Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases.

It has previously been shown that maximal activation of Raf-1 is produced by synergistic signals from oncogenic Ras and activated tyrosine kinases. This synergy arises because Ras-GTP translocates Raf-1 to the plasma membrane where it becomes phosphorylated on tyrosine residues 340 and 341 by membrane-bound tyrosine kinases (Marais, R., Light, Y.

p21Ras activation by the guanine nucleotide exchange factor Sos, requires the Sos/Grb2 interaction and a second ligand-dependent signal involving the Sos N-terminus.

It has been suggested that a key event in growth factor-induced p21Ras activation by the guanine nucleotide exchange factor Sos, is the recruitment of Sos to the plasma membrane by its interaction with the adaptor protein Grb2. However, other evidence argues that the sub cellular localisation of Sos is independent of Grb2, and that the Sos/Grb2 interaction can be dispensed with for p21Ras activation. To clarify the role of the Sos/Grb2 interaction in ligand-stimulated p21Ras activation, we have utilised the observation that overexpression of the Sos C-terminal domain can effectively inhibit p21Ras-dependent signalling in three different mammalian systems.

A member of the MAP kinase phosphatase gene family in mouse containing a complex trinucleotide repeat in the coding region.

We have identified a novel mouse gene encoding a protein that shows high homology to the dual-specificity tyrosine/threonine phosphatase family of proteins. The gene encodes a 5 kb transcript which is expressed predominantly in brain and lung and contains a translated complex trinucleotide repeat within the coding region. Using interspecific mouse backcross analysis, the gene has been localised to distal mouse chromosome 7.

Phospholipase C delta 1 requires a pleckstrin homology domain for interaction with the plasma membrane.

The structural requirements of phospholipase C delta 1 for interaction with the plasma membrane were analysed by immunofluorescence after microinjection into living cells. Microinjection of deletion mutants revealed that the region required for membrane attachment and binding of inositol 1,4,5-trisphosphate in vitro corresponded to the pleckstrin homology domain, a structural module described in more than 90 proteins.

Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation.

A central feature of signal transduction downstream of both receptor and oncogenic tyrosine kinases is the Ras-dependent activation of a protein kinase cascade consisting of Raf-1, Mek (MAP kinase kinase) and ERKs (MAP kinases). To study the role of tyrosine kinase activity in the activation of Raf-1, we have examined the properties of p74Raf-1 and oncogenic Src that are necessary for activation of p74Raf-1. We show that in mammalian cells activation of p74Raf-1 by oncogenic Src requires pp60Src to be myristoylated and the ability of p74Raf-1 to interact with p21Ras-GTP.

Ultrastructural localization of ras-related proteins using epitope-tagged plasmids.

To determine the ultrastructural distribution of H-ras, the rho proteins rho-A, rho-B, rho-C, and the rac1 protein (members of the ras GTP-binding protein family), we used cDNA expression plasmids in which a short sequence coding for the epitope recognized by the anti c-myc monoclonal antibody 9E10 has been inserted at the N-terminus. Each of the expressed proteins has this epitope as a tag, allowing its localization by light and electron microscopy by the same antibody. After nuclear microinjection of these plasmids into MDCK or Rat 2 cells, expression of the protein (6-18 hr later) was confirmed by immunofluorescence labeling with 9E10 imaged by confocal microscopy.

Reversion of a human tumour cell line containing oncogenic p21ras is associated with a defect in the post-translational processing of the ras protein.

Correct post-translational modifications of the ras proteins are essential for their membrane localisation and functioning. The flat revertant cell lines 1aCB and 8b, derived from the human bladder carcinoma cell line EJ, contain the transforming gene V12Ha-ras and are resistant to retransformation by ras protein or DNA, but still do require the presence of ras for proliferation. Both revertant cell lines demonstrated reduced levels of membrane associated p21ras when compared to their parental EJ cell lines.

Ectoenzyme regulation by phenotypically distinct fibroblast sub-populations isolated from the human mammary gland.

Inter- and intralobular mammary fibroblasts have been separated from normal human breast tissue and cultured to study the differential expression of ectoenzymes present within the stroma of the normal gland and associated with breast cancers. Specific ectoenzymes were identified by indirect immunofluorescence and quantified by flow cytometry and semi-quantitative PCR. A consistent difference was noted between the two fibroblast sub-populations at early passage in respect of dipeptidyl peptidase IV (DPP IV) and aminopeptidase N (APN) expression.

EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor.

Background: Mitogen-activated protein (MAP) kinase is the central component of a signal transduction pathway that is activated by growth factors interacting with receptors that have protein tyrosine kinase activity. The stimulation of PC12 phaeochromocytoma cells with nerve growth factor leads to the sustained activation and nuclear translocation of the p42 and p44 isoforms of MAP kinase and induces the differentiation of these chromaffin cells to a sympathetic-neuron-like phenotype. In contrast, stimulation with epidermal growth factor induces a transient activation of p42 and p44 MAP kinases without pronounced nuclear translocation and does not trigger cell differentiation.

A single autophosphorylation site confers oncogenicity to the Neu/ErbB-2 receptor and enables coupling to the MAP kinase pathway.

