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Biomedical subjects

C J Marshall

Publications and source records attributed to C J Marshall.

At least 73 records · Page 4Linked to original sources
PubMed1995

Regulation of GATA-2 phosphorylation by mitogen-activated protein kinase and interleukin-3.

GATA-2 is a member of a family of transcription factors which bind a common DNA sequence motif (WGA-TAR) through an evolutionarily conserved zinc finger domain. An essential role for GATA-2 in the development of hematopoietic stem cells has recently been shown in gene targeting experiments in mice. Here we show that GATA-2 exists in hematopoietic progenitor cells as a phosphoprotein. Stimulation of progenitors with interleukin-3 (IL-3) results in enhanced phosphorylation of GATA-2 which occurs within 5 min. IL-3 is known to signal in part through mitogen-activated protein (MAP) kinase, and evidence for MAP kinase signaling in the control of GATA-2 phosphorylation was obtained by genetically manipulating the MAP kinase pathway in COS cells using either constitutively activating or interfering mutants of MAP kinase kinase. Furthermore, using an interfering mutant of MAP kinase kinase, we directly demonstrated a critical role for the MAP kinase pathway in the IL-3-dependent phosphorylation of GATA-2 in hematopoietic progenitor cells. Finally, in vitro phosphorylation experiments using recombinant GATA-2 raise the possibility that MAP kinase itself may phosphorylate GATA-2. Our results provide evidence for phosphorylation via the MAP kinase pathway constituting a cytoplasmic link between GATA-2 and growth factor receptors and are consistent with the hypothesis that GATA-2 is involved in the growth factor responsiveness and proliferation control of hematopoietic progenitor cells.

Animals↗
PubMed1995

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. This reduced level in 1aCB was reflected by an increase in nuclear associated p21ras, as seen by immunofluorescence of endogenous and introduced ras. In addition, 1aCB had a reduced ratio of ras in the detergent to aqueous phases after triton X114 partitioning, suggesting a defect in Step I processing of the p21ras in the cell line. This was not however due to defects in the Step I enzymes farnesyltransferase or carboxymethyltransferase whose activities were not reduced.

Alkyl and Aryl Transferases↗
PubMed1995

Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation.

A number of different intracellular signaling pathways have been shown to be activated by receptor tyrosine kinases. These activation events include the phosphoinositide 3-kinase, 70 kDa S6 kinase, mitogen-activated protein kinase (MAPK), phospholipase C-gamma, and the Jak/STAT pathways. The precise role of each of these pathways in cell signaling remains to be resolved, but studies on the differentiation of mammalian PC12 cells in tissue culture and the genetics of cell fate determination in Drosophila and Caenorhabditis suggest that the extracellular signal-regulated kinase (ERK-regulated) MAPK pathway may be sufficient for these cellular responses. Experiments with PC12 cells also suggest that the duration of ERK activation is critical for cell signaling decisions.

Animals↗
PubMed1995

Evolutionary analysis of aspartate aminotransferases.

Aspartate aminotransferase isoenzymes are located in both the cytosol and organelles of eukaryotes, but all are encoded in the nuclear genome. In the work described here, a phylogenetic analysis was made of aspartate aminotransferases from plants, animals, yeast, and a number of bacteria. This analysis suggested that five distinct branches are present in the aspartate aminotransferase tree. Mitochondrial forms of the enzyme form one distinct group, bacterial aspartate aminotransferase formed another, and the plant and vertebrate cytosolic isoenzymes each formed a distinct group. Plant cytosolic isozymes formed a further group of which the plastid sequences were a member. The yeast mitochondrial and cytosolic aspartate aminotransferases formed groups separate from other members of the family.

Amino Acid Sequence↗
PubMed1995

Phylogenetic relationships among transposon-like elements in human and primate DNA.

A THE-1 sequence in intron 7 of the human dystrophin gene has been found to represent a new subfamily of THE-1 elements. The sequence is closely related to the MstII family of repetitive sequences and is more like single-copy sequences found in the galago genome than any other THE-1 sequence previously reported. This new THE-1 sequence has been compared with two other complete THE-1 sequences and three related long-terminal repeat elements that we have previously found in intron 7 of the dystrophin gene, and with members of the same family from elsewhere in the primate genome. Parsimony and deletion analysis show that the cluster of THE-1 sequences in intron 7 of the dystrophin gene has arisen from at least three individual insertion events, rather than from the insertion and duplication of a single progenitor sequence.

Animals↗
PubMed1995

Opportunities for pharmacological intervention in the ras pathway.

The delineation of a ras-dependent signalling pathway from tyrosine kinases to mitogen-activated protein kinases, which is common to a variety of effectors of cell proliferation, suggests that this pathway may provide targets for agents to inhibit cell proliferation. Considerable investigation is needed to determine which points of the pathway are the most suitable for inhibition in terms of effectiveness and selectivity. The use of interfering mutants derived from each component in the pathway is one approach to determining at which point it is best to inhibit.

