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P Chardin

Publications and source records attributed to P Chardin.

At least 19 recordsLinked to original sources
PubMed1996

Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation.

Lethal toxin (LT) from Clostridium sordellii is one of the high molecular mass clostridial cytotoxins. On cultured cells, it causes a rounding of cell bodies and a disruption of actin stress fibers. We demonstrate that LT is a glucosyltransferase that uses UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in vitro and in vivo. LT glucosylates Ras, Rap, and Rac. In Ras, threonine at position 35 was identified as the target amino acid glucosylated by LT. Other related members of the Ras GTPase superfamily, including RhoA, Cdc42, and Rab6, were not modified by LT. Incubation of serum-starved Swiss 3T3 cells with LT prevents the epidermal growth factor-induced phosphorylation of mitogen-activated protein kinases ERK1 and ERK2, indicating that the toxin blocks Ras function in vivo. We also demonstrate that LT acts inside the cell and that the glucosylation reaction is required to observe its dramatic effect on cell morphology. LT is thus a powerful tool to inhibit Ras function in vivo.

3T3 Cells↗
PubMed1996

Myristoylation-facilitated binding of the G protein ARF1GDP to membrane phospholipids is required for its activation by a soluble nucleotide exchange factor.

We have investigated the role of N-myristoylation in the activation of bovine ADP-ribosylation factor 1 (ARF1). We previously showed that myristoylation allows some spontaneous GDP-to-GTP exchange to occur on ARF1 at physiological Mg2+ levels in the presence of phospholipid vesicles (Franco, M., Chardin, P., Chabre, M., and Paris, S. (1995) J. Biol. Chem. 270, 1337-1341). Here, we report that this basal nucleotide exchange can be accelerated (by up to 5-fold) by addition of a soluble fraction obtained from bovine retinas. This acceleration is totally abolished by brefeldin A (IC50 = 2 microM) and by trypsin treatment of the retinal extract, as expected for an ARF-specific guanine nucleotide exchange factor. To accelerate GDP release from ARF1, this soluble exchange factor absolutely requires myristoylation of ARF1 and the presence of phospholipid vesicles. The retinal extract also stimulates guanosine 5'-3-O-(thio)-triphosphate (GTP gamma S) release from ARF1 in the presence of phospholipids, but in this case myristoylation of ARF is not required. These observations, together with our previous findings that both myristoylated and non-myristoylated forms of ARF GTP-gamma S but only the myristoylated form of ARFGDP bind to membrane phospholipids, suggest that (i) the retinal exchange factor acts only on membrane-bound ARF, (ii) the myristate is not involved in the protein-protein interaction between ARF1 and the exchange factor, and (iii) N-myristoylation facilitates both spontaneous and catalyzed GDP-to-GTP exchange on ARF1 simply by facilitating the binding of ARFGDP to membrane phospholipids.

ADP-Ribosylation Factor 1↗
PubMed1995

The Grb2 adaptor.

Grb2 is an 'adaptor' protein made of one SH2 and two SH3 domains. The SH3 domains bind to prolinerich motifs in the C-terminal part of the ras exchange factor Sos. Binding of the Grb2 SH2 domain to phosphotyrosine motifs on receptors, or other adaptor proteins such as Shc, recruits this Grb2/Sos complex at the plasma membrane where Sos stimulates nucleotide exchange on ras, then ras activates raf and leads to MAP kinase activation. The structure of Grb2, the precise motifs recognised by its SH2 and SH3 domains, the way Grb2 performs its function, a possible regulation of its association with Sos, and its ability to complex with other proteins in vivo, are discussed.

Adaptor Proteins, Signal Transducing↗
PubMed1995

A soluble protein negatively regulates phospholipase D activity. Partial purification and characterization.

Phosphatidylcholine-specific phospholipase D (PLD) is an important signalling phospholipase in mammalian cells. Recently, PLD activity has been shown to be positively regulated by the GTP-binding protein ARF (ADP-ribosylating factor). In the present work, we document the presence of a factor negatively regulating PLD activity in bovine brain cytosol. The inhibitory factor is characterized as a large protein or a complex of proteins with a molecular mass higher than 300 kDa. Using permeabilized and pre-permeabilized HL-60 cells depleted of their cytosol, we demonstrate that the inhibitor acts on GTP[S]-stimulated PLD activity. This effect is immediate, persistent and dose dependent for GTP[S]-stimulated PLD. Different possibilities for a mechanism of action of the inhibitory factor on the regulation of GTP binding of ARF were investigated. This inhibitory factor is not the guanine-dissociating inhibitor (GDI) for the small G-binding proteins Rho (Rho-GDI), reported to be a PLD inhibitor, since specific antibodies against this protein did not recognize a protein in the peak containing the inhibitory factor for PLD activity. Furthermore, the inhibitory factor does not prevent the binding of GTP[S] to ARF in the presence of HL-60 membranes. This excludes its possible role as an inhibitor of an ARF/guanine exchange factor. The inhibitory factor not only inhibits a pathway of PLD through GTP[S] activation in particular of the small GTP-binding protein, ARF, but it also inhibits PLD activated via either protein kinase C (PKC) or tyrosine kinase activation. The inhibitory factor also decreases PLC activity and this effect seems to be secondary to the inhibition of PLD activity. We discuss a mechanism of action of the inhibitor on PLD and the importance of this enzyme activity for membrane traffic.