The transforming potential of the Neu/ErbB-2 receptor tyrosine kinase undergoes inactivation by deletion of the non-catalytic C-terminal tail, which contains five autophosphorylation sites. To determine which site is essential for oncogenicity, we tailed the C-terminally-deleted mutant with individual autophosphorylation sites. Complete restoration of the transforming action in vitro and in vivo was conferred by a stretch of 12 amino acids that contained the most C-terminal tyrosine autophosphorylation site (Y1253).

N-terminally myristoylated Ras proteins require palmitoylation or a polybasic domain for plasma membrane localization.

Plasma membrane targeting of Ras requires CAAX motif modifications together with a second signal from an adjacent polybasic domain or nearby cysteine palmitoylation sites. N-terminal myristoylation is known to restore membrane binding to H-ras C186S (C-186 is changed to S), a mutant protein in which all CAAX processing is abolished. We show here that myristoylated H-ras C186S is a substrate for palmitoyltransferase, despite the absence of C-terminal farnesylation, and that palmitoylation is absolutely required for plasma membrane targeting of myristoylated H-ras.

Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells.

The MAP kinase pathway is activated by a wide variety of external signals leading to cell proliferation or differentiation. However, it is not clear whether activation of this pathway is required for cellular responses or whether it is only one branch point in signal transduction. To investigate these questions, we generated constitutively activated and interfering mutants of MAP kinase kinase 1.

Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane.

A conserved tyrosine kinase-activated signal transduction pathway has recently been identified that comprises the plasma membrane-bound small guanine-nucleotide-binding protein Ras and the protein kinases Raf, MAP-kinase kinase and MAP kinase. GTP-bound Ras interacts directly with the amino-terminal regulatory domain of Raf, but although Ras and Raf can be coimmunoprecipitated from ligand-stimulated cells, Ras-GTP does not stimulate the kinase activity of Raf in vitro. Furthermore, we have failed to detect Ras in preparations of active detergent-solubilized Raf, demonstrating that once it is activated, Raf does not require Ras.

The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization.

C-terminal lipid modifications are essential for the interaction of Ras-related proteins with membranes. While all Ras proteins are farnesylated and some palmitoylated, the majority of other Ras-related proteins are geranylgeranylated. One such protein, Rab6, is associated with the Golgi apparatus and has a C-terminal CXC motif that is geranylgeranylated on both cysteines.

rho family GTPase activating proteins p190, bcr and rhoGAP show distinct specificities in vitro and in vivo.

rho family GTPases link extracellular signals to changes in the organization of cytoskeletal actin. Serum stimulation of quiescent Swiss 3T3 fibroblasts leads to rho-dependent actin stress fibre formation and focal adhesions, whilst several growth factors initiate signalling pathways leading to rac-dependent actin polymerization at the plasma membrane, and membrane ruffling. The product of the breakpoint cluster region gene bcr, rho GTPase accelerating protein (rhoGAP) and rasGAP-associated p190 share structurally related rho GAP domains, and possess GAP activity for rho family members in vitro.

CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms.

A novel sequence-specific DNA-binding protein, CTCF, which interacts with the chicken c-myc gene promoter, has been identified and partially characterized (V. V. Lobanenkov, R.

Specific association of activated MAP kinase kinase kinase (Raf) with the plasma membranes of ras-transformed retinal cells.

Plasma membrane-enriched fractions were prepared from human embryonic retinal cells transformed with either adenovirus E1A and oncogenic ras DNA, or E1A and E1B DNA. Ras comprised 5-10% of the membrane protein from the E1A/ras transformed cells, whereas the membranes from E1A/E1B transformed cells did not overexpress Ras. The membranes from E1A/ras cells contained MAP kinase kinase kinase (MAPKKK) activity, even after washing in 0.

Salmonella typhimurium induces membrane ruffling by a growth factor-receptor-independent mechanism.

Invasive Salmonella typhimurium induces dramatic actin rearrangements on the membrane surface of mammalian cells as part of its entry mechanism. These changes, which are best characterized as membranous ruffles, closely resemble the membrane changes that occur when a growth factor binds to its receptor. Recently, inhibition of the function of the small GTPases rac and rho in quiescent serum-starved fibroblasts was demonstrated to abolish growth factor-mediated ruffling and stress-fiber formation, respectively.

Cellular responses regulated by rho-related small GTP-binding proteins.

Rho-related proteins are members of the ras superfamily of small GTP-binding proteins. Their function in fibroblasts has been analysed using microinjection of living cells. Rho appears to link plasma membrane receptors to the assembly of focal adhesions and actin stress fibres.

The proto-oncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis.

Apoptosis plays an important role in regulating cell numbers in a wide variety of tissues during development. The product of the bcl-2 gene inhibits apoptosis in certain cells of the myeloid and lymphoid lineages and is expressed in many cells that have an extended life span. To assess the role of bcl-2 in neuronal apoptosis, we microinjected a bcl-2 expression vector into neurotrophic factor-deprived embryonic neurons.

Management of enuresis in children.

A postal survey was conducted to gather information on the enuresis service of Oxfordshire Health Authority Community Unit. This article discusses the size of the problem, treatment and knowledge and expertise. The findings are summarized in the form of a list of recommendations.

Different structural organization of Ras and Rho effector domains.

Ras regulates proliferation and differentiation signals in cells, and activation of the protein can lead to malignant transformation. Activation of the related protein, Rho, affects cell morphology, and it has been suggested that it may also have some oncogenic potential. We show here that Rho does not induce a malignant phenotype in NIH3T3 cells but instead is a potent activator of actin stress fibre formation.