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R Insall

Publications and source records attributed to R Insall.

18 recordsLinked to original sources
PubMed2000

Purification and cloning of phosphatidylinositol transfer proteins from Dictyostelium discoideum: homologues of both mammalian PITPs and Saccharomyces cerevisiae sec14p are found in the same cell.

Soluble phosphatidylinositol transfer proteins (PITPs) have important roles in lipid-mediated signalling as well as in membrane traffic. Two PITPs (alpha and beta) have been cloned from mammalian cells, which are unrelated in sequence to yeast PITP (the product of the SEC14 gene). However, all three PITPs can perform interchangeably to reconstitute function in mammalian cells. We have now purified the major PITP from the cytoplasm of Dictyostelium discoideum and cloned the gene. This protein, DdPITP1, is homologous with mammalian PITPalpha and PITPbeta. We have also cloned a second gene (DdPITP2) related in sequence to DdPITP1. In addition, an independently cloned cDNA encodes a relative of the SEC14 family of yeast PITPs. DdPITP1, DdPITP2 and DdSec14 proteins were all able to mediate the transfer of PtdIns from one membrane compartment to another; they thus exhibited the hallmark of PITPs. Secondly, all three PITPs were able to rescue phospholipase C-mediated phosphoinositide hydrolysis in PITP-depleted HL60 cells, indicating that all three PITPs were capable of stimulating phosphoinositide synthesis. The identification of PITPs related to both mammalian PITPs and yeast Sec14p in a single organism will provide a unique opportunity to examine the functions of this class of protein with genetic approaches.

Amino Acid Sequence↗
PubMed2000

Functional overlap of the dictyostelium RasG, RasD and RasB proteins.

Disruption of the rasG gene in Dictyostelium discoideum results in several distinct phenotypes: a defect in cytokinesis, reduced motility and reduced growth. Reintroduction of the rasG gene restores all of the properties of the rasG(-) cells to those of the wild type. To determine whether the defects are due to impaired interactions with a single or multiple downstream effectors, we tested the ability of the highly related but non identical Dictyostelium ras genes, rasD and rasB, to rescue the defects. Introduction of the rasD gene under the control of the rasG promoter into rasG null (rasG(-)) cells corrected all phenotypes except the motility defect, suggesting that motility is regulated by a RasG mediated pathway that is different to those regulating growth or cytokinesis. Western blot analysis of RasD protein levels revealed that vegetative rasG(- )cells contained considerably more protein than the parental AX-3 cells, suggesting that RasD protein levels are negatively regulated in vegetative cells by RasG. The level of RasD was enhanced when the rasD gene was introduced under the control of the rasG promoter, and this increase in protein is presumably responsible for the reversal of the growth and cytokinesis defects of the rasG(- )cells. Thus, RasD protein levels are controlled by the level of RasG, but not by the level of RasD. Introduction of the rasB gene under the control of the rasG promoter into rasG(-) cells produced a complex phenotype. The transformants were extremely small and mononucleate and exhibited enhanced motility. However, the growth of these cells was considerably slower than the growth of the rasG(-) cells, suggesting the possibility that high levels of RasB inhibit an essential process. This was confirmed by expressing rasB in wild-type cells; the resulting transformants exhibited severely impaired growth. When RasB protein levels were determined by western blot analysis, it was found that levels were higher in the rasG(- )cells than they were in the wild-type parental, suggesting that RasG also negatively regulates rasB expression in vegetative cells. Overexpression of rasB in the rasG(- )cells also reduced the level of RasD protein. In view of the fact that alternate Ras proteins correct some, but not all, of the defects exhibited by the rasG(-) cells, we propose that RasG interacts with more than one downstream effector. In addition, it is clear that the levels of the various Ras proteins are tightly regulated in vegetative cells and that overexpression can be deleterious.

Animals↗
PubMed1999

A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.

Adenylyl Cyclases↗
PubMed1997

The right stuff.

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Assertiveness↗
PubMed1997

The Dictyostelium mitogen-activated protein kinase ERK2 is regulated by Ras and cAMP-dependent protein kinase (PKA) and mediates PKA function.

