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J D Lambeth

Publications and source records attributed to J D Lambeth.

At least 37 records · Page 2Linked to original sources
PubMed1997

A peptide with unique receptor specificity: stimulation of phosphoinositide hydrolysis and induction of superoxide generation in human neutrophils.

Previously, we identified peptides that stimulate phosphoinositide hydrolysis in several leukocyte cell lines from mixtures of random hexapeptide sequences. Moreover, the peptides activate phospholipase C via a pertussis toxin-sensitive G protein-coupled receptor. We now investigate the structure-activity relationship of the peptides with the goal of improving the activity of the peptides, as well as the biologic function of the peptides. Substitution of the L-methionine at the C terminus of peptides with D-methionine markedly increased the effectiveness of the peptides. The half-maximal effective concentrations of MKYMPm-NH2 and WKYMVm-NH2 for stimulation of phosphoinositide hydrolysis in U266 cells were 30 and 0.5 nM, respectively. By BIAcore analysis we confirmed the existence of a receptor for WKYMVm-NH2. Furthermore, the intracellular calcium concentration increase induced by WKYMVm-NH2 was not inhibited by several chemoattractants (FMLP, IL-8, platelet-activating factor, C5a, granulocyte-macrophage CSF, and granulocyte CSF) suggests that WKYMVm-NH2 has a unique cell surface receptor on leukocytes. WKYMVm-NH2 stimulated the phosphoinositide hydrolysis in U937, HL60, and U266 cells, as well as in human neutrophils. Moreover, WKYMVm-NH2 is more effective than FMLP in the production of superoxide in human neutrophils. The data suggest that WKYMVm-NH2 may have the ability to activate the microbicidal functions of human neutrophils.

Amino Acids↗
PubMed1996

Membrane association of Rac is required for high activity of the respiratory burst oxidase.

NADPH-dependent superoxide generation can be reconstituted in a cell-free system using recombinant cytosolic factors (p47-phox, p67-phox, and Rac) plus flavocytochrome b558. Rac1 and Rac2 are closely related small GTPases, differing primarily in the C-terminal 10 residues where Rac1 but not Rac2 contains a polybasic sequence. In their nonisoprenylated forms, Rac1 was highly effective in reconstituting NADPH oxidase activity (low EC50, high Vmax), whereas Rac2 was only minimally effective (high EC50, low Vmax). In contrast, low concentrations of isoprenylated Rac1 and Rac2 both supported high rates of superoxide generation. Like full length Rac2, truncated forms of both Rac1 and Rac2 in which the C-terminal 10 residues were eliminated were poorly activating, pointing to the C terminus of Rac1 as a determinant of activity. Mutation of single positively charged residues in the C terminus of nonisoprenylated Rac1 markedly reduced its ability to support superoxide generation, affecting both its EC50 and the Vmax. In contrast, mutation or truncation of the C terminus failed to affect the activation of PAK, a Rac-regulated protein kinase. The EC50 for Rac1 increased with increasing salt concentrations, whereas that of Rac2 was independent of salt, implicating the involvement of electrostatic forces for the former. Using flavocytochrome b558 reconstituted into phosphatidylcholine vesicles, the EC50 for Rac1 but not Rac2 decreased (increased binding) when an acidic phospholipid (phosphatidylinositol) was present, supporting a role for the Rac1 polybasic C terminus in binding to the membrane. A model in which Rac must associate simultaneously both with p67-phox and with the membrane to activate the NADPH oxidase can account for the above observations.

Amino Acid Sequence↗
PubMed1996

Inhibition of phospholipase D by a protein factor from bovine brain cytosol. Partial purification and characterization of the inhibition mechanism.

