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.

Transformation of chick embryo neuroretinal cells by Rous sarcoma virus in vitro: induction of cell proliferation.

Neuroretinal cells from 7-day-old chick embryos are transformed and induced to proliferate after infection with Rous sarcoma virus in vitro. Susceptibility of neuroretinal cells to the virus is also dependent on the stage of development since infection of cells from 10-day-old embryos is uneffective.

Stable expression of intracellular Notch suppresses v-Src-induced transformation in avian neural cells.

Understanding how disruption of differentiation contributes to the cancer cell phenotype is required to identify alterations essential for malignant transformation and provide experimental basis for their correction. We investigated whether primary quail neuroretina cells, transformed by a conditional v-Src mutant (QNR/v-src(ts)), could revert to a normal phenotype, in response to the stable expression of constitutively active Notch1 intracellular domain (ICN). This model system was chosen because Notch signaling plays an instructive role in cell fate determination during NR development, and because the intrinsic capacity of QNR cultures to differentiate is blocked by v-Src. We report that stable ICN expression results in suppression of QNR/v-src(ts) cell transformation in the presence of an active oncoprotein. This phenotypic reversion coincides with a major switch in cell identity, as these undifferentiated cells acquire glial differentiation traits. Both changes appear to be mediated by CBF, a transcription factor that binds to ICN and activates target genes. Cells restored to a normal and differentiated phenotype have undergone changes in the functioning of signaling effectors, essentially regulating cell morphology and cytoskeleton organization. This dominant interference may be partially mediated by an autocrine/paracrine mechanism, as revertant cells secrete a factor(s), which inhibits transformation properties of QNR/v-src(ts) cells.

Murine Ksr interacts with MEK and inhibits Ras-induced transformation.

BACKGROUND: Ksr (kinase supressor of Ras) was identified as a regulator of the Ras-MAP kinase (mitogen-activated protein kinase) pathway by genetic screens in Drosophila and Caenorhabditis elegans. Ksr is a kinase with similarities to the three conserved regions of Raf kinases, especially within the kinase domain. To investigate whether these structural similarities correlated with common functional properties, we examined the ability of mKsr-1, the murine homolog of Ksr, to interact with components of the vertebrate MAP kinase pathway. RESULTS: In the yeast two-hybrid interaction assay, mKsr-1 did not bind to either Ras, B-Raf or Raf-1, but interacted strongly with both MEK-1 and MEK-2, activators of MAP kinase. The Ksr-MEK interaction was confirmed by co-immunoprecipitation experiments. Ectopically expressed mKsr-1 co-precipitated with endogenous MEK-1 in COS-1 cells, and endogenous Ksr and MEK co-precipitated from PC12 cells. Phosphorylation of MEK by mKsr-1 was not detected, however. In contrast, the MEK subpopulation complexed with mKsr-1 in COS-1 cells or PC12 cells did not display kinase activity. This ability of Ksr to block MEK in an inactive form correlated with a biological response: mKsr-1 did not transform NIH3T3 cells, and, furthermore, mKsr-1 reduced Ras-induced transformation. Similarly, mKsr-1 inhibited the proliferation of embryonic neuroretina cells induced by Ras and B-Raf but not that induced by MEK. CONCLUSIONS: Our results suggest a novel mechanism for Ksr in regulating the MAP kinase pathway, at least in part through an ability to interact with MEK.

Transcriptional downregulation of the retina-specific QR1 gene by pp60v-src and identification of a novel v-src-responsive unit.

The embryonic avian neuroretina (NR) is part of the central nervous system and is composed of various cell types: photoreceptors and neuronal and Müller (glial) cells. These cells are derived from proliferating neuroectodermal precursors which differentiate after terminal mitosis and become organized in cell strata. Proliferation of differentiating NR cells can be induced by infection with Rous sarcoma virus (RSV) and requires the expression of a functional v-src gene. To understand the mechanisms involved in the regulation of neural cell growth and differentiation, we studied the transcriptional regulation of QR1, a gene specifically expressed in postmitotic NR cells. Transcription of this gene is detected primarily in Müller cells and is strongly downregulated by the v-src gene product. Moreover, QR1 expression takes place only during the late phase of retinal development and is shut off abruptly at hatching. We have isolated a promoter region(s) of the QR1 gene that confers v-src responsiveness. By transfection of QR1-CAT constructs into quail NR cells infected with the temperature-sensitive mutant of RSV, PA101, we have identified a v-src-responsive region located between -1208 and -1161 upstream of the transcription initiation site. This sequence is able to form two DNA-protein complexes, C1 and C2. Formation of complex C2 is specifically induced in cells expressing an active v-src product, while formation of C1 is detected mainly in nonproliferating quail NR cells upon pp60v-src inactivation. C1 is also a target for regulation during development. We have identified the DNA binding site for the C1 complex, a repeated GCTGAC sequence, and shown that mutations in this element abolish binding of this factor as well as transcription of the gene at the nonpermissive temperature. Neither formation of C1 nor that of C2 seems to involve factors known to be targeted in the pp60v-src cascade. Our data suggest that C1 could be a novel target for both developmental control and oncogene-induced cell growth regulation.