The chemoattractant cAMP, acting through serpentine cAMP receptors, results in a rapid and transient stimulation of the Dictyostelium mitogen-activated protein kinase ERK2 activity (). In this study we show that other pathways required for aggregation, including Ras and cAMP-dependent protein kinase (PKA), are important regulators of ERK2 activation and adaptation. By examining both the level and kinetics of activation and adaptation of ERK2, we show that Ras is a negative regulator of ERK2. Activated Ras or disruption of a Ras GAP gene results in reduced ERK2 activation whereas disruption of putative Ras GEF or expression of dominant negative Ras proteins have a more rapid, higher, and extended activation. CRAC, a PH domain-containing protein required for adenylyl cyclase activation, is also required for proper ERK2 adaptation. PKA overexpression results in a more rapid, higher level of activation, whereas pka null cells show a lower level but more extended ERK2 activation. Furthermore, we show that constitutive expression of PKA catalytic subunit bypasses the requirement of ERK2 for aggregation and later development, indicating that PKA lies downstream from ERK2 and that ERK2 may regulate one or more components of the signaling pathway required for mediating PKA function, possibly by directly regulating PKA R or a protein controlling the intracellular level of cAMP.

Adenylyl Cyclases↗
PubMed1996

Seven helix chemoattractant receptors transiently stimulate mitogen-activated protein kinase in Dictyostelium. Role of heterotrimeric G proteins.

Mitogen-activated protein (MAP) kinases are involved in controlling a cell's responses to a variety of stimuli and can be activated by both protein tyrosine kinase and G protein-coupled receptors. It was shown previously that Dictyostelium MAP kinase ERK2 is required for normal activation of adenylyl cyclase and erk2 null cells are aggregation-deficient. In this manuscript, we show that the Dictyostelium MAP kinase ERK2 is rapidly and transiently activated in response to the chemoattractant cAMP. This response requires cAMP receptors, but is independent of the coupled G alpha2 subunit and the only known G beta subunit. These data indicate that ligand-mediated receptor activation of adenylyl cyclase requires two receptor-dependent pathways, one of which requires heterotrimeric G proteins, including G alpha2 and the only known G beta subunit, and the second of which requires ERK2. Our results suggest that ERK2 may be activated by a novel receptor-mediated pathway.

Adenylyl Cyclases↗
PubMed1994

CRAC, a cytosolic protein containing a pleckstrin homology domain, is required for receptor and G protein-mediated activation of adenylyl cyclase in Dictyostelium.

Adenylyl cyclase in Dictyostelium, as in higher eukaryotes, is activated through G protein-coupled receptors. Insertional mutagenesis into a gene designated dagA resulted in cells that cannot activate adenylyl cyclase, but have otherwise normal responses to exogenous cAMP. Neither cAMP treatment of intact cells nor GTP gamma S treatment of lysates stimulates adenylyl cyclase activity in dagA mutants. A cytosolic protein that activates adenylyl cyclase, CRAC, has been previously identified. We trace the signaling defect in dagA- cells to the absence of CRAC, and we demonstrate that dagA is the structural gene for CRAC. The 3.2-kb dagA mRNA encodes a predicted 78.5-kD product containing a pleckstrin homology domain, in agreement with the postulated interaction of CRAC with activated G proteins. Although dagA expression is tightly developmentally regulated, the cDNA restores normal development when constitutively expressed in transformed mutant cells. In addition, the megabase region surrounding the dagA locus was mapped. We hypothesize that CRAC acts to connect free G protein beta gamma subunits to adenylyl cyclase activation. If so, it may be the first member of an important class of coupling proteins.

Adenylyl Cyclases↗
PubMed1992

Differentiation-inducing-factor dechlorinase, a novel cytosolic dechlorinating enzyme from Dictyostelium discoideum.