A specific protein inhibitor of partially purified bovine brain phospholipase D (PLD) was identified from bovine brain cytosol. The PLD inhibitor has been enriched through several chromatographic steps and characterized with respect to size and mechanism of inhibition. The inhibitor showed an apparent molecular mass of 30 kDa by Superose 12 gel exclusion chromatography and inhibited PLD activity with an IC50 of 7 nM. The inhibitor had neither proteolytic activity nor phospholipid-hydrolyzing activity. Because phosphatidylinositol 4,5-bisphosphate (PIP2), which is included in substrate vesicles, is an essential cofactor for PLD, we examined whether the inhibition might be mediated by sequestration of PIP2. PIP2 hydrolysis by phospholipase C (PLC)-beta1 was not affected by the inhibitor and the inhibitor did not bind to substrate vesicles containing PIP2. In contrast, a PH domain derived from PLC-delta1, which could bind to PIP2, showed a nearly identical inhibition of both PLC-beta1 and PLD activities. Thus, the PLD inhibition by the inhibitor is due to the specific interaction with not PIP2 but PLD. The suppression of PLD activity by the inhibitor was largely eliminated by the addition of ADP-ribosylation factor (ARF) and GTPgammaS. We propose that the inhibitor plays a negative role in regulation of PLD activity by PIP2 and ARF.

Animals↗
PubMed1996

NADPH oxidase activity is independent of p47phox in vitro.

The neutrophil superoxide generating NADPH oxidase is activated by the assembly of cytosolic protein components with a membrane-associated flavocytochrome. The activity can be reconstituted in vitro using purified cytosolic factors p47(phox), p67(phox), and Rac plus the phospholipid-reconstituted flavocytochrome b558. Here, we demonstrate that activity is reconstituted in the absence of p47(phox) when high concentrations of p67(phox) and Rac are used. Vmax values were the same in the presence or absence of p47(phox), yet p47(phox) increases the affinity of both p67(phox) and Rac for the oxidase complex by nearly 2 orders of magnitude. p67(phox)-(1-246), a truncated form of the protein which eliminates SH3 domains involved in binding to p47(phox), also supports superoxide generation, both in the presence and absence of p47(phox), providing further evidence for p47(phox) independent activity. In the absence of p47(phox), p67(phox)-(1-246) binds to the NADPH oxidase complex 3-fold more tightly than does native p67(phox), indicating that the C terminus contains a region which masks binding to the oxidase complex. Results indicate that p47(phox) does not play a direct role in regulating electron transfer. Rather, its function is to serve as an adaptor protein to enhance the assembly of the other cytosolic components with the flavocytochrome and possibly to unmask a binding region in the N terminus of p67(phox) by binding to its C-terminal domains. p67(phox) and/or Rac play a more direct role in regulating electron transfer.

Arachidonic Acid↗
PubMed1996

Rac "insert region" is a novel effector region that is implicated in the activation of NADPH oxidase, but not PAK65.

The small GTPase Rac assembles with the cytosolic p47(phox) and p67(phox) and the membrane-associated flavocytochrome b558 to form the multicomponent respiratory burst oxidase. Mutation of amino acids in a region of Rac (residues 26-45), homologous to an effector region in Ras, was previously shown to interfere with Rac binding to the oxidase. Herein we have elucidated an additional region in Rac involved in regulating oxidase activity. Rho family small GTPases contain a 12-amino acid "insert" region (residues 124-135) that is not present in Ras. Point mutations in and deletion of this region were constructed and used for in vitro studies of the activation of PAK65 and NADPH oxidase. Apparent binding constants (based on EC50 values) of the mutant Rac proteins for the oxidase are at least 13-25-fold higher than for wild-type Rac. Mutations in the insert region versus the 26-45 effector region resulted in distinct kinetic consequences, pointing to different roles for these two protein regions: mutations in the insert region but not the 26-45 effector region resulted in an increase in the EC50 for p67(phox). Although mutations in the 26-45 amino acid effector region showed markedly diminished activation of both PAK and the NADPH oxidase, insert region mutations did not affect activation of PAK. We propose that the combinatorial use of the 26-45 effector region and the insert region provides the Rho family GTPases with versatility in their specificity for several downstream targets.