Developmental control of transcription of a retina-specific gene, QR1, during differentiation: involvement of factors from the POU family.

Developmental control of gene expression often results from the coupling of growth arrest with the establishment of differentiation programs. QR1 is a gene specifically expressed in retinas during the late phase of embryogenesis. At this stage neuroectodermal precursors have reached terminal mitosis and are undergoing differentiation into distinct cell types. Transcription of the QR1 gene is tightly regulated during retinal development: this gene is expressed between embryonic day 9 (ED9) and ED17 and is completely repressed at hatching in quail. Moreover, QR1 transcription is downregulated when postmitotic neural retina cells are induced to proliferate by pp60v-src. We studied the stage-dependent transcriptional control of this gene during quail neural retina (QNR) cell development. Transient transfection experiments with QR1/CAT constructs at various stages of development showed that a region located between -935 and -1265 bp upstream of the transcription start site is necessary to promote transcription in retina cells during the late phase of embryonal development (QNR9, corresponding to ED9). By in vivo footprinting assays we identified at least two elements that are occupied by DNA-protein complexes in QNR cells: the A and B boxes. The A box allows formation of several biochemically distinct complexes: C1, C2, C3, and C4. Formation of the C2 complex mainly during early stages (ED7) and of C2, C3, and C4 complexes during postnatal life correlates with repression of QR1 transcription, whereas the C1 complex is strongly induced at ED11 when the QR1 gene is expressed. We previously showed that C1 was involved in downregulation of QR1 transcription by pp60v-src. Several complexes are also formed on the B box. We show that these complexes are exclusively present in neural tissues and that they involve members of the POU family of transcription factors. Mutations of each one of the two regions which abolish the binding of the C1 factor(s) on the A box and of the POU factor(s) on the B box also prevent stimulation of QR1 transcription in QNR9. Therefore, both elements appear to be required for the stage-specific transcription of the QR1 gene. We also show that the regulatory region from position -1265 to position -935 is able to confer stage-specific transcription upon a heterologous promoter (thymidine kinase). Indeed, this region stimulates transcription in differentiating retinas (QNR9) and represses transcription in terminally differentiated retinas (QNR17, corresponding to postnatal life). Our results suggest that cell growth regulation and developmental control are coordinated through the A and B boxes in regulating QR1 transcription during retinal differentiation.

Transcriptional stimulation of the retina-specific QR1 gene upon growth arrest involves a Maf-related protein.

The avian neural retina (NR) is derived from proliferating neuroectodermal precursors which differentiate after terminal mitosis and become organized in cell strata. Proliferation of postmitotic NR cells can be induced by infection with Rous sarcoma virus (RSV) and requires the expression of a functional v-Src protein. QR1 is a retina-specific gene expressed exclusively at the stage of growth arrest and differentiation during retinal development. In NR cells infected with tsPA101, an RSV mutant conditionally defective in pp60v-src mitogenic capacity, QR1 expression is downregulated in proliferating cells at 37 degrees C and is fully restored when the cells become quiescent as a result of pp60v-src inactivation at 41 degrees C. We were able to arrest proliferation of tsPA101-infected quail NR cells expressing an active v-Src protein by serum starvation at 37 degrees C. This allowed us to investigate the role of cell growth in regulating QR1 transcription. We report that QR1 transcription is stimulated in growth-arrested cells at 37 degrees C compared with that in proliferating cells maintained at the same temperature. Growth arrest-dependent stimulation of QR1 transcription requires the integrity of the A box, a previously characterized cis-acting element responsible for QR1 transcriptional stimulation upon v-Src inactivation and during retinal differentiation. We also show that formation of the C1 complex on the A box is increased upon growth arrest by serum starvation in the presence of an active v-Src oncoprotein. Thus, the C1 complex represents an important link between cell cycle and developmental control of QR1 gene transcription during NR differentiation and RSV infection. By using antibodies directed against different Maf proteins of the leucine zipper family and competition with Maf consensus site-containing oligonucleotides in a gel shift assay, we show that the C1 complex is likely to contain a Maf-related protein. We also show that a purified bacterially expressed v-Maf protein is able to bind the A box and that the level of a 43-kDa Maf-related protein is increased upon growth arrest in infected retinal cells. Moreover, ectopic expression of c-mafI, c-mafII, and mafB cDNAs in quiescent tsPA101-infected quail NR cells is able to stimulate transcription of a QR1 reporter gene through the A box. Therefore, QR1 appears to be the first target gene for a Maf-related protein(s) in the NR.