Differentiation-inducing factor 1 (DIF-1) is a dichlorinated alkyl phenone (1-[(3,5-dichloro-2,6-dihydroxy-4-methoxy)phenyl]hexan-1-one) from Dictyostelium discoideum, that induces amoebae to differentiate into stalk cells. It was shown previously that DIF-1 is rapidly metabolized into a series of more polar compounds by living cells [Traynor, D. & Kay, R.R. (1991) J. Biol. Chem. 266, 5291-5297]. The first step in DIF-1 metabolism is the formation of DIF metabolite 1 (now known to be DIF-3) by a monodechlorination. We report here the discovery of the enzyme activity catalyzing this dechlorination. A very sensitive enzyme assay was developed, using [3H]DIF-1 and a TLC system to separate DIF-1 from the product, DIF-3. DIF-1 3(5)-dechlorinase is present in the high-speed supernatant of cell lysates, and uses glutathione, at physiological concentrations, as cofactor. Kinetic measurements indicate a Km for DIF-1 of about 70 nM. The enzyme activity is inhibited by DIF-2 (the pentan-1-one analogue of DIF-1), with a median inhibitor concentration (IC50) of 1 microM, and DIF-3 (IC50 = 5 microM), which presumably act as substrates, but other compounds structurally related to DIF-1 were much less effective. Aurothioglucose, an inhibitor of selenocysteine enzymes, inhibited DIF-1 3(5)-dechlorinase with IC50 = 100 nM. DIF-1 3(5)-dechlorinase activity is developmentally regulated. It is essentially absent from growing cells and increases at the end of aggregation to reach a first peak of activity at the first finger stage, with a further rise at culmination.

Animals↗
PubMed1992

DIF-1 induces its own breakdown in Dictyostelium.

DIF-1 is a novel chlorinated alkyl phenone which induces differentiation of prestalk cells in Dictyostelium discoideum. It is broken down and inactivated by a cytoplasmic enzyme, DIF-1 3(5)-dechlorinase (hereafter referred to as DIF-1 dechlorinase), which is found only in prestalk cells. We show that DIF-1 dechlorinase levels are induced at least 50-fold when cells are treated with DIF-1. This response is rapid--enzyme activity doubles within 15 min and is fully induced within an hour--and occurs early in development, before other prestalk markers can be induced by DIF-1. Maximum inducibility is seen towards the end of aggregation, when DIF-1 dechlorinase is barely detectable in uninduced cells. The dose-dependence reveals a threshold concentration of DIF-1 (15 nM) below which almost no response is seen. Cyclic AMP, which is the chemoattractant during aggregation and plays a key role in later development, suppresses the induction of DIF-1 dechlorinase by DIF-1. We conclude that induction of DIF-1 dechlorinase is one of the first steps on the developmental pathway which leads to prestalk cell differentiation, and suggest that the resulting negative feedback on DIF-1 levels is an important part of the mechanism by which cells decide whether to become prestalk or prespore cells.

Animals↗
PubMed1992

A G-rich sequence element common to Dictyostelium genes which differ radically in their patterns of expression.

Removal of a G-rich element from the DIF-inducible, prestalk-, and stalk-specific ecmB gene reduces expression but cell-type specificity is retained. The ecmB element will functionally substitute for a homologous sequence upstream of CP2, a cAMP-inducible gene and is bound by GBF, the factor which interacts with the CP2 G box. These results suggest that the G box may play a similar stimulatory role in these two independently regulated genes where it presumably interacts with different ancillary promoter elements.

Animals↗
PubMed1992

Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium.

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.

Adenylyl Cyclases↗
PubMed1990

A specific DIF binding protein in Dictyostelium.

Differentiation Inducing Factor (DIF-1), a small chlorinated organic molecule which is produced during Dictyostelium development, is believed to be the morphogen that controls the stalk-specific pathway of differentiation. We describe the identification and characterization of a protease-sensitive activity from cell lysates which binds tritiated DIF-1 with the properties expected of a DIF receptor. Scatchard and linear subtraction plots show a single class of binding sites, of high affinity (Kd = 1.8 nM) and low abundance (1100 sites/cell). The activity elutes from heparin-agarose as a single peak. Various DIF-1 analogues compete for binding in proportion to their activities in a stalk cell differentiation bioassay. The amount of binding activity is developmentally regulated, peaking shortly before the appearance of the prestalk-prespore pattern and before the developmental rise in DIF concentration; the rise occurs at the same time that prestalk-specific genes become DIF inducible. Addition of cyclic AMP to aggregated cells, which induces post-aggregative gene expression in general, also induces the binding activity.

Carrier Proteins↗