Amino Acid Sequence↗
PubMed1996

Biochemistry and cell biology of phospholipase D in human neutrophils.

Neutrophils play a major role host defense against invading microbes. Recent studies have emphasized the importance of the phospholipase D (PLD) in the signalling cascade leading to neutrophil activation. Phospholipase D catalyzes the hydrolysis of phospholipids to generate phosphatidic acid with secondarily generation of diradylglycerol; both of these products have been implicated as second messengers. Herein, we discuss the regulation and the biochemistry of the receptor-regulated PLD in human neutrophils. In vivo and in vitro studies suggest an activation mode in which initial receptor-linked activation of phospholipase C generates diacylglycerol and inositol trisphosphate. The resulting calcium flux along with the diacylglycerol activate a conventional isoform of protein kinase C (PKC), probably PKC beta 1. This PKC, in turn phosphorylates a plasma membrane component resulting in PLD activation and a second outpouring of diradylglycerol. The small GTP-binding proteins, RhoA and ARF, also participate in this process, and synergize with a 50 kDa cytosolic regulatory factor.

Animals↗
PubMed1995

RhoA and a cytosolic 50-kDa factor reconstitute GTP gamma S-dependent phospholipase D activity in human neutrophil subcellular fractions.

Receptor activation of phospholipase D has been implicated in signal transduction in a variety of cells. Reconstitution of cell-free guanosine 5'-O-(3-thiotriphosphate)(GTP gamma S)-dependent phospholipase D activity from human neutrophils requires protein factors in both the plasma membrane and the cytosol. We previously proposed that one of the factors is a Ras-family small molecular weight GTPase of the Rho subtype (Bowman, E. P., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509-21512). Herein, we have used RhoGDI (GDP dissociation inhibitor), an inhibitory Rho-binding protein, to selectively extract Rho-type GTPases from the plasma membrane, and have used immunoprecipitation as well as chromatographic methods to remove cytosolic Rho. Depletion of RhoA from either the plasma membrane or the cytosol resulted in a partial loss in GTP gamma S dependent activity, while removal of RhoA from both fractions resulted in a nearly complete loss in activity. Activity was nearly completely restored by adding purified recombinant RhoA, which showed an EC50 of 52 nM, while Rac1 showed little activity. Cytosol fractionated using DEAE-cellulose chromatography separated ADP-ribosylation factor and Rho from the major activating fraction. Gel exclusion chromatography of this fraction revealed an activating factor of 50 kDa apparent molecular mass. Using RhoA-depleted membranes, reconstitution of phospholipase D activity required both RhoA and the 50-kDa factor. Thus, RhoA along with a non-Rho, non-ADP-ribosylation factor 50-kDa cytosolic factor are both required to reconstitute GTP gamma S-dependent phospholipase D activity by neutrophil plasma membranes.

Adult↗
PubMed1995

Regulation of phospholipase D by protein kinase C in human neutrophils. Conventional isoforms of protein kinase C phosphorylate a phospholipase D-related component in the plasma membrane.

In a variety of intact cells, phorbol esters are known to activate phospholipase D. In a cell-free system consisting of plasma membrane and cytosol from human neutrophils, phorbol esters activated phospholipase D in an adenosine nucleotide triphosphate-dependent manner. ATP gamma S (adenosine 5'-O-(thiotriphosphate)) was 2-3-fold more effective than ATP, while ADP and AppNHp (adenyl-5'-yl imidodiphosphate) were ineffective, and activation was blocked by the kinase inhibitor staurosporine. In cytosol deplete of protein kinase C by chromatography on threnoine-Sepharose, phorbol ester-dependent activation was lost, but was restored upon addition of purified rat brain protein kinase C. The target for phosphorylation was shown to be the plasma membrane plasma membrane was phosphorylated using ATP gamma S/phorbol 12,13-dibutyrate and protein kinase C and was reisolated to remove activators. Upon adding nucleotide-depleted cytosol, activator-independent phospholipase D activity was seen. Using this prephosphorylation protocol, PKC-dependent activation of plasma membranes was found to require micromolar calcium, implicating a conventional protein kinase C. Using recombinant isoforms of protein kinase C, only the conventional isoforms showed significant activation, with the following rank order of potency: beta 1 > alpha > gamma; the beta 2, delta, epsilon, eta, and sigma isoforms showed little or no activity. Thus, conventional isoform(s) of protein kinase C activate neutrophil phospholipase D by phosphorylating a target protein located in the plasma membrane.