Down regulation by p60v-src of genes specifically expressed and developmentally regulated in postmitotic quail neuroretina cells.

The avian neuroretina (NR) is composed of photoreceptors and different neurons that are derived from proliferating precursor cells. Neuronal differentiation takes place after terminal mitosis. We have previously shown that differentiating NR cells can be induced to proliferate by infection with Rous sarcoma virus (RSV) and that cell multiplication requires expression of a functional v-src gene. We speculated that the quiescence of NR cells could be determined by specific genes. Cell proliferation could then result from the negative regulation of these genes by the v-src protein. By differential hybridization of a cDNA library, we isolated eight clones corresponding to genes expressed in postmitotic NR cells from 13-day-old quail embryos, transcriptional levels of which are significantly reduced in NR cells induced to proliferate by tsNY68, an RSV mutant with temperature-sensitive mitogenic activity. Partial sequencing analysis indicated that one RNA encoded the calmodulin gene, whereas the other seven showed no similarity to known sequences. By using v-src mutants that induce NR cell proliferation in the absence of transformation, we showed that transcription of six genes was negatively regulated by the v-src protein and that of four genes was correlated with NR cell quiescence. We also report that a subset of genes are specifically transcribed in neural cells and developmentally regulated in the NR. These results indicate that the v-src protein regulates expression of genes likely to play a role in the control of neural cell growth or differentiation.

Induction of postmitotic neuroretina cell proliferation by distinct Ras downstream signaling pathways.

Ras-induced cell transformation is mediated through distinct downstream signaling pathways, including Raf, Ral-GEFs-, and phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathways. In some cell types, strong activation of the Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) cascade leads to cell cycle arrest rather than cell division. We previously reported that constitutive activation of this pathway induces sustained proliferation of primary cultures of postmitotic chicken neuroretina (NR) cells. We used this model system to investigate the respective contributions of Ras downstream signaling pathways in Ras-induced cell proliferation. Three RasV12 mutants (S35, G37, and C40) which differ by their ability to bind to Ras effectors (Raf, Ral-GEFs, and the p110 subunit of PI 3-kinase, respectively) were able to induce sustained NR cell proliferation, although none of these mutants was reported to transform NIH 3T3 cells. Furthermore, they all repressed the promoter of QR1, a neuroretina growth arrest-specific gene. Overexpression of B-Raf or activated versions of Ras effectors Rlf-CAAX and p110-CAAX also induced NR cell division. The mitogenic effect of the RasC40-PI 3-kinase pathway appears to involve Rac and RhoA GTPases but not the antiapoptotic Akt (protein kinase B) signaling. Division induced by RasG37-Rlf appears to be independent of Ral GTPase activation and presumably requires an unidentified mechanism. Activation of either Ras downstream pathway resulted in ERK activation, and coexpression of a dominant negative MEK mutant or mKsr-1 kinase domain strongly inhibited proliferation induced by the three Ras mutants or by their effectors. Similar effects were observed with dominant negative mutants of Rac and Rho. Thus, both the Raf-MEK-ERK and Rac-Rho pathways are absolutely required for Ras-induced NR cell division. Activation of these two pathways by the three distinct Ras downstream effectors possibly relies on an autocrine or paracrine loop, implicating endogenous Ras, since the mitogenic effect of each Ras effector mutant was inhibited by RasN17.

Crystallin gene expression and lentoid body formation in quail embryo neuroretina cultures transformed by the oncogenic retrovirus Mill Hill 2 or Rous sarcoma virus.