Animals↗
PubMed1995

Myristoylation of ADP-ribosylation factor 1 facilitates nucleotide exchange at physiological Mg2+ levels.

Recombinant N-myristoylated bovine ADP-ribosylation factor 1 (myr-rARF1) has been expressed in bacteria and purified to near homogeneity with a high (85%) myristoylation efficiency. Myr-rARF1 and nonmyristoylated rARF1 have been compared with respect to their kinetics of guanine nucleotide exchange and their interactions with phospholipids. Myristoylation is shown to allow the release of bound GDP at physiological (mM) concentrations of Mg2+. GDP dissociation is slow in the absence of phospholipids but is accelerated 2-fold in the presence of phospholipid vesicles. On the contrary, myristoylation decreases 10-fold the rate of dissociation of GTP or guanosine 5'-O-(thiotriphosphate) (GTP gamma S) in the presence of phospholipids. As a result, myr-ARF1 can be spontaneously activated by GTP or GTP gamma S (t1/2 approximately 30 min at 37 degrees C) at 1 mM Mg2+, in the sole presence of phospholipid membranes without the need for a nucleotide exchange factor. In contrast to the nonacylated protein, the GDP-bound form of myr-ARF1 interacts with phospholipids, as demonstrated by its cosedimentation with phospholipid vesicles and its comigration with phospholipid/cholate micelles on gel filtration. The interaction is, however, weaker than for the GTP-bound form, suggesting that only the myristate in myr-ARF1GDP interacts with phospholipids, whereas both the myristate and the amino-terminal hydrophobic residues in myr-ARF1GTP bind to phospholipids.

ADP-Ribosylation Factor 1↗
PubMed1994

Prenylation of Ras proteins is required for efficient hSOS1-promoted guanine nucleotide exchange.

We have studied whether hSOS1, a mammalian guanine nucleotide exchange factors responsible for activating Ras in response to growth factor stimulation, requires post-translational processing of Ras proteins to promote guanine nucleotide exchange. Our results showed that full-length hSOS1 catalyzed guanine nucleotide exchange on prenylated K-Ras(4B) but with a much lower efficiency on unprocessed K-Ras(4B). The apparent Km of hSOS1 for prenylated K-Ras(4B) was 225 (+/- 25) nM with a Vmax of 0.7 (+/- 0.1) mmol/min/mmol. The activity of hSOS1 against unprocessed K-Ras(4B) was too low to measure Km and Vmax. Consistent with these observations, full-length hSOS1 formed a complex with nucleotide-depleted prenylated K-Ras(4B) but not with unprocessed K-Ras(4B). A geranylgeranylated mutant of K-Ras(4B) was an equally good substrate for hSOS1 as wild-type farnesylated K-Ras. Similarly hSOS1 promoted guanine nucleotide exchange on prenylated Ha-Ras but showed minimal activity toward unprocessed Ha-Ras. Neither the polybasic domain of K-Ras (4B) or palmitoylation of Ha-Ras were required for hSOS1-promoted guanine nucleotide exchange. We attempted to identify a minimal region of hSOS1 capable of promoting guanine nucleotide exchange on both prenylated and unprocessed K-Ras. However, a truncated form of hSOS1 comprising only the CDC25 homology domain retained preferential catalytic activity against prenylated K-Ras, whereas the cognate domain from CDC25 was more active against unprocessed K-Ras.

Amino Acid Sequence↗
PubMed1994

Binding of the Grb2 SH2 domain to phosphotyrosine motifs does not change the affinity of its SH3 domains for Sos proline-rich motifs.