Adenosine Triphosphate↗
PubMed1995

ADP-ribosylation factor functions synergistically with a 50-kDa cytosolic factor in cell-free activation of human neutrophil phospholipase D.

Proteins in both the cytosol and plasma membrane are needed to reconstitute cell-free phospholipase D activity from phagocytes (Olson, S., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-17242); membrane factors include a small GTP-binding protein in the Rho family (Bowman, E., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509-21512). ADP-ribosylation factor (ARF) was recently implicated as the cytosolic factor, as it activates phospholipase D in HL-60 membranes. Herein, we show that ion exchange chromatography separates ARF from the major phospholipase D-stimulating cytosolic factor. Both bovine brain ARF and recombinant human ARF-1 stimulated a small amount of phospholipase D activity in the absence of cytosol (about 10% of the response seen with cytosol). With a high concentration of ARF-depleted cytosol, ARF did not further activate. However, at low cytosol, ARF caused marked activation. Thus, ARF synergizes with the cytosolic factor in phospholipase D activation.

ADP-Ribosylation Factors↗
PubMed1995

On the mechanism of inhibition of the neutrophil respiratory burst oxidase by a peptide from the C-terminus of the large subunit of cytochrome b558.

A peptide (RGVHFIF) from near the carboxyl terminus (residues 559-565) of gp91-phox, the large subunit of cytochrome b558, was previously shown to inhibit activation of the respiratory burst oxidase [Kleinberg, M. E., Malech, H. L., & Rotrosen, D. (1990) J. Biol. Chem. 265, 15577-15583]. The peptide has been proposed to compete with gp91-phox binding to p47-phox, one of the cytosolic oxidase components. In the present studies, we have used a semirecombinant system consisting of recombinant cytosolic factors (p47-phox, p67-phox, and Rac1) along with isolated plasma membrane to investigate the mechanism by which the peptide inhibits oxidase activation. In an in vitro translocation model, the peptide inhibited arachidonate-activated translocation of both p47-phox and p67-phox to the plasma membrane. The kinetic mechanism of inhibition was examined. Inhibition was noncompetitive or mixed with respect to not only Rac and p67-phox but also to p47-phox. We suggest that the peptide, rather than competing for cytochrome-p47-phox interactions, inhibits indirectly, perhaps by binding to and altering the conformation of cytochrome b558.

Adult↗
PubMed1994

Ras effector-homologue region on Rac regulates protein associations in the neutrophil respiratory burst oxidase complex.

Rac, a small molecular weight GTPase in the Ras superfamily, participates in the activation of the multicomponent superoxide-generating NADPH oxidase of human neutrophils. Rac is 30% identical to Ras overall, but is 75% identical within the sequence corresponding to the effector region of Ras, which regulates mitogenesis through interactions with the protein kinase Raf1. We investigated the role of this region in Rac1 using site-directed mutagenesis. In a cell-free semirecombinant NADPH oxidase system, mutants in the 26, 33, 38, and 45 amino acids showed 20-110-fold reduced binding to the oxidase complex as judged by EC50 values and reduced (44-80%) maximal activities in superoxide generation. Only the GTP gamma S-bound form associated, since the GDP-bound form of Rac neither activated alone nor competed with GTP gamma S-Rac. EC50 values for neither p47-phox nor p67-phox were affected when mutant Racs were used in place of Rac. Data indicate direct binding of the Rac effector region to one or more components of the respiratory burst oxidase. Results indicate a general role for conserved effector-equivalent regions in small GTPases in the regulation of protein-protein interactions.

