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C Pears

Publications and source records attributed to C Pears.

At least 19 recordsLinked to original sources
PubMed2001

A Dictyostelium nuclear phosphatidylinositol phosphate kinase required for developmental gene expression.

The generation of diacylglycerol (DAG) in response to receptor stimulation is a well-documented signalling mechanism that leads to activation of protein kinase C (PKC). Putative alternative effectors contain sequences that interact with DAGs, but the mechanisms of signal transduction are unknown. We have identified a Dictyostelium gene encoding a novel protein which contains a domain with high identity to the DAG-binding domain of PKC. It does not encode a PKC homologue as the conservation does not extend outside this region. We confirm that the proposed DAG-binding domain is sufficient to mediate interaction of a fusion protein with vesicles containing DAG. The protein also shows significant homology to mammalian phosphatidylinositol phosphate (PIP) kinases and we show that this domain has PIP kinase activity. The protein, PIPkinA, is enriched in the nucleus and abrogation of gene function by homologous recombination inhibits early developmental gene expression, blocking development at an early stage. Thus, we have identified a PIP kinase from Dictyostelium which is required for development, is a candidate effector for DAG and has the potential to synthesize nuclear PIP(2).

Animals↗
PubMed1999

Cell cycle-dependent regulation of early developmental genes.

Cell cycle phase at the onset of development in Dictyostelium influences cell fate. Cells in the G2 phase, which tend to become spores, show a more rapid induction of expression of the cell surface receptor involved in the chemotaxis. We show that differential induction of developmental expression is restricted to some transcripts, including those encoding proteins required for chemotaxis, and thus is not due to general transcriptional repression during mitosis. We also show that cells showing rapid induction of one such gene are preferentially located at the centre of early aggregates. These results are consistent with cells derived from G2 phase being at the centre of early aggregates because selective differences in gene regulation render them more efficient at aggregation.

Animals↗
PubMed1999

Protein kinase C-alpha is essential for Ramos-BL B cell survival.

The Ramos-Burkitt lymphoma (BL) B cell line is driven into growth arrest and apoptosis by cross-linking surface immunoglobulin. We demonstrate that protein kinase C (PKC) activity is required for Ramos B cell proliferation and survival. A variety of PKC inhibitors trigger a significant decrease in [(3)H]thymidine incorporation with a concomitant increase in cell death. Antisense depletion of expression of the PKC-alpha isoform is sufficient to trigger cell death in the absence of any other signal, demonstrating a requirement for this isoform for survival of Ramos-BL B cells. Cross-linking surface immunoglobulin also leads to depletion of PKC-alpha levels, suggesting that this may be one mechanism by which this signals for cell death in Ramos-BL B cells.

Antibodies, Anti-Idiotypic↗
PubMed1998

Negative influence of RasG on chemoattractant-induced ERK2 phosphorylation in Dictyostelium.

The Dictyostelium ERK2 protein is transiently activated when cells are treated with the chemotactic agents cAMP or folic acid. Activating phosphorylation is markedly inhibited in strains overexpressing the constitutively activated RasG protein. This is in marked contrast to mammalian cells where the highly related mitogen-activated protein kinases (MAPKs) are stimulated by Ras activation.

Animals↗
PubMed1997

Cells at the center of Dictyostelium aggregates become spores.

The cellular slime mold Dictyostelium discoideum undergoes a developmental life cycle on starvation to generate a fruiting body consisting of a mass of spores supported on a stalk of dead, vacuolated cells. The choice between alternative cell fates is influenced by a variety of factors including cell cycle position at the onset of starvation. We present evidence to suggest that the cell cycle position influences cell fate by determining the position of cells in the early aggregate. The existence of a strain which cannot initiate development on its own but which can respond to signals generated by nonmutant cells has allowed us to investigate the eventual cell fate of the initiating cells which are, by definition, at the center of the early aggregate. Cells which have a propensity to become prespore cells show an increased efficiency in initiating development of this strain. Labeling the initiating cells by the expression of green fluorescent protein reveals that these cells become spores. The higher levels of expression of genes characteristic of early development in cells with a prespore tendency are consistent with the earlier expression of the components of relay in prespore cells.

Animals↗
PubMed1997

A protein kinase C-like activity involved in the chemotactic response of Dictyostelium discoideum.

During the developmental life cycle of the cellular slime mould Dictyostelium discoideum cells aggregate in response to pulses of extracellular cAMP. This chemotactic agent stimulates a number of signalling pathways in the cell including the activation of a phospholipase C activity leading to the transient generation of inositol 3,4,5-trisphosphate and diacylglycerol. The role of diacylglycerol in chemotactic response and development of Dictyostelium is not known. We have evidence to suggest that two protein kinase C-like enzymes exist in Dictyostelium due to the different cellular responses to two inhibitors specific for protein kinase C. One enzyme is preferentially sensitive to D-erythro-sphingosine, a diacylglycerol analogue, and is required for growth. A second is preferentially inhibited by bisindolylmaleimide GF109203X and is required for chemotaxis. We have identified protein kinase C-like kinase activity in Dictyostelium cell extracts which appears as the cells aggregate. This activity is stimulated by diacylglycerol, especially biologically relevant diacylglycerol species, and phosphorylates a peptide substrate which is an efficient substrate for mammalian protein kinase Cs. This activity is a candidate for the effector of diacylglycerol generated during the aggregative phase of Dictyostelium development and defines a role for diacylglycerol in the chemotactic response.

