PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase.

The ERK 1/2 MAP kinase pathway controls cell growth and survival and modulates integrin function. Here, we report that PEA-15, a protein variably expressed in multiple cell types, blocks ERK-dependent transcription and proliferation by binding ERKs and preventing their localization in the nucleus. PEA-15 contains a nuclear export sequence required for its capacity to anchor ERK in the cytoplasm. Genetic deletion of PEA-15 results in increased ERK nuclear localization with consequent increased cFos transcription and cell proliferation. Thus, PEA-15 can redirect the biological outcome of MAP kinase signaling by regulating the subcellular localization of ERK MAP kinase.

A novel calmodulin-binding protein, belonging to the WD-repeat family, is localized in dendrites of a subset of CNS neurons.

A rat brain synaptosomal protein of 110,000 M(r) present in a fraction highly enriched in adenylyl cyclase activity was microsequenced (Castets, F., G. Baillat, S. Mirzoeva, K. Mabrouk, J. Garin, J. d'Alayer, and A. Monneron. 1994. Biochemistry. 33:5063-5069). Peptide sequences were used to clone a cDNA encoding a novel, 780-amino acid protein named striatin. Striatin is a member of the WD-repeat family (Neer, E.J., C.J. Schmidt, R. Nambudripad, and T.F. Smith. 1994. Nature (Lond.). 371:297-300), the first one known to bind calmodulin (CaM) in the presence of Ca++. Subcellular fractionation shows that striatin is a membrane-associated, Lubrol-soluble protein. As analyzed by Northern blots, in situ hybridization, and immunocytochemistry, striatin is localized in the central nervous system, where it is confined to a subset of neurons, many of which are associated with the motor system. In particular, striatin is conspicuous in the dorsal part of the striatum, as well as in motoneurons. Furthermore, striatin is essentially found in dendrites, but not in axons, and is most abundant in dendritic spines. We propose that striatin interacts, through its WD-repeat domain and in a CaM/Ca(++)-dependent manner, with one or several members of a surrounding cluster of molecules engaged in a Ca(++)-signaling pathway specific to excitatory synapses.

Glutamate induces phosphorylation of Elk-1 and CREB, along with c-fos activation, via an extracellular signal-regulated kinase-dependent pathway in brain slices.

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 (V. Sgambato, P. Vanhoutte, C. Pagès, M. Rogard, R. A. Hipskind, M. J. Besson, and J. Caboche, J. Neurosci. 18:214-226, 1998). 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, we used a semi-in vivo system 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.

Chromosomal assignment of two human B-raf(Rmil) proto-oncogene loci: B-raf-1 encoding the p94Braf/Rmil and B-raf-2, a processed pseudogene.

The B-raf gene is the human homolog of the avian c-Rmil proto-oncogene encoding a 94-kDa serine/threonine kinase detected in avian cells. We have previously shown that this protein contains amino-terminal sequences not found in other proteins of the mil/raf gene family. These sequences are encoded by three exons in the avian genome. We report that these three exons are conserved in the human B-raf gene and that they encode an amino acid sequence similar to that of the avian c-Rmil gene, indicating that in both avian and mammalian species the product of the B-raf/c-Rmil gene is a 94-kDa protein. We also identified two human B-raf loci: B-raf-1, located on chromosome 7q34, which encodes the functional B-raf/Rmil gene product, and B-raf-2, an inactive processed pseudogene located on chromosome Xq13.

Quail neuroretina c-Rmil(B-raf) proto-oncogene cDNAs encode two proteins of 93.5 and 95 kDa resulting from alternative splicing.

c-Rmil is the cellular allele of the v-Rmil oncogene transduced during in vitro passaging of Rous-associated virus type 1 in chicken embryonic neuroretina (NR) cells. The c-Rmil proto-oncogene is the avian homolog of the mammalian B-raf gene and belongs to the mil/raf oncogene family of serine/threonine protein kinases. The c-Rmil/B-raf gene is preferentially expressed in avian and mammalian neural tissues. Two c-Rmil cDNA species, resulting from an alternative splicing mechanism, were isolated from quail embryonic NR cDNA libraries. They encode two proteins of 767 and 807 amino acids that differ by the presence of an alternative exon, located upstream of the kinase domain. Expression of these cDNAs in COS-1 cells leads to the synthesis of two proteins with apparent molecular weights of 93.5 and 95 kDa, recognized by an Rmil-specific antiserum. Both proteins are phosphorylated in an immune complex kinase assay. A protein of 94 kDa is also immunoprecipitated in avian NR cells and is identical to the 93.5-kDa protein expressed in COS-1 cells, as shown by Staphylococcus aureus V8 protease mapping. The c-Rmil proteins contain the three conserved regions previously identified in mil/raf protein kinases. In addition, they contain amino-terminal sequences that are not present in the other mil/raf proteins identified to date. These additional sequences may define a novel functional domain for c-Rmil/B-raf and could play a role in signal transduction in neural cells.

Expression and activation of B-Raf kinase isoforms in human and murine leukemia cell lines.

The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serine/threonine protein kinases. It encodes multiple protein isoforms previously shown to be expressed predominantly in neural tissues. We report here that B-Raf proteins of 95 and 72 kDa are also expressed in various human and murine hematopoietic cell lines. Their relative level of expression is variable depending on the cell line examined. The highest level of expression of p95B-raf was found in UT-7 cells, a human pluripotent cell line established from a patient with a megakaryoblastic leukemia. These cells are able to differentiate toward erythroid or myeloid lineage phenotypes in presence of erythropoietin (EPO) or granulocyte-macrophage colony-stimulating factor (GM-CSF) respectively. We show that treatment of UT-7 cells with EPO, GM-CSF or stem cell factor (SCF) rapidly induces phosphorylation of p95B-raf as indicated by a shift of electrophoretic mobility. This increase in phosphorylation is correlated with a three-fold increase of B-Raf kinase activity. B-Raf activation also increases in a dose-dependent manner in response to EPO and GM-CSF. We also show that both p95B-raf and p72B-raf can be activated by IL-3 in murine BAF-3 pro-B cells and by anti-CD3 in human Jurkat cells, respectively. These observations provide the first evidence that the B-Raf kinase is involved in signal transduction pathways regulating proliferation and differentiation of hematopoietic cells of both myeloid and lymphoid lineages.

B-Raf protein isoforms interact with and phosphorylate Mek-1 on serine residues 218 and 222.

The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serine/threonine protein kinases. It encodes multiple protein isoforms resulting from alternative splicing of two exons located upstream of the kinase domain. Recent studies suggested that B-Raf could be the intermediate molecule between Ras and Mek-1 (MAP Kinase Kinase) in signalling pathways specific of neural cells. However, there has been no evidence for a direct interaction between B-Raf and Mek-1. We report here that different B-Raf isoforms can be co-immunoprecipitated with anti-Mek-1 antisera in COS-1 cells and that the kinase activity of B-Raf is not required for its interaction with Mek-1. We also show that all B-Raf isoforms tested phosphorylate Mek-1 in a time-dependent manner, whereas kinase defective mutants fail to do so. Finally, we demonstrate that the constitutively activated S218D, S222D and S218D/S222D mutants of Mek-1 interact similarly with B-Raf. However, only the S218D and S222D mutants, and not the S218D/S222D double mutant, can be phosphorylated by B-Raf isoforms. Therefore, serine residues 218 and 222, previously shown to regulate Mek-1 activity, appear to be the major phosphorylation sites by B-Raf in vitro.

Differential subcellular distribution and transcriptional activity of sigmaE3, sigmaE4, and sigmaE3-4 isoforms of the rat estrogen receptor-alpha.

E3, E4, and E3-4 are naturally occurring estrogen receptor (ER) isoforms, generated through differential splicing of the ERalpha primary transcript and abundantly expressed in embryonic rat pituitary. Studies in COS cells transfected with full-length ERalpha or its three splice variants fused to green fluorescent protein (GFP), revealed a different subcellular localization for each isoform. In the absence of estradiol, full-length ERalpha-GFP was predominantly nuclear, and E3-GFP and E4-GFP were present both in cytoplasm and nucleus, whereas E3-4-GFP was predominantly cytoplasmic. Upon hormone treatment, a dramatic redistribution of full-length ERalpha-GFP and E3-GFP, from a diffuse to punctate pattern, occurred within the nucleus. In contrast, the distribution of E4-GFP and E3-4-GFP was unaffected. Nuclear fractionation studies showed that full-length ER-alpha and E3 displayed the same hormone-induced ability to tether to nuclear matrix, whereas nuclear E4 appeared to remain loosely associated to functional nuclear constituents. When cotransfected with an estrogen-inducible reporter plasmid (VIT-TK-CAT) in ER-negative (CHO k1) and ER-positive pituitary (GH4 C1) cells, E3-4 exhibited a very weak estrogen-dependent transactivation activity, whereas E3 had an inhibitory effect on full-length ER action. Conversely, E4 displayed estrogen-independent transcriptional activity in ER-negative cells, and in ER-positive cells, enhanced the estrogen-induced gene expression as efficiently as full-length ERalpha. In a gel mobility shift assay, phosphorylated E4 was able to form a specific complex with a consensus ERE, while E3 and E3-4 never did bind by themselves. The observed inhibitory action of E3 on estrogen-dependent transcription would rather involve protein-protein interactions such as formation of heterodimers with full-length ERalpha, as suggested by immunoprecipitation followed by Western blotting. These data suggest that E3 and E4 may play a physiologically relevant role as negative or constitutively positive modulators of transcription, in the developing rat pituitary.

Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP.

In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44mapk independently of these events. Secondly, we studied the role of B-Raf in this process. We observed that NGF, PMA and cAMP induce the phosphorylation of B-Raf as well as an upward shift in its electrophoretic mobility. We show that B-Raf is activated following NGF and PMA treatment of PC12 cells, and that it can phosphorylate and activate MEK-1. However, cAMP inhibits B-Raf autokinase activity as well as its ability to phosphorylate and activate MEK-1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B-Raf in vitro. Further, we show that B-Raf binds to p21ras, but more important, this binding to p21ras is virtually abolished with B-Raf from PC12 cells treated with CPT-cAMP. Hence, these data indicate that the PKA-mediated phosphorylation of B-Raf hampers its interaction with p21ras, which is responsible for the PKA-mediated decrease in B-Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP kinase through the activation of an unidentified MEK kinase and/or the inhibition of a MEK phosphatase.

Effect of PRL on MAPK activation: negative regulatory role of the C-terminal part of the PRL receptor.

Prolactin induces cell proliferation and cell differentiation through well-known MAPK Erk, and JAK2/STAT5 pathways depending on the cell line. The aim of the present study was to delineate the functional domains of the PRL receptor involved in PRL induced MAPK regulation. Using various PRL-R mutants of the cytoplasmic domain we found, that the membrane proximal domain is necessary for PRL induced MAPK activation and that the C-terminal part of the receptor exerts a negative regulatory role. A pharmacological approach, using different types of inhibitors, provided evidence that PRL induced MAPK activation requires both a MEK dependent pathway and a PI3K dependent pathway. The negative regulation induced by the carboxy-terminal part of the receptor involves a combination of tyrosine phosphatases and serine/threonine phosphatases as concluded from the actions of the phosphatase inhibitors: pervanadate, PAO and okadaic acid. The mechanism by which these phosphatases are recruited or are induced by the last 141 cytoplasmic residues of the receptor remains to be determined. Finally the negative regulatory role of the carboxy-terminal part of the receptor, first demonstrated in the present study, is discussed in terms of the regulation of different effects of PRL on growth and differentiation.

New case of c-src gene transduction: the generation of virus PR2257.

PR2257 is a new replication-defective avian sarcoma virus which harbours in addition to the spliced version of the c-src gene also about 950 bp of no-coding cellular sequences located downstream from the c-src stop codon (Geryk et al., 1989). Comparison of the 950 bp region transduced by PR2257 with the chicken c-src cDNA (Dorai et al., 1991) and genomic sequences of the c-src 3' non-coding region from chicken and quail has shown that there are no additional introns. The c-src 3' non-coding region represents the largest c-src exon (No. 12) comprising about 2 kb. Absence of conserved open reading frames within this region in chicken and quail genomic DNAs excludes the possibility for coding a protein by these sequences. Also, the possibility was excluded that numerous endogenous virus-derived sequences identified in molecularly cloned PR2257 provirus played a role in the c-src transduction. After serial passaging of PR2257 virus in vivo a variant PR2257/16 was isolated. In PR2257/16, the size of the env gene was increased due to homologous recombination with a helper virus. In addition to mutations in the viral leader and the v-src coding region, a large deletion in transduced c-src 3' non-coding sequences was found in the PR2257/16 genome. The significance of genome modifications for selective advantage of this viral variant in vivo is discussed.

[Experimental study of mechanisms of neuronal death in the course oh HIV infection]. / Etude expérimentale des mécanismes de la mort neuronale au cours de l'infection à VIH.

The HIV1 virus and its envelope glycoprotein gp120 are toxic for human neurones in vitro. This neurotoxicity is, at least partially, of an apoptotic nature, resulting from the interaction of gp120 with the neuronal membrane which leads to perturbations of intracellular signaling systems. These latter bring about on the one hand a raising of [Ca2+]i partly due to the potentiation of the NMDA receptor response to endogenous glutamate and on the other hand the activation of certain MAP kinases (ERK and JNK) which lead to the initiation of the cell death program.

Mouse 89 kD heat shock protein. Two polypeptides with distinct developmental regulation.

Unstressed early mouse embryos have been previously shown [1] to synthesize at very high rates 70 and 89 kD proteins belonging to the heat shock protein (HSP) family. But it was not clear whether expression of heat shock-inducible or non-inducible (cognate) genes accounted for this spontaneous synthesis. In this report we show that the 89 kD mouse HSP can be separated into two proteins by high resolution PAGE. These two components show distinct but related peptide pattern after limited proteolysis. They are synthesized from distinct mRNAs. One of these proteins--HSP89f--is synthesized at a high rate by unstressed cells and its synthesis is rather insensitive to stress, whereas synthesis of the other protein--HSP89s--is strongly stimulated by heat shock in fibroblasts. Both HSP89f and HSP89s are major proteins synthesized in unstressed mouse preimplantation embryos and embryonal carcinoma (EC) cells. After in vitro differentiation of the EC cells the spontaneous synthesis of HSP89s decreases. Thus spontaneous expression of a mammalian inducible HSP is developmentally regulated.

[B-raf gene encodes for multiple isoforms with Mek-1 kinase activity]. / Le gène B-raf code pour de multiples isoformes ayant une activité Mek-1 kinase.

The c-Rmil/B-raf proto-oncogene belongs to the mil/raf family encoding serine/threonine protein kinases shown to be involved in signal transduction from the membrane to the nucleus. We previously showed that the avian c-Rmil gene encodes two proteins of 94 and 95 kDa resulting from the alternative splicing of a 120 bp exon encoding 40 aminoacids (exon 10). We isolated from a mouse brain library B-raf cDNAs containing this exon 10 and a previously unidentified 36 bp insert which constitutes an additional alternatively spliced exon designated exon 8b. These two exons are located between the CR2 region and the catalytic domain of the protein. By using specific sera generated against different regions of the B-Raf protein, we identified 10 B-Raf isoforms and we defined their structure and their expression pattern in adult mouse tissues. The B-Raf proteins are mainly expressed in neural tissues and, interestingly, isoforms containing aminoacids encoded by exon 10 are specifically expressed in these tissues. We also show that several B-Raf isoforms interact with the Mek-1 protein (MAP kinase kinase) and phosphorylate this protein on serine residues 218 and 222.

gamma-Hexachlorocyclohexane inhibition of the calcium fluxes at the desensitized mouse neuromuscular junction.

The peripheral neurotoxicity of lindane was studied in vitro on mouse phrenic-diaphragm preparation. The experiments were performed on normal and denervated preparations. Twitches evoked by direct or indirect electrical stimulations and contractions evoked by external application of cholinergic agonists were compared in the presence or absence of lindane in the bath medium. Histochemical localizations of calcium were made on fibers after chemical stimulations. The results indicated that, at the normal endplate or at extrajunctionnal sites after denervation, there was a change in the intracellular Ca2+ distribution in response to external application of concentrated cholinergic agonists. This modification caused a transient contracture and could be visualized either by 45Ca autoradiography or by Alizarin Red S intracellular precipitations. Lindane inhibited the ACh-dependent contracture (I50 less than 10(-5) M) and suppressed the histochemical localizations of calcium. These lindane effects were observed on both normal and denervated muscles. It was concluded that lindane inhibited the Ca2+ influx presented by the desensitized ACh-sensitive areas of the muscle.

Common mechanism of retrovirus activation and transduction of c-mil and c-Rmil in chicken neuroretina cells infected with Rous-associated virus type 1.

We previously described the isolation of the IC10 retrovirus which transduced the v-Rmil oncogene, a new member of the mil/raf gene family. This virus was generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina (NR) cells and was selected for its ability to induce proliferation of these nondividing cells. IC10 was isolated after six passages of culture supernatants but was not detected in proliferating NR cells during early virus passages. In this study, we molecularly cloned and sequenced another v-Rmil-containing provirus, designated IC11, from NR cells infected at the third virus passage of the same experiment. Both IC11 and IC10 transduced only the serine/threonine kinase domain of c-Rmil. Comparison of v-Rmil and c-Rmil sequences indicated that amino-terminal truncation is sufficient to activate the mitogenic properties of c-Rmil. IC11 and IC10 have identical 3' ends but differ by their 5' RAV-1-Rmil junctions. The 3' ends of both viruses were generated by recombination between Rmil and env genes, involving partial sequence identity. The 5' RAV-1-Rmil junction of IC11 was formed by a splicing process between the RAV-1 leader and a 37-bp c-Rmil exon located upstream of the kinase domain. NR cells infected with this virus synthesize a unique Rmil protein. IC10 contains most of the gag gene recombined with v-Rmil and encodes a gag-Rmil hybrid protein. Serial passaging of IC11 in NR cells led to the formation of a gag-Rmil-containing retrovirus. These results indicate that IC11 represents an early step in transduction and that this virus further recombined with RAV-1 to generate IC10. They confirm our previously proposed model for the multistep generation of v-mil-transducing retroviruses. Therefore, activation and transduction of c-mil and c-Rmil, in NR cells infected with RAV-1, result from a common mechanism.

The mouse B-raf gene encodes multiple protein isoforms with tissue-specific expression.

The c-Rmil/B-raf proto-oncogene is a member of the mil/raf family encoding serine/threonine protein kinases shown to be involved in signal transduction from the membrane to the nucleus. We isolated from a mouse brain library B-raf cDNAs containing a previously unidentified 36-base pair alternatively spliced exon located between exons 8 and 9 and, therefore, designated exon 8b. Human and mouse B-raf mRNAs also contain the 120-base pair alternatively spliced exon 10 previously described in the avian c-Rmil gene. Independent splicing of these two exons, located between the conserved region 2 (CR2) and the catalytic domain (CR3) gives rise to mRNAs potentially encoding four distinct proteins. By using specific sera generated against different portions of B-Raf, we identified at least 10 protein isoforms in adult mouse tissues. Some isoforms, in the range of 69-72 kDa, are not recognized by antisera directed against peptides encoded by exons 1 and 2, indicating the existence of B-Raf proteins with two different NH2 extremities. The other isoforms, in the range of 79-99 kDa, contain the amino acids encoded by exons 1 and 2, by either or both of the alternatively spliced exons, and, possibly, by another of the unidentified exon. Analysis of B-raf mRNA expression by reverse transcriptase-polymerase chain reaction and immunocharacterization of B-Raf proteins in different tissues of the adult mouse showed a tissue-specific pattern of B-Raf isoforms expression. Interestingly, isoforms containing amino acids encoded by exon 10 are specifically expressed in neural tissues. Taken together, these results suggest that distinct B-Raf proteins could be involved, in a tissue-specific manner, in signal transduction pathways.

Genomic organization and nucleotide sequence of the coding region of the chicken c-Rmil(B-raf-1) proto-oncogene.

c-Rmil is the cellular allele of the v-Rmil oncogene, transduced during in vitro passaging of Rous associated virus type 1 in chicken embryonic neuroretina (NR) cells. The c-Rmil proto-oncogene is the avian homolog of the mammalian B-raf gene and belongs to the mil/raf oncogene family of serine/threonine protein kinases. We recently reported that the avian c-Rmil gene encodes two proteins of 94 and 95 kDa, resulting from an alternative splicing mechanism. We describe here the exon-intron organization of the coding region of the chicken c-Rmil locus. We show that c-Rmil proteins are encoded by 19 exons lying within about 100 kbp of genomic DNA. Comparison of the organization of this gene with those of the other mil/raf genes shows strong similarities within three conserved domains previously identified in mil/raf protein kinases. However, c-Rmil contains two additional 5' coding exons that are not present in the other mil/raf genes.

Phosphorylation by PKA of a site unique to B-Raf kinase.

The Raf kinases serve as central intermediates to relay signals from Ras to ERK. Cell-specific effects of these signals on growth, differentiation and survival can be observed due to the recruitment of different isoenzymes of the Raf family. The in vitro phosphorylation of a site unique to B-Raf (Ser429) has been proposed to be responsible for the negative regulation of the isoenzyme by Akt. Using phosphopetide mapping and site-directed mutagenesis we showed that Ser429 is phosphorylated upon cAMP elevation in PC12 cells and proposed that PKA is a major kinase phosphorylating the B-Raf-specific site in vivo.

High constitutive transcription of HSP86 gene in murine embryonal carcinoma cells.

In order to investigate HSP86 heat-shock gene expression in embryonal carcinoma cell lines (EC), a partial mouse HSP86 cDNA clone was isolated and characterized. As observed for the corresponding protein, HSP86 RNA is shown to be constitutively more abundant in PCC4 and undifferentiated F9 EC cells than in fibroblasts, while its amount decreases upon F9 differentiation. Although mRNA stabilization is suggested to account in part of the high constitutive expression of the heat-shock-like protein HSC73 in F9 cells, HSP86 RNA appears as stable in fibroblasts as in F9 cells. Using run-on experiments we have established that high HSP86 expression in undifferentiated F9 cells in mainly due to enhanced transcription of the gene. Possible mechanisms responsible for this high level of transcription are discussed.