Antineoplastic Agents↗
PubMed1994

Loss of heterozygosity in familial tumors from three BRCA1-linked kindreds.

BRCA1, a breast-ovarian cancer susceptibility gene which has been localized to 17q21, appears to be a tumor suppressor gene based on evidence from loss of heterozygosity (LOH) studies. We analyzed 14 ovarian and breast tumors from BRCA1 carriers and 1 sporadic breast tumor from 3 kindreds for 17q21 LOH. Thirteen of the 14 tumors from gene carriers exhibited LOH of the wild-type allele. Tumors from one gene carrier and the sporadic breast case did not exhibit any LOH in the region. There was loss of the wild-type allele from both maternally and paternally derived chromosomes, therefore excluding the possibility of genomic imprinting and providing further evidence that BRCA1 is a tumor suppressor. Three tumors showed interstitial LOH in the region, and thus established the utility of familial tumors in refining a region surrounding a tumor suppressor gene in a manner analogous to using genetic recombinants.

Adult↗
PubMed1994

The sevenmaker gain-of-function mutation in p42 MAP kinase leads to enhanced signalling and reduced sensitivity to dual specificity phosphatase action.

A mammalian mutant MAP kinase, D319N ERK2, analogous to Drosophila melanogaster sevenmaker (rlsem) gain-of-function mutation was shown to have an increased sensitivity to low levels of signalling in vivo. However, the mutation does not lead to an elevated basal kinase activity and still requires activation by MAP kinase kinase (MAPKK) as does wild type ERK2. This increased responsiveness seen in vivo is not due to an increased ability to phosphorylate substrates but appears to reflect a reduced sensitivity to a MAP kinase phosphatase CL100.

3T3 Cells↗
PubMed1994

Interleukin-1 activates a novel protein kinase that phosphorylates the epidermal-growth-factor receptor peptide T669.

We have isolated from KB cells stimulated with interleukin-1 (IL-1) a protein kinase that phosphorylates a peptide (T669) based on the sequence around T669 of the epidermal growth factor (EGF) receptor. The enzyme, which had an apparent molecular mass of 45 kDa on gel-filtration chromatography, was purified 170,000-fold from cytosolic extracts by sequential chromatography on Mono Q, Mono S, phenyl-Sepharose, Superose 12, ATP-Sepharose and Mono Q. The enzyme activity co-chromatographed at the last step with a 45 kDa protein band that stained for phosphotyrosine. This peak fraction also contained some actin and a 60 kDa protein that stained weakly for phosphotyrosine. The T669 peptide is a substrate for mitogen-activated protein (MAP) kinase. Amounts of IL-1-induced T669 kinase and activated recombinant p42 MAP kinase having equal activity on T669 peptide were compared on commonly used MAP kinase substrates. T669 kinase was two or three orders of magnitude less active on myelin basic protein or microtubule-associated protein-2 than was MAP kinase. The IL-1-induced T669 kinase did not react with antiserum to p42/p44 MAP kinase. It was inactivated by treatment with protein phosphatase 2A or protein phosphotyrosine phosphatase 1B, so it may be regulated by dual phosphorylation in similar fashion to MAP kinase. The dephosphorylated enzyme was not re-activated by MAP kinase kinase. This novel enzyme could lie on a kinase cascade induced by IL-1. It may be responsible for phosphorylating T669 of the EGF receptor.

Amino Acid Sequence↗
PubMed1994

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. We have examined whether the differential activation of MAP kinases forms the basis of the differential response of the cells to the two factors. RESULTS: By overexpressing either wild-type or mutant receptors for epidermal growth factor in PC12 cells, we found that p42 and p44 MAP kinase activity remains elevated for longer in cells that overexpress receptors than in untransfected cells. Epidermal growth factor promotes both a striking nuclear translocation of p42 MAP kinase and the differentiation of the overexpressing cells. CONCLUSIONS: Our results strongly suggest that the distinct effects of nerve growth factor and epidermal growth factor on PC12 cell differentiation can be explained by differences in the extent and duration of activation of p42 and p44 MAP kinases in response to the two factors, without invoking a signal transduction pathway specific to nerve growth factor.

Animals↗
PubMed1994

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). Reconstitution of transformation was specific to this amino acid sequence because none of the other autophosphorylation sites, when grafted individually, caused transformation, and replacement of the tyrosine with a phenylalanine residue significantly reduced the oncogenic potential of both the full-length and the tailed proteins. When present alone the most C-terminal sequence enabled coupling to a biochemical pathway that includes Ras, MAP kinase and transactivation of Jun. These results indicate that the multiplicity of autophosphorylation sites on a receptor tyrosine kinase is not essential for transformability, and implicate the MAP kinase pathway in transduction of the oncogenic signal of Neu/ErbB-2.

Animals↗
PubMed1994

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. The activated mutants stimulated PC12 cell neuronal differentiation and transformed NIH 3T3 cells. The interfering mutants inhibited growth factor-induced PC12 differentiation, growth factor stimulation of proliferation, and reverted v-src- and ras-transformed cells. These results therefore show that, depending on cellular context, activation of MAP kinase kinase is necessary and sufficient for cell differentiation or proliferation.

3T3 Cells↗
PubMed1994

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. Whereas Raf is normally cytosolic, in cells expressing active Ras, Raf is associated with the plasma membrane. This led us to investigate whether Ras is required to localize Raf to the plasma membrane in order for Raf to become activated. We fused the membrane localization signal of K-Ras(4B) to the carboxy terminus of Raf. This protein is constitutively active and can be further activated by epidermal growth factor, independently of Ras. Our results indicate that Ras functions as a regulated, membrane-bound anchor for Raf, and that other signal(s) also contribute to Raf activation.

Amino Acid Sequence↗
PubMed1994

Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1.

Many growth factors whose receptors are protein tyrosine kinases stimulate the MAP kinase pathway by activating first the GTP-binding protein Ras and then the protein kinase p74raf-1. p74raf-1 phosphorylates and activates MAP kinase kinase (MAPKK). To understand the mechanism of activation of MAPKK, we have identified Ser217 and Ser221 of MAPKK1 as the sites phosphorylated by p74raf-1. This represents the first characterization of sites phosphorylated by this proto-oncogene product. Ser217 and Ser221 lie in a region of the catalytic domain where the activating phosphorylation sites of several other protein kinases are located. Among MAPKK family members, this region is the most conserved, suggesting that all members of the family are activated by the phosphorylation of these sites. A 'kinase-dead' MAPKK1 mutant was phosphorylated at the same residues as the wild-type enzyme, establishing that both sites are phosphorylated directly by p74raf-1, and not by autophosphorylation. Only the diphosphorylated form of MAPKK1 (phosphorylated at both Ser217 and Ser221) was detected, even when the stoichiometry of phosphorylation by p74raf-1 was low, indicating that phosphorylation of one of these sites is rate limiting, phosphorylation of the second then occurring extremely rapidly. Ser217 and Ser221 were both phosphorylated in vivo within minutes when PC12 cells were stimulated with nerve growth factor. Analysis of MAPKK1 mutants in which either Ser217 or Ser221 were changed to glutamic acid, and the finding that inactivation of maximally activated MAPKK1 required the dephosphorylation of both serines, shows that phosphorylation of either residue is sufficient for maximal activation.

Amino Acid Sequence↗
PubMed1994

Characterization of two different forms of mitogen-activated protein kinase kinase induced in polymorphonuclear leukocytes following stimulation by N-formylmethionyl-leucyl-phenylalanine or granulocyte-macrophage colony-stimulating factor.

Incubation of polymorphonuclear leukocytes with chemoattractants, granulocyte-macrophage colony-stimulating factor (GM-CSF), or phorbol 12-myristate 13-acetate (PMA) activated both mitogen-activated protein kinase kinase (MAPKK) and mitogen-activated protein kinase (MAPK). Activation by chemoattractants was rapid and transient, being maximal by 1 min and decreasing by 10 min. The order of efficacy was formyl-met-leu-phe > C5a > > LTB4 > interleukin 8 > platelet-activating factor. In contrast, activation by GM-CSF or PMA was slow and sustained being maximal at 5 min and with little decrease by 30 min. Sustained MAPK activation required continuous activation of the MAPKK. The MAPKK induced by N-formylmethionyl-leucyl-phenylalanine, GM-CSF, or PMA was resolved into two forms by anion exchange chromatography (Mono Q). Both corresponded to a 45-kDa MAPKK antigen by Western blotting and were inactivated by serine/threonine protein phosphatase 2A. Rechromatography of both forms after dephosphorylation resulted in the antigen's eluting slightly earlier on the Mono Q gradient than when in the active state. However, the two peaks remained separate, suggesting that they are not merely different phosphoforms of the same enzyme. The MAPK cascade is a signaling pathway common to many polymorphonuclear leukocyte stimulants, which may be activated transiently or in a sustained manner.

Blotting, Western↗
PubMed1994

The threonine residues in MAP kinase kinase 1 phosphorylated by MAP kinase in vitro are also phosphorylated in nerve growth factor-stimulated rat phaeochromocytoma (PC12) cells.

The residues on MAP kinase kinase-1 (MAPKK1) phosphorylated by MAP kinase in vitro have been identified as Thr-291 and Thr-385. Both threonines are phosphorylated in PC12 cells and the 32P-labelling of each residue increases after stimulation with nerve growth factor (NGF). The results establish that MAPKK1 is a physiological substrate for MAP kinase. The two active forms of MAPKK that are resolved by Mono Q chromatography of PC12 cell extracts are both phosphorylated at Thr-291 and Thr-385, demonstrating that neither species is the MAPKK2 isoform which lacks Thr-291.

Amino Acid Sequence↗