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Biomedical subjects

E G Lapetina

Publications and source records attributed to E G Lapetina.

At least 19 recordsLinked to original sources
PubMed1995

Identification of alpha-subunits of trimeric GTP-binding proteins in human platelets by RT-PCR.

In a search for new alpha-subunits of trimeric GTP-binding proteins in human platelets, we prepared leucocyte-free platelet concentrates and analyzed total RNA for areas homologous to known alpha-subunits. RT-PCR based on two degenerate primers revealed the expected band of 495 base pairs and an additional band of 540 base pairs reflecting the alternative splice product of Gs alpha. Following subcloning in pGEM-T vector and sequencing, we identified the alpha-subunits Gi alpha-2 and Gs alpha-S of the regulating GTP-binding proteins of adenyl cyclase as well as Gz alpha whose function is unknown, confirming earlier immunological identification. In addition, we identified Gs alpha-L (differing from Gs alpha-S by an insertion of 45 base pairs), G16 alpha, (a member of the pertussis toxin insensitive Gq-family), and two new variants of both Gs alpha-S and Gs alpha-L each containing a C-A-G triplet. With G16 we have identified another candidate for pertussis-toxin insensitive signal transduction in platelets. The C-A-G containing sequences of Gs alpha lead to an insertion of a Ser-residue, which results in the consensus sequence of a phosphorylation site for protein kinase C (Ser-X-Lys), making these variants candidates for protein kinase C-sensitive cyclic AMP formation.

Amino Acid Sequence↗
PubMed1995

Regulation of platelet glycoprotein IIb/IIIa (integrin alpha IIB beta 3) function via the thrombin receptor.

Binding sites on glycoprotein (GP) IIb/IIIa exposed by 0.5 unit/ml alpha-thrombin are insensitive to prostaglandin I2 (PGI2), in contrast with sites exposed by ADP or platelet-activating factor. Here we show that the thrombin receptor agonist peptide (TRAP) (SFLLRN; 15 microM) opens almost the same number of GPIIb/IIIa molecules as 0.5 unit/ml alpha-thrombin (64840 +/- 8920 compared with 81050 +/- 6030 molecules of fibronectin bound/platelet), but these sites rapidly close on addition of PGI2. To investigate whether alpha-thrombin and TRAP initiate different signalling pathways, we measured phospholipase C (PLC)-mediated control of GPIIb/IIIa and its sensitivity to cyclic AMP. Optimal concentrations of alpha-thrombin and TRAP activated PLC maximally, but TRAP induced only about 50% protein kinase C PKC) activation after 10 min stimulation compared with alpha-thrombin. These concentrations also suppressed PGI2-induced cyclic AMP accumulation, with alpha-thrombin inducing complete inhibition and TRAP about 10% less. Direct activation of PKC by phorbol 12-myristate 13-acetate confirmed earlier observations that PGI2-induced cyclic AMP accumulation is partly inhibited via PKC. Applying different concentration of alpha-thrombin, TRAP or a combination of alpha-thrombin and the thrombin receptor inhibitory peptide (TRIP) (Mpr-F-Cha-Cha-RKPNDK-NH2; 800 microM) (Mpr, 3-mercaptopropionic acid; Cha, cyclohexylalanine), we show that the different means of stimulating the thrombin receptor all suppressed PGI2-induced cyclic AMP accumulation via (i) activation of PKC and (ii) activation of the heterotrimeric G-protein, Gi. We conclude that complete inhibition of cyclic AMP accumulation requires activation of both PKC and Gi, as observed with 0.5 unit/ml alpha-thrombin. Although TRAP almost fully exposes GPIIb/IIIa, its activation of PKC is incomplete, enabling PGI2 to raise cyclic AMP concentration from 1.4 +/- 0.7 to 4.1 +/- 1.3 nmol/10(11) platelets (P < 0.005) which is sufficient to close exposed GPIIb/IIIa molecules.

Adenosine Diphosphate Ribose↗
PubMed1995

Cyclic AMP-dependent activation of Rap1b.

Rap1 proteins belong to the Ras superfamily of small molecular weight GTP-binding proteins. Although Rap1 and Ras share approximately 50% overall amino acid sequence identity, the effector domains of the two proteins are identical, suggesting either similar or antagonistic signaling roles. Several pathways leading to Ras activation have been defined, including those initiated by agonist binding to tyrosine kinase or Gi-coupled receptors. Nothing is known about such events for Rap1 proteins. The cAMP-mediated inhibition of Ras-dependent MAP kinase activation is well documented and resembles that caused by expression of GTPase-deficient Rap1. We have developed a system whereby signals leading to Rap1b activation, i.e. an increase in Rap1b-bound GTP/GDP ratio, can be measured. We report here that treatment of cells with agents that elevate intracellular cAMP levels result in Rap1b activation. These results demonstrate for the first time agonist-dependent activation of Rap1 proteins.

3T3 Cells↗
PubMed1995

Protein thiol modification of glyceraldehyde-3-phosphate dehydrogenase as a target for nitric oxide signaling.

Nitric oxide signaling is achieved through cGMP-dependent and -independent mechanisms. The latter are exemplified by the NAD(+)-dependent automodification of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The experimental post-translational, covalent modification of the enzyme by [32P]NAD+ is achieved using NO-releasing compounds and an active constitutive or inducible NO-synthase. Potential roles for NO in this covalent enzyme modification can be grouped as follows: S-Nitrosylation of GAPDH by NO+ NAD(+)-dependent, post-translational covalent automodification of GAPDH. Oxidative modification of GAPDH by NO-related compounds, probably ONOO. GAPDH modification by one of the proposed mechanisms would lead to inhibition of enzyme catalysis. It is likely that the NAD(+)-dependent automodification process occurs in vitro, in intact cells, and in whole animals. Besides its normal function in glycolysis, GAPDH not only is a target for NO-mediated direct and indirect modifications but also is ADP-ribosylated in the presence of brefeldin A (90). The relation of such ADP-ribosylation to enzyme activity is so far unknown. GAPDH also may be involved in one of the following functions unrelated to its glycolytic activity (81 and refs. therein; 90): binding and transport of tRNA associated with nuclear localization of GAPDH. DNA-repair activity, i.e., uracil DNA glycosylase. Activation of transcription in neurons. Interaction with tubulin and microtubules. The transport of nitric oxide. Serves as a substrate for brefeldin A stimulated ADP-ribosylation. Because some of these alternative functions of GAPDH, just like NO-mediated modification of the enzyme, are related to the NAD+ binding site of the protein, we are interested in searching for the significance of these activities in relation to NO actions. In recent years, several functions of NO have been linked to direct, cGMP-independent actions. Modification of GAPDH is probably just one interesting target related to NO-redox chemistry and active-site thiol modification. It will be challenging to investigate NO biochemistry in closer detail and to elucidate how NO targets biological systems, especially in relation to the patho-physiological role of NO in medically related conditions.

Amino Acid Oxidoreductases↗
PubMed1995

Nitric oxide-induced apoptosis in RAW 264.7 macrophages is antagonized by protein kinase C- and protein kinase A-activating compounds.

Endogenously generated or exogenously applied nitric oxide (NO) redox species induce apoptotic cell death in murine RAW 264.7 macrophages. Activation of the inducible NO synthase by incubation of cells with a combination of lipopolysaccharide and interferon-gamma produced internucleosomal DNA fragmentation and morphological alterations, i.e., chromatin condensation, indicative of apoptotic cell death. These alterations, reflecting the production of NO, were prevented by an inhibitor of NO synthase, NG-monomethyl-L-arginine. Moreover, NO derived from endogenous or exogenous sources caused accumulation of the tumor suppressor gene p53. Proposing a link between NO generation and DNA fragmentation, we investigated interfering biochemical signaling pathways. Therefore, we tested the ability of four NO-releasing compounds [sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP), and S-nitrosoglutathione (GSNO)] to cause specific DNA fragmentation. All NO donors induced DNA fragmentation in a time- and concentration-dependent manner. However, substance-specific differences became obvious. After an 8-hr incubation period, GSNO proved to be the strongest apoptotic inducer, whereas SIN-1 was much less active. Apoptosis was rapid with GSNO and SNP, yielding specific DNA fragments after 4 hr and 5 hr, respectively. In contrast, SNAP and SIN-1 produced DNA fragmentation after considerable lag times of 9 hr and 14 hr, respectively. Furthermore, an inhibitory effect of protein kinase C (PKC) and cAMP-dependent protein kinase became apparent. 12-O-Tetradecanoylphorbol-13-acetate, an activator of PKC, inhibited DNA fragmentation by all four NO donors, whereas PKC inhibitors such as staurosporine and calphostin C sensitized macrophages to apoptosis induced by SNP and GSNO. Lipophilic cAMP analogues suppressed SNP-, SIN-1, and SNAP-induced DNA fragmentation. Thus, our study suggests the existence of specific down-modulatory mechanisms related to NO-induced apoptotic DNA fragmentation.

Animals↗
PubMed1994

Glycoprotein IIb-IIIa and the translocation of Rap2B to the platelet cytoskeleton.

The stimulation of human platelets with physiological agonists results in the incorporation of several proteins into the cytoskeleton, fibrinogen binding, and platelet aggregation. We recently demonstrated that the Ras-related low molecular weight GTP-binding protein Rap2B associates with the cytoskeleton in activated platelets and that this interaction requires platelet aggregation. In the present study we demonstrate that agonist-induced actin polymerization is necessary for the translocation of Rap2B to the cytoskeleton, suggesting that Rap2B interacts with the newly formed actin filaments. Moreover, the association of Rap2B with Triton X-100-insoluble material from platelets was totally blocked by treatment of intact platelets with monoclonal antibodies against the fibrinogen receptor glycoprotein IIb-IIIa. Platelets from patients affected by Glanzmann thrombastenia, a genetic disorder in which platelet plasma membranes lack glycoprotein IIb-IIIa but possess normal levels of Ras-related proteins, failed to incorporate Rap2B into the cytoskeleton upon activation by thrombin. Comparative immunoblotting revealed that the translocation of Rap2B to the cytoskeleton during platelet aggregation was accompanied by the simultaneous translocation of glycoprotein IIb-IIIa. Moreover, the cytoskeleton from aggregated platelets contained Rap2B and glycoprotein IIb-IIIa in comparable amounts. These results demonstrate the association of Rap2B and glycoprotein IIb-IIIa and their translocation to the cytoskeleton in aggregated human platelets.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

Nitric oxide: a signal for ADP-ribosylation of proteins.

Nitric oxide (NO), a highly reactive gas, is now established as a major messenger molecule regulating blood vessel dilation, immune functions and serving as a neurotransmitter in brain and peripheral nervous system. NO can also act as a tumoricidal and bactericidal molecule. The effect of NO to dilate blood vessels is largely explained by stimulation of soluble guanylate cyclase (a heme-iron containing protein) leading to formation of cGMP and protein phosphorylation. This is considered to be the main physiological signaling mechanism of NO. NO also binds to non-heme iron-containing proteins and this has been considered as a pathophysiological or cytotoxic action of NO. Furthermore, NO, more correctly nitrosonium (NO+) which can be formed by the removal of one electron, reacts with protein SH-groups to cause the S-nitrosylation of proteins. We have recently established a link between NO and the S-nitrosylation and mono-ADP-ribosylation of the enzyme glyceraldehyde 3-monophosphate dehydrogenase, which adds a further protein modification mechanism for NO action. This links the formation of the second messenger molecule NO to post-translational protein modification and adds a new dimension to NO in the communication of intracellular signals.

Adenosine Diphosphate Ribose↗
PubMed1994

Insulin-like growth factor-1-mediated association of p85 phosphatidylinositol 3-kinase with pp 185: requirement of SH2 domains for in vivo interaction.

Insulin-like growth factor-I (IGF-1) stimulates the production of 3-phosphoinositides and increases the phosphatidylinositol 3-kinase activity that is immunoprecipitated by antiphosphotyrosine antibodies, a small portion of which are also associated with the IGF-1 receptor. In vitro reconstitution experiments showed that p85 associates with high affinity to the IGF-1 receptor and this interaction is mediated through the p85 SH2 groups. Moreover, in vitro, p85 is a substrate for the IGF-1 receptor tyrosine kinase activity. In this study, we analyzed the in vivo association of p85 with tyrosyl- phosphorylated proteins and its tyrosyl phosphorylation state, in response to IGF-1. After stimulation with IGF-1, the major tyrosylphosphorylated protein that was associated with p85 was a 185-kilodalton protein, identified as IRS-1. Only a small fraction of p85 was associated with the IGF-1 receptor. In contrast, the PDGF receptor was the major protein associated with p85 upon stimulation. Neither ligand stimulated the tyrosyl phosphorylation of p85 in vivo. In order to determine whether the SH2 domains of p85 were involved in its association with p185 in vivo after IGF-1 stimulation, different SH2-constructs of p85 were expressed in COS-1 cells. After stimulation with IGF-1, the expressed SH2 proteins were immunoprecipitated with specific antibodies, and associated p185 was detected on Western blots. These results show that both the p85 N-SH2 and N+C-SH2 associate with IRS-1 after IGF-1 stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Glyceraldehyde-3-phosphate dehydrogenase is required for the transport of nitric oxide in platelets.

Nitric oxide (NO) or NO-generating compounds like sodium nitroprusside (SNP) increase cellular levels of cGMP and produce S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase [GAPDH; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12]. In search of a reagent that could discriminate between these two effects, we used the sesquiterpene antibiotic koningic acid, which binds to GAPDH at the Cys-149 of the active site. Koningic acid inhibited basal and sodium nitroprusside-stimulated NAD-dependent covalent modification of purified rabbit muscle GAPDH in a dose-dependent manner. Furthermore, we tested the effect of koningic acid on human platelets. Approximately 90% of GAPDH is present in the cytosol of human platelets, and the exposure of platelet cytosol to koningic acid inhibited GAPDH activity, while the soluble guanylyl cyclase (basal and sodium nitroprusside-stimulated) activity remained unaltered. Pretreatment of intact platelets with koningic acid slowed the rate of aggregation induced by a submaximal concentration of thrombin. In addition, the antibiotic also inhibited the cGMP increases triggered by SNP, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosyndomidine (SIN-1) but failed to prevent an increase in cGMP caused by nitrosylated albumin. Under the same conditions, koningic acid also inhibited basal and SNP- SNAP-, and SIN-1-stimulated NAD-dependent modification of GAPDH and its enzymatic activity. These results suggest that the mechanism of delivery of NO from SNP, SNAP, and SIN-1 to platelets may require the active form of GAPDH. When NO is delivered by nitrosylated albumin, active GAPDH was not necessary.

Biological Transport↗
PubMed1993

Association of the low molecular weight GTP-binding protein rap2B with the cytoskeleton during platelet aggregation.

The intracellular distribution of the low-molecular-weight GTP-binding protein rap2B was investigated in resting and agonist-activated human platelets. In both cases, platelets were lysed by Triton X-100, and cell fractions were obtained by differential centrifugations. Using a specific polyclonal antiserum, we found that rap2B in resting platelets was completely detergent-soluble. When platelets were aggregated with thrombin, the thromboxane analogue U46619, or the Ca(2+)-ATPase inhibitor thapsigargin, a significant amount of rap2B became associated with the cytoskeleton. This association was paralleled by a decrease of rap2B in the Triton X-100-soluble fraction. Translocation of rap2B to the cytoskeleton strictly depended on platelet aggregation, and maximal incorporation was found when approximately 50% aggregation was measured. Inhibition of fibrinogen binding to the glycoprotein IIb-IIIa complex completely prevented the interaction of rap2B with the cytoskeleton. These results clearly demonstrate that changes in the intracellular localization of rap2B occur during platelet activation and represent evidence that this low molecular weight GTP-binding protein may be involved in platelet function.

Blood Platelets↗
PubMed1993

Microinjection of Rap2B protein or RNA induces rearrangement of pigment granules in Xenopus oocytes.

Rap2B, a member of the ras superfamily of low-molecular-mass GTP-binding proteins, induced a characteristic rearrangement of the pigment granules in Xenopus oocytes following its microinjection, resulting in numerous unpigmented spots on the animal hemisphere. This phenomenon, termed 'mottling', was also induced by microinjection of in vitro-transcribed Rap2B RNA or of purified recombinant Rap2A. Following the microinjection of Rap2B, more than 90% of the oocytes showed signs of mottling within 10 h. The time course of mottling paralleled the association of the recombinant Rap2B with an oocyte membrane fraction. Like other members of the ras superfamily, Rap2B possesses a C-terminal CAAX motif that serves as a signal for post-translational processing. Mutation of the cysteine residue in the CAAX motif to serine prevents the association of Rap2B with oocyte membranes, and also prevents mottling. This result suggests that post-translational processing of Rap2B is required for the observed effect. Mottling was blocked by boiling Rap2B prior to its microinjection or by co-injection of the cytoskeletal reagent phalloidin.

Animals↗
PubMed1993

Mutational analysis of the cAMP-dependent protein kinase-mediated phosphorylation site of Rap1b.

Rap1b, a member of the Ras superfamily of low molecular weight GTP-binding proteins, can be phosphorylated by cAMP-dependent protein kinase (protein kinase A). The experiments presented here were undertaken to determine the precise site of this phosphorylation. Because the Rap1 proteins are highly homologous, there are no specific antibodies able to discriminate between them. To overcome this problem, we used a transient expression system of a fused protein containing in the NH2 terminus an epitope for a known antibody. Using this system, the transfected protein was expressed at a high level and was localized in a perinuclear structure, as previously reported for the endogenous Rap1 proteins. The mutational analysis of Rap1b revealed Ser179 as the residue involved in the protein kinase A-mediated phosphorylation. The presence of a Lys179 instead of the wild-type Ser179 (resembling the Rap1a sequence) rendered Ser180 a better substrate for phosphorylation caused by protein kinase A. The mobility shift of Rap1b in SDS gels, observed in cells that were stimulated with agonists that increase cAMP, was caused, at least in part, by the phosphorylation of Rap1b.

Animals↗
PubMed1993

Hepatocytes and macrophages express an identical cytokine inducible nitric oxide synthase gene.

Polymerase chain reaction amplification of reverse transcribed RNA (RNA-PCR) was used to isolate overlapping cDNA clones encoding an inducible form of nitric oxide synthase (NOS) from purified rat hepatocytes. NOS gene expression in hepatocytes is dependent upon bacterial lipopolysaccharide (LPS) and cytokine treatment. The predicted amino acid sequence of rat hepatocyte inducible NOS is 94% identical with that from the mouse macrophage cell line, RAW264.7. Differences between the two sequences occur as sporadic amino acid substitutions with no major gaps or insertions. These differences are most likely the result of species variability which suggests that hepatocytes and macrophages express the same NOS gene upon induction.

Amino Acid Oxidoreductases↗
PubMed1993

Prenyl group identification of rap2 proteins: a ras superfamily member other than ras that is farnesylated.

Rap proteins comprise a subset of the large family of ras-related proteins. They contain the C-terminal tetrapeptide sequence motif Cys-Ali-Ali-Xaa (Ali is an aliphatic amino acid and X is any amino acid), which has been found to be the site of membrane attachment via isoprenylation for ras, nuclear lamins and the gamma subunits of the heterotrimeric G-proteins. To investigate the isoprenylation of rap2a and rap2b, human cDNAs coding for these proteins were expressed in COS cells incubated in the presence of [3H]mevalonolactone. Both proteins incorporated a product of [3H]mevalonolactone, as judged by Western blot analysis. To identify the specific isoprenoid attached to each protein, the cDNAs were transcribed in vitro and the rap2 specific RNA was translated in a rabbit reticulocyte lysate system in the presence of [3H]mevalonolactone. The translation products were treated with methyl iodide and the released isoprenoid groups were analysed by h.p.l.c. Rap2b, which terminates in Cys-Val-Ile-Leu, is geranylgeranylated as predicted while rap2a, which terminates in Cys-Asn-Ile-Gln, incorporated farnesyl. A mutant construct generated by site-directed mutagenesis of rap2a cDNA yielding a protein terminating in leucine instead of glutamine incorporated geranylgeranyl, lending further support to the notion that isoprenoid specificity is governed by the terminal amino acid. In addition, when the CAAX motif cysteine at position 180 of rap2a was replaced by a serine residue no isoprenoid incorporation was observed. Thus rap2a and rap2b, despite showing 90% sequence identity, incorporate different isoprenoid groups. Thus glutamine is a signal for farnesylation, and rap2a is the first non-ras member of the ras superfamily that is farnesylated.

Amino Acid Sequence↗
PubMed1992

Agonist-induced association of the p21ras GTPase-activating protein with phosphatidylinositol 3-kinase.

The signal transduction properties of the 21-kDa GTP-binding proteins, encoded by the ras genes, are only partly known. In a recent report, we demonstrated that the signaling pathway of p21ras, like that of several growth factors, is closely associated with phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity. We showed that insulin-like growth factor-1 (IGF-1) and insulin increased the phosphatidylinositol 3-kinase activity in immunoprecipitates obtained with anti-phosphotyrosine and anti-ras antibodies in Ha-ras-transformed epithelial cells. Several findings in this previous study suggested that an additional protein was likely to be associated with the PtdIns 3-kinase. The suggestion that p21ras GTPase-activating protein (GAP) acts not only as a regulator of p21ras activity but also as a direct downstream target in the signaling pathway of p21ras led us to investigate the possible association of PtdIns 3-kinase with GAP. The stimulation of Ha-ras-transformed epithelial cells with IGF-1 caused an increased association of PtdIns 3-kinase activity with GAP, as seen by immunoprecipitation with anti-p21ras and anti-GAP antibodies. The 85-kDa regulatory subunit of PtdIns 3-kinase was present in immunoprecipitates obtained with antibodies against GAP and p21ras of IGF-1 stimulated cells. These data suggest that GAP acts as a downstream target for p21ras via its association with PtdIns 3-kinase.

Animals↗