Amino Acid Sequence↗
PubMed1994

p67-phox enhances the binding of p47-phox to the human neutrophil respiratory burst oxidase complex.

The neutrophil respiratory burst oxidase consists of both the plasma membrane-associated flavocytochrome b558 and cytosolic regulatory proteins including p47-phox, p67-phox, and a small GTP-binding protein (Rac1 and/or Rac2). Oxidase activation is thought to result from the assembly of the cytosolic components on the cytochrome. A model has been proposed in which p47-phox binds directly to the cytochrome, while p67-phox binding occurs indirectly by binding to p47-phox. We have carried out a steady state kinetic analysis using a cell-free semirecombinant activation system (isolated plasma membrane plus recombinant cytosolic factors) to analyze the effects of p47-phox and p67-phox on one another's association in the active oxidase complex. As predicted from the model, increasing p47-phox concentration markedly lowered the EC50 for p67-phox, indicating that p67-phox is dependent upon p47-phox for binding. Unexpectedly, increasing p67-phox concentration also produced a significant enhancement of p47-phox binding. Thus, a more complex binding model must be invoked in which p47-phox and p67-phox mutually enhance one another's binding.

Cytochrome b Group↗
PubMed1994

Participation of the small molecular weight GTP-binding protein Rac1 in cell-free activation and assembly of the respiratory burst oxidase. Inhibition by a carboxyl-terminal Rac peptide.

NADPH-dependent superoxide generation was activated by anionic amphiphiles plus GTP gamma S in a cell-free system consisting of plasma membranes plus recombinant p47-phox, p67-phox, and the small GTP-binding protein Rac1. Rac1 was expressed in Escherichia coli both as the native form and as a mutant form (Rac1(C189S)) lacking the prenylation site. When preloaded with GTP gamma S, both Rac proteins supported activity to a level comparable to that seen using cytosol. A peptide corresponding to the carboxyl-terminal region of Rac1 was used to investigate oxidase assembly and activation. Rac1(178-188), but not several control peptides, inhibited activity. The peptide inhibited competitively (Ki = 15 microM) with respect to Rac1(C189S), while inhibition was noncompetitive or mixed with respect to p47-phox and p67-phox. This indicated specific inhibition of the interaction of the Rac protein with its target, possibly cytochrome b558. The peptide was effective only when added prior to activation with arachidonic acid, suggesting that it affects assembly rather than activity. Consistent with this possibility, the peptide prevented translocation of p47-phox and p67-phox to the plasma membrane. Thus, Rac plays a central role in the assembly of the neutrophil NADPH oxidase.

Amino Acid Sequence↗
PubMed1993

A novel ecto-phosphatidic acid phosphohydrolase activity mediates activation of neutrophil superoxide generation by exogenous phosphatidic acid.

Phosphatidic acid (PA) added to intact cells activates a variety of processes including mitogenesis in fibroblasts and superoxide generation in neutrophils. We have investigated the mechanism of activation of superoxide generation in intact human neutrophils by a short-chain (dioctanoyl) PA (diC8PA). After a lag, diC8PA caused a high rate of superoxide production (19.6 nmol of cytochrome c reduced/min/10(6) cells). Activation did not require extracellular Ca2+ and coincided with near quantitative conversion of diC8PA to dioctanoylglycerol (diC8-glycerol). diC8PA also activated cellular phospholipase D with release of long-chain PA and secondary production of long-chain diradylglycerol (sn-1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol). The metabolism of diC8PA to diC8-glycerol was catalyzed by a novel PA phosphohydrolase on the outer leaflet of the plasma membrane as demonstrated by the exclusive release of Pi into the extracellular medium. This enzyme also showed activity toward PA containing long-chain unsaturated fatty acids. The ecto-PA phosphohydrolase differed from the intracellular PA phosphohydrolase based on its relative insensitivity to desipramine and N-ethylmaleimide. The enzyme was also present in Chinese hamster ovary (CHO) cells and its activity did not change in transfected CHO cells expressing the two membrane-associated isoforms of alkaline phosphatase, indicating that the PA phosphohydrolase was not alkaline phosphatase. Non-hydrolyzable phosphonate analogs of diC8PA poorly stimulated superoxide production. Activation of superoxide generation by diC8PA was inhibited by staurosporine, suggesting a protein kinase C-dependent mechanism. We suggest that the action of a novel ecto-PA phosphohydrolase permits exogenously added short-chain PA to serve as "timed-release diacylglycerol" and that its biological effects in neutrophils are secondary to diacylglycerol-mediated protein kinase C activation.