The lens-specific proteins alpha and delta crystallins and lentoid bodies, structures that follow a differentiation pathway similar to that of the lens, regularly appear after 4 to 5 weeks in quail embryo neuroretina monolayer cultures. We have investigated the effects of the avian oncogenic retroviruses Mill Hill 2 and Rous sarcoma virus on this process. Quail embryo neuroretina cells transformed by Mill Hill 2 virus were established into permanent cultures that synthesized alpha and delta crystallins and contained stem cells for the production of lentoid bodies. In contrast, transformation with the Rous sarcoma virus mutant tsNY-68 blocked the appearance of mRNA crystallins, but cytoplasmic alpha and delta crystallin mRNA and alpha crystallin appeared 44 h after a shift to the nonpermissive temperature. However, delta crystallins and lentoid bodies were only present after 7 days. The crystallins of transformed quail neuroretina cultures were immunologically indistinguishable from those of quail lenses and of normal quail embryo neuroretina cultures.

Phosphorylation of MafA is essential for its transcriptional and biological properties.

We previously described the identification of quail MafA, a novel transcription factor of the Maf bZIP (basic region leucine zipper) family, expressed in the differentiating neuroretina (NR). In the present study, we provide the first evidence that MafA is phosphorylated and that its biological properties strongly rely upon phosphorylation of serines 14 and 65, two residues located in the transcriptional activating domain within a consensus for phosphorylation by mitogen-activated protein kinases and which are conserved among Maf proteins. These residues are phosphorylated by ERK2 but not by p38, JNK, and ERK5 in vitro. However, the contribution of the MEK/ERK pathway to MafA phosphorylation in vivo appears to be moderate, implicating another kinase. The integrity of serine 14 and serine 65 residues is required for transcriptional activity, since their mutation into alanine severely impairs MafA capacity to activate transcription. Furthermore, we show that the MafA S14A/S65A mutant displays reduced capacity to induce expression of QR1, an NR-specific target of Maf proteins. Likewise, the integrity of serines 14 and 65 is essential for the MafA ability to stimulate expression of crystallin genes in NR cells and to induce NR-to-lens transdifferentiation. Thus, the MafA capacity to induce differentiation programs is dependent on its phosphorylation.

Identification of drm, a novel gene whose expression is suppressed in transformed cells and which can inhibit growth of normal but not transformed cells in culture.

Using differential display analysis, we compared the expression of RNA in v-mos-transformed cells and their flat revertant and isolated a novel gene, drm (down-regulated in mos-transformed cells), whose expression is down-regulated in parental v-mos-transformed cells but which is expressed at a high level in the revertant and normal rat fibroblasts (REF-1 cells). Analysis of different oncogene-transformed cells revealed that drm gene expression was also suppressed in REF-1 cells transformed by v-ras, v-src, v-raf, and v-fos. The drm cDNA contains a 184-amino-acid-protein-encoding open reading frame which shows no significant homologies to known genes in DNA databases. Polyclonal antibodies raised against drm peptide detect a protein with the predicted size of 20.7 kDa in normal cells and under nonpermissive conditions in cells conditionally transformed by v-mos but not in parental v-mos-transformed cells. Northern analysis of normal adult tissues shows that drm is expressed as a 4.4-kb message in a tissue-specific manner, with high expression in the brain, spleen, kidney, and testis and little or no expression in the heart, liver, and skeletal muscle. In situ hybridization analysis in adult rat tissue reveals good correlation with this pattern and indicates that drm mRNA is most highly expressed in nondividing and terminally differentiated cells, such as neurons, type 1 lung cells, and goblet cells. Transfection of a drug-selectable drm expression vector dramatically reduced the efficiency of colony formation in REF-1 and CHO cells, and the drm-transfected REF-1 survivors expressed low or nondetectable levels of exogenous drm mRNA. The toxic effects of drm can be overcome by cotransfection with constructs expressing oncogenic ras; furthermore, cells expressing high levels of drm and conditionally transformed with mos-expressing Moloney murine sarcoma virus rapidly undergo apoptosis when shifted to the nonpermissive temperature. Taken together, our data suggest that cells expressing high levels of drm undergo apoptotic death in the absence of oncogene-induced transformation and that drm represents a novel gene with potential roles in cell growth control or viability and tissue-specific differentiation.

Cell division is required for expression of v-myc transforming properties in chicken embryonic neuroretina cells.

We previously reported that avian retroviruses carrying the v-myc oncogene alone fail to induce sustained proliferation and transformation of non-dividing chicken neuroretina (CNR) cells from 7-day-old embryos. However, v-myc is capable of transforming CNR cells which have been induced to multiply by the v-mil oncogene. These results suggest that entry into the cell cycle is required for the transformation of CNR cells by v-myc. To further assess the role of cell division, we investigated the transforming properties of v-myc in CNR cells conditionally induced to divide by the v-src gene or by modified culture conditions. We show that v-myc transforms CNR cells infected with Rous sarcoma virus mutants which induce cell proliferation in the absence of transformation. Expression of these transforming properties in CNR cells infected with temperature-sensitive v-src mutants depends on the continuous mitogenic activity of p60v-src. We also report that v-myc is able to transform CNR cells and to increase their growth potential under culture conditions which allow transient multiplication of uninfected cells. However, these v-myc-transformed cells rapidly cease to divide when returned to culture conditions that restrict the growth of normal cells. Taken together, these results indicate that transformation of CNR cells by the v-myc oncogene continuously depends on their ability to enter the cell cycle.

Identification of signalling proteins interacting with B-Raf in the yeast two-hybrid system.

Recent studies suggested the existence of Ras/B-Raf/ MEK-1 complexes and a critical role for B-Raf in regulating the MAP kinase/ERKs signalling pathway. We report, here, that both Ras and MEK-1 proteins interact physically with B-Raf proteins in the yeast two-hybrid system. In addition, by screening a mouse brain cDNA library, we isolated additional B-Raf interacting proteins. These include three members of the 14-3-3 proteins family (eta, theta and zeta) and the MEK-2 protein. We also show that c-Raf-1, previously reported to interact with beta and zeta 14-3-3 proteins, also interacts with eta and theta 14-3-3 proteins in the two-hybrid system. By using different portions of the B-Raf protein, we mapped the regions of the protein involved in these interactions. Specifically, we have characterized B-Raf specific sequences required for an efficient interaction with MEK proteins. We show that, consequently, B-Raf interacts with MEK-1 and MEK-2 with a better affinity than does c-Raf-1, thus strengthening the notion that B-Raf is a stronger MEK activator than c-Raf-l. Our results also suggest that a MEK specific sequence, not present in MAP kinase kinases which are not activated by members of the Raf family, is required for the interaction with Raf proteins.

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.

Characterization of a novel quiescence responsive element downregulated by v-Src in the promoter of the neuroretina specific QR1 gene.

The neuroretina is a functional unit of the central nervous system which arises through successive steps of division, growth arrest and differentiation of neuroectodermal precursors. Postmitotic quail neuroretina (QNR) cells are conditionally induced to divide upon infection with temperature sensitive mutants of Rous sarcoma virus (RSV), since QNR cell division can be arrested by either inactivating p60v-Src at the nonpermissive temperature (41 degrees C) or by serum deprivation at 37 degrees C. We are studying the transcriptional control of QR1, a neuroretina specific gene, whose expression is down-regulated in proliferating cells at 37 degrees C and is fully restored when these cells are made quiescent. We previously showed that this quiescence specific upregulation implicates a promoter region named A box, which binds Maf transcription factors. We report the identification of the C box, a second promoter sequence that activates QR1 transcription in non dividing cells. This sequence is able to form two DNA-protein complexes, one of which (C4) is predominantly detected in growth arrested NR cells. We identified the DNA binding site for C4 and described mutations that abolish both C4 binding and promoter activity in quiescent cells. Moreover, we show that a multimerized C box is able to stimulate a heterologous promoter in non dividing cells and constitutes, therefore, a novel quiescence responsive enhancer. Finally, we report that QR1 transcriptional response to cell quiescence requires cooperation between the C box and A box.

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.

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.

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.

mafA, a novel member of the maf proto-oncogene family, displays developmental regulation and mitogenic capacity in avian neuroretina cells.

Transcription factors of the Maf proto-oncogene family have been shown to participate in the regulation of several differentiation specific genes. We previously reported that a member(s) of this family is involved in the regulation of the neuroretina specific gene, QR1, through a promoter region, designated the A box, that is closely related to the Maf recognition element (MARE). We undertook an identification of Maf family genes expressed in the quail neuroretina (QNR) and we report the isolation of mafA, a gene encoding a novel member of the large Maf proteins subgroup. Expression of this gene is developmentally regulated in the neuroretina. MafA is able to bind to MARE sequence and to heterodimerize with v-Maf, MafB, Jun and Fos, but not with the small MafF and MafK proteins. Accordingly, it is able to transactivate the QR1 promoter A box. We also show that increased expression of mafA induces sustained proliferation of postmitotic QNR cells.