Georges Calothy

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 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.

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.

Modulation of Kinase Activity and Oncogenic Properties by Alternative Splicing Reveals a Novel Regulatory Mechanism for B-Raf

Members of the raf oncogene family encode serine/threonine protein kinases, which activate the mitogen-activated protein kinase kinase MEKs (MAPK orERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.

Phosphorylation of MafA Is Essential for Its Transcriptional and Biological Properties

ABSTRACT 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.

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.

Choline acetyl transferase activity in chick embryo neuroretinas during development in ovo and in monolayer cultures

Abstract The specific activity of the enzyme choline acetyl transferase (CAT) in chick neuroretinas was investigated during in ovo development and in monolayer cultures. The enzyme activity was barely detectable on the 6th day of incubation but increased markedly between the 7th and 11th days. The activity increased sharply between the 15th and 17th days and then slowly until hatching. When cell suspensions from 6- to 7-day neuroretinas were cultured as monolayers, CAT specific activity increased rapidly. After 4–5 days in culture, the activity of the enzyme was identical to that found in the neuroretina on the 11th day of incubation. Cells from 9-day neuroretinas also differentiate in monolayer cultures, but with a more irregular pattern. These data show that cholinergic neurons from chick embryo neuroretina differentiate in monolayer cultures without a lag and at the same rate as in vivo.

Induction of Postmitotic Neuroretina Cell Proliferation by Distinct Ras Downstream Signaling Pathways

ABSTRACT 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.

Differential expression of TrkC catalytic and noncatalytic isoforms suggests that they act independently or in association.

Members of the trk gene family encode neurotrophin receptors. The trkC locus encodes multiple neurotrophin‐3 catalytic and noncatalytic receptor isoforms. We report the molecular cloning and characterization of mouse cDNAs encoding two noncatalytic TrkC receptors: novel isoforms designated as TrkC NC1 and TrkC NC2, the mouse homologue of the TrkC truncated form previously identified in rat (Tsoulfas et al. [1993] Neuron 10:975–990; Valenzuela et al. [1993] Neuron 10:963–974). We extensively analyzed the transcription pattern of these two noncatalytic isoforms and that of the catalytic isoforms by Northern blotting and in situ hybridization. We did not detect trkC NC1 transcripts in embryos, but we found that trkC NC1 expression is restricted to specific areas in adult brain. In contrast, trkC NC2 transcripts are readily detected early during embryogenesis and are expressed predominantly in adult brain and gonads. We also provide the first evidence for the existence of TrkC NC2 protein by using polyclonal antibodies that specifically recognize this isoform. By using in situ hybridization, we show for the first time that trkC NC2 transcripts are found in differentiating fields of maturing neurons and in mature neurons of laminar structures of adult brain. We also report a similarity of localization between trkC NC2 transcripts and markers of oligodendrocyte progenitors in the embryonic spinal cord. Furthermore, our results also show that trkC NC2 and trkC catalytic transcripts could be either codistributed (in the central and peripheral nervous system) or independently expressed, especially outside the nervous system. These results suggest that the TrkC NC2 isoform acts either independently or in association with its catalytic counterpart. Finally, we show that TrkC NC2 is expressed in dendrites of pyramidal neurons of hippocampus and cerebral cortex. We propose that this receptor is involved in proliferation of oligodendrocyte progenitors, neuronal differentiation, and synaptic plasticity and that it may also play a fundamental role in mediating neurotrophin‐3 effects outside the nervous system. J. Comp. Neurol. 401:47–64, 1998.

Growth stimulation of chick embryo neuroretinal cells infected with Rous sarcoma virus: Relationship to viral replication and morphological transformation

Abstract Neuroretinal (NR) cells from 7-day-old chick embryos infected with Rous sarcoma virus (RSV) are morphologically transformed, synthesize virus, and are induced to proliferate for several generations. By contrast, uninfected cells have a limited growth capacity and cannot be propagated in vitro . The relationship of induction of cell multiplication to viral replication and morphological transformation was analyzed by infecting NR cells with conditional and nonconditional transformation defective viruses. NR cells infected with a temperature-sensitive mutant of RSV defective for cell transformation, but not for virus replication, were induced to multiply at both nonpermissive and permissive temperatures, although expression of the transformed phenotype, as tested by several parameters, was suppressed in these cells at nonpermissive temperature. Nonconditional, nontransforming viruses replicated normally in NR cells, but failed to induce their multiplication. These results indicate that only transforming viruses can induce NR cell multiplication and that viral replication alone does not account for the growth changes in infected NR cells. These data also suggest that expression of the transformed phenotype and induction of NR cell proliferation may depend on distinct viral informations.

D53 is a novel endosomal SNARE-binding protein that enhances interaction of syntaxin 1 with the synaptobrevin 2 complex in vitro

Synaptobrevin 2 (Sb2), syntaxin1 (Stx1), and synaptosomal-associated protein of 25 kDa (SNAP-25) are the main components of the soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex involved in fusion of synaptic vesicles with the presynaptic plasma membrane. We report the characterization of D53, a novel SNARE-binding protein preferentially expressed in neural and neuro-endocrine cells. Its two-dimensional organization, established by the hydrophobic cluster analysis, is reminiscent of SNARE proteins. D53 contains two putative helical regions, one of which includes a large coiled-coil domain involved in the interaction with Sb2 in vitro. Following subcellular fractionation, endogenous D53 was specifically detected in the membrane-containing fraction of PC12 cells, where it co-immunoprecipitated with Sb2. Analysis by confocal microscopy showed that, in these cells, endogenous D53 co-localized partially with the transferrin receptor in early endosomes. In vitro assays revealed that binding properties of D53 to Stx1 and Sb2 are comparable with those of SNAP-25. Furthermore, D53 forms Sb2/Stx1/D53 complexes in vitro in a manner similar to SNAP-25. We propose that D53 could be involved in the assembly or disassembly of endosomal SNARE complexes by regulating Sb2/Stx interaction.