Animals↗
PubMed1993

Neutrophil phospholipase D is activated by a membrane-associated Rho family small molecular weight GTP-binding protein.

Phospholipase D in human neutrophil lysates is activated by GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)), implying the participation of a GTP-binding protein. Reconstitution of GTP gamma S-dependent activity requires protein factors in both the plasma membrane and the cytosol (Olson, S. C., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-17242). The location of the GTP-binding protein was investigated by preincubating either cytosol or plasma membrane with GTP gamma S, followed by removal of all but tightly bound nucleotide and reconstituting activity with the complementing untreated subcellular fraction. This approach indicated that the GTP-binding protein was membrane-associated. A low magnesium requirement for GTP gamma S prebinding, as well as a failure of aluminum fluoride to activate, suggested a Ras-like small M(r) GTP-binding protein. smg GDP dissociation stimulator, which stimulates the exchange of GDP for GTP on a variety of small GTP-binding proteins, stimulated GTP-dependent phospholipase D activity. Rho GDP dissociation inhibitor, a regulatory protein that binds specifically to and inhibits the functions of Rho family small GTP-binding proteins, inhibited GTP gamma S-dependent activity. Thus, neutrophil phospholipase D is regulated by a membrane-associated small molecular weight GTP-binding protein, likely to be a member of the Rho family.

Adult↗
PubMed1993

Cholesterol trafficking in steroidogenic cells. Reversible cycloheximide-dependent accumulation of cholesterol in a pre-steroidogenic pool.

Peptide hormones activate steroid hormone biosynthesis in responsive tissues by stimulating the delivery of cholesterol to a steroidogenic pool, thought to be located in the inner mitochondrial membrane. At this site, it is metabolized to pregnenolone, the precursor of the steroid hormones, by side-chain-cleaving cytochrome P-450 (cytochrome P-450scc). In the presence aminoglutethimide (an inhibitor of cytochrome P-450scc) and an activating stimulus, cholesterol accumulates in the steroidogenic pool, and increased pregnenolone generation is observed upon removal of the inhibitor. Using Y-1 adrenocortical cells and MA-10 Leydig tumor cells, we now provide evidence for a distinct, functionally relevant cholesterol pool which precedes the steroidogenic pool, which we designate the pre-steroidogenic pool. This pool was defined by activating the cells with 8-bromo-adenosine 3',5'-cyclic monophosphoric acid in the presence of cycloheximide, an inhibitor of steroidogenesis. Following a wash procedure, which removed 8-bromo-adenosine 3',5'-cyclic monophosphoric acid and cycloheximide, augmented pregnenolone synthesis was observed. Unlike synthesis from the steroidogenic pool, pregnenolone formation from pre-steroidogenic pool in Y-1 cells indicates that this pool is somewhat smaller than the steroidogenic pool. The results support a cholesterol-trafficking model in which cycloheximide-sensitive transport from the pre-steroidogenic pool to the steroidogenic pool precedes metabolism, and is regulated by cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

The respiratory burst oxidase of human neutrophils. Guanine nucleotides and arachidonate regulate the assembly of a multicomponent complex in a semirecombinant cell-free system.

We recently characterized a "semirecombinant" cell-free NADPH-oxidase system, comprised of plasma membