identify functionally similar yet non-homologous molecules such as different families of cysteine-rich neurotrophic factors in different phyla. As long as comprehensive experimental studies of these questions are not feasible, additional perspectives for evolutionary and genomic studies will be very helpful. Cephalopod mollusks represent the most complex nervous systems outside the vertebrate lineage, thus we suggest that genome sequencing of different mollusk models will provide useful insights into the evolution of complex brains.