Phosphotyrosine peptide binding to Grb2 induces tryptophan fluorescence changes in the Src homology 2 (SH2) domain. Affinities are in the nanomolar range, the Shc peptide having the highest affinity, followed by peptides mimicking Grb2 binding sites on EGF and HGF receptors, the putative sites on insulin and IGF-1 receptors having much lower affinities. Proline-rich peptide binding to the SH3 domains induces fluorescence changes mainly in the C-terminal SH3. Affinities are in the micromolar range, the highest affinity peptides mimicking the first proline-rich motif of the Sos C-terminus. Additional residues before this PVPPPVPP motif provide a minor contribution to the binding, but the two residues after this motif are important and may contribute to specificity. The affinity of each SH3 for each proline-rich motif is too low to account for the high stability of the Grb2-Sos complex, suggesting that Grb2 recognizes other structural features in the Sos C-terminus. Binding of a phosphotyrosine peptide to the SH2 has no effect on the SH3s. Thus the binding of Grb2 to a receptor or to an associated protein phosphorylated on tyrosines is unlikely to activate the exchange factor activity of Sos through a conformational change transmitted from the SH2 to the SH3 domains.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Myristoylation is not required for GTP-dependent binding of ADP-ribosylation factor ARF1 to phospholipids.

Membrane binding of ADP-ribosylation factors (ARFs) is GTP-dependent and seems to require amino-terminal myristoylation. Recently it has been proposed that myristoylation is needed not for the activation of ARF by GTP but for its subsequent association to membranes. Here we show that unmyristoylated bovine ARF1, expressed in bacteria, can be efficiently loaded with GTP gamma S (guanosine 5'-O-(thio)triphosphate) at 1 microM free Mg2+, in the presence of phospholipids. Unmyristoylated ARFGTP gamma S cosediments with phospholipid vesicles and totally binds to phospholipid-cholate micelles, as seen by gel filtration chromatography. We therefore propose that, in vivo, myristoylation is required for the interaction of ARFGDP with its membrane-bound exchange factor rather than for the association of ARFGTP with lipid membranes. Phospholipid-bound ARFGTP gamma S can also stably interact with and activate the catalytic subunit of cholera toxin, suggesting that ARFGTP provides a membrane anchor for cholera toxin and thereby facilitates its access to membrane-bound substrates.

ADP-Ribosylation Factor 1↗
PubMed1993

Interaction between the retinal cyclic GMP phosphodiesterase inhibitor and transducin. Kinetics and affinity studies.

In the retinal cyclic GMP phosphodiesterase (PDE), catalysis by the alpha beta-heterodimer is inhibited in the dark by two identical gamma-subunits and stimulated in the light by the GTP-bearing alpha-subunit of the heterotrimeric G-protein transducin (T beta gamma-T alpha GDP). Two T alpha GTP molecules, dissociated from T beta gamma, bind to and displace the PDE gamma subunits from their inhibitory sites on PDE alpha beta. With GTP gamma S in lieu of GTP, this association becomes persistent. Under physiological conditions, the PDE alpha beta (gamma T alpha)2 active complex stays on the membrane. But in low-salt buffers, it becomes soluble and dissociates into a partially active PDE alpha beta catalytic moiety and two PDE gamma-T alpha GTP gamma S complexes. This indicates that T alpha binds preferentially to PDE gamma. We have studied the interaction of recombinant bovine PDE gamma with purified T alpha in solution or with retinal rod outer segments (ROS) containing both T beta gamma-T alpha GDP and PDE alpha beta gamma 2. When added to dark ROS, recombinant PDE gamma did not bind to inactive PDE alpha beta gamma 2 but extracted T alpha GDP from membrane-bound holo-transducin to form a soluble PDE gamma-T alpha GDP complex. PDE gamma also bound to purified T alpha GDP in solution. The kinetics and affinity of the interaction between PDE gamma and T alpha GDP or T alpha GTP gamma S were determined by monitoring changes in the proteins' tryptophan fluorescence. The Kd's for the binding of recombinant PDE gamma to soluble T alpha GTP gamma S and T alpha GDP are < or = 0.1 and 3 nM, respectively. PDE gamma-T alpha GDP falls apart in 3 s. This slow dissociation means that, in situ, T alpha-PDE gamma cannot physically leave the active PDE alpha beta, since after GTP hydrolysis, an isolated T alpha-PDE gamma complex would dissociate too slowly to allow a fast PDE reinhibition by the liberated PDE gamma. When recombinant PDE gamma was added to PDE that had been persistently activated by T alpha GTP gamma S, reinhibition occurred and T alpha GTP gamma S, complexed to the native PDE gamma, was released, indicating that both had hitherto stayed bound to PDE alpha beta. The mutation W70F does not prevent recombinant PDE gamma from inhibiting PDE alpha beta but diminishes its affinity for T alpha GTP and T alpha GDP 100-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2.

A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling.

Many of the actions of receptor tyrosine kinases are mediated by the protein Ras, including the activation of various downstream serine/threonine kinases and the stimulation of growth and differentiation. The human protein Grb2 binds to ligand-activated growth factor receptors and downstream effector proteins through its Src-homology (SH) domains SH2 and SH3, respectively, and like its homologue from Caenorhabditis elegans, Sem-5, apparently forms part of a highly conserved pathway by which these receptors can control Ras activity. Here we show that the SH3 domains of Grb2 bind to the carboxy-terminal part of hSos1, the human homologue of the Drosophila guanine-nucleotide-releasing factor for Ras, which is essential for control of Ras activity by epidermal growth factor receptor and sevenless. Moreover, a synthetic 10-amino-acid peptide containing the sequence PPVPPR specifically blocks the interaction. These results indicate that the Grb2/hSos1 complex couples activated EGF receptor to Ras signalling.

3T3 Cells↗
PubMed1993

Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor.

In fibroblasts, stimulation of receptor tyrosine kinases results in the activation of the extracellular signal-regulated kinase 2 (ERK2). The major signalling pathway employed by these receptors involves the activation of p21ras and raf-1 kinase. Here we show that in NIH3T3 and rat-1 fibroblasts, elevation of the intracellular cAMP level results in the inhibition of ERK2 activation induced by PDGF, EGF and insulin treatment. Analysis of various signalling intermediates shows that cAMP interferes at a site downstream of p21ras, but upstream of raf-1 kinase. Inhibition by cAMP depends on both the cAMP concentration and the absolute amount of p21ras molecules bound to GTP, suggesting a mechanism of competitive inhibition. Also TPA-induced, p21ras-independent, activation of raf-1 kinase and ERK2 is inhibited by cAMP. We have used the inhibitory effect of cAMP to investigate whether phosphorylation of mSos, a p21ras nucleotide exchange factor, is dependent on the activity of the raf-1 kinase/ERK2 pathway. We found that phosphorylation of mSos, as monitored by a mobility shift, is delayed with respect to p21ras and ERK2 activation and is inhibited by cAMP in a similar cell type- and concentration-dependent manner as the inactivation of ERK2. These results provide evidence for a model of p21ras-directed signalling towards ERK2 that feeds back on mSos by regulating its phosphorylation status and that can be negatively modulated by protein kinase A and positively modulated by protein kinase C action.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

GTP hydrolysis mechanisms in ras p21 and in the ras-GAP complex studied by fluorescence measurements on tryptophan mutants.

We have substituted leucine 56 or tyrosine 64 of p21 ras with a tryptophan. The intrinsic fluorescence of this tryptophan was used as an internal conformational probe for time-resolved biochemical studies of the ras protein. The slow intrinsic GTPase, GDP/GTP exchange induced by the SDC25 "exchange factor", and the fast GTP hydrolysis induced by GAP were studied. Tryptophan fluorescence of mutated ras is very sensitive to magnesium binding, GDP/GTP exchange, and GTP hydrolysis (changes in tyrosine fluorescence of wild-type ras are also observed but with a lower sensitivity). Nucleotide affinities, exchange kinetics, and intrinsic GTPase rates of the mutated ras could be measured by this method and were found to be close to those of wild-type ras. The SDC25 gene product enhances GDP/GTP exchange in both mutants. In both mutants, a slow fluorescence change follows the binding of GTP gamma S; its kinetics are close to those of the intrinsic GTPase, suggesting that a slow conformational change precedes the GTPase and is the rate-limiting step, as proposed by Neal et al. (1990) (Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565). GAP interacts with both mutant ras proteins and accelerates the GTPase of (L56W)ras but not that of (Y64W)ras, suggesting a role for tyrosine 64 in GAP-induced GTP hydrolysis. However, GAP does not accelerate the slow conformational change following GTP gamma S binding in either of the mutated ras proteins. This suggests that the fast GAP-induced catalysis of GTP hydrolysis that is observed with (L56W)ras bypasses the slow conformational change associated with the intrinsic GTPase and therefore might proceed by a different mechanism.

Amino Acid Sequence↗
PubMed1991

The KUP gene, located on human chromosome 14, encodes a protein with two distant zinc fingers.

We have isolated a human cDNA (kup), encoding a new protein with two distantly spaced zinc fingers of the C2H2 type. This gene is highly conserved in mammals and is expressed mainly in hematopoietic cells and testis. Its expression was not higher in the various transformed cells tested than in the normal corresponding tissues. The kup gene is located in region q23-q24 of the long arm of human chromosome 14. The kup protein is 433 a.a. long, has a M.W. close to 50 kD and binds to DNA. Although the structure of the kup protein is unusual, the isolated fingers resemble closely those of the Krüppel family, suggesting that this protein is also a transcription factor. The precise function and DNA motif recognized by the kup protein remain to be determined.

Amino Acid Sequence↗