Animals↗
PubMed1997

Isoform specificity of activators and inhibitors of protein kinase C gamma and delta.

Expression of certain mammalian protein kinase C (PKC) isoforms inhibits the proliferation of Schizosaccharomyces pombe (Goode et al., Mol. Biol. Cell 5 (1994) 907-920). We have taken advantage of this fact to determine the in vivo isoform preference of a number of PKC inhibitors, using a microtitre plate assay which allows rapid screening. This in vivo model has revealed previously unreported preferences; calphostin C is a more efficient inhibitor of the novel PKCS than chelerythrine chloride whereas the efficiencies are reversed for inhibition of the classical PKCgamma. We have also shown that the anti-leukaemic agent bryostatin 1 inhibits or activates in vivo in an isoform-specific manner.

Acetophenones↗
PubMed1992

Studies on the phosphorylation of protein kinase C-alpha.

A kinase-defective protein kinase C-alpha mutant is shown to be a phosphoprotein when expressed in COS-1 cells, indicating that intramolecular phosphorylation does not fully account for the phosphate content of protein kinase C-alpha. Furthermore, evidence is presented that the intermolecular phosphorylation of protein kinase C-alpha is due to an activity other than protein kinase C-alpha itself, and this phosphorylation appears to be necessary for protein kinase C-alpha activity. By contrast, the characteristic shift in apparent molecular mass consequent on phosphorylation in vivo can be accounted for by autophosphorylation, as demonstrated in vitro. The relationship between these phosphorylated protein kinase C-alpha species is discussed.

Acid Phosphatase↗
PubMed1991

Down-regulation of a kinase defective PKC-alpha.

A kinase defective mutant of PKC-alpha down-regulates in response to phorbol esters as effectively as the wild-type protein when introduced into COS-1 cells. This demonstrates that intramolecular autophosphorylation is not a prerequisite for down-regulation.

Animals↗
PubMed1991

The regulatory domain of protein kinase C-epsilon restricts the catalytic-domain-specificity.

Protein kinase C (PKC) consists of a family of closely related enzymes that can be divided into two subfamilies (alpha, beta and gamma and delta, epsilon and zeta) on the basis of primary sequence. Functional differences have also been described; thus PKC-alpha, PKC-beta and PKC-gamma readily phosphorylate histone IIIS in vitro, whereas PKC-epsilon will not employ this substrate efficiently. We have previously demonstrated, however, that proteolytic cleavage of PKC-epsilon generates a constitutive kinase activity that is an efficient histone IIIS kinase [Schaap, Hsuan, Totty & Parker (1990) Eur. J. Biochem. 191, 431-435]. In order to investigate the structural basis for this switch in specificity, we have constructed a chimaeric protein containing the regulatory domain of PKC-epsilon fused to the catalytic domain of PKC-gamma. When this is expressed in COS1 cells the chimaeric kinase shows a substrate-specificity similar to that of PKC-epsilon rather than to that of PKC-gamma. This demonstrates a role for the regulatory domain in substrate selection of PKC-epsilon.

Animals↗
PubMed1990

A developmentally regulated trans-acting factor recognizes dissimilar G/C-rich elements controlling a class of cAMP-inducible Dictyostelium genes.

Transcriptional response elements involved in the cAMP-inducible and developmentally regulated expression of the Dictyostelium aggregate-stage gene pst-cath/CP2 have been shown to include a G/C-rich sequence element [G-box regulatory element (GBRE)]. We have recently identified a trans-acting factor, GBF (GBRE binding factor), that specifically interacts with this sequence and have shown that the binding activity of GBF to GBRE is developmentally regulated and inducible by cAMP. Here, we examine further the possible role of GBF in the regulation of pst-cath/CP2 and three other coordinately regulated, cAMP-inducible aggregate-stage genes. We show that GBF itself (or other closely related factors) recognizes dissimilar G/C-rich elements present in the 5'-flanking regions of these genes and that the ability of the individual, distinct G/C-rich elements to confer regulated expression on a promoter deletion mutant of the pst-cath/CP2 gene is correlated with the relative affinity for GBF. G/C-rich elements carrying point mutations that prevent in vitro binding of GBF to two of the G/C-rich elements fail to activate expression in vivo. An analysis of major points of contact between the GBF protein and two distinctly different binding sites suggests that binding of GBF to these sequence elements involves a considerable degree of flexibility in DNA-protein interactions. These results suggest that the regulated expression of a class of aggregate-stage cAMP-inducible genes involves the interaction of GBF or homologous factors with dissimilar G/C-rich sequence elements and that induction of GBF activity or that of homologous factors by cAMP may thus be a limiting step in the induction of this temporally coordinate set of genes during Dictyostelium development.

Base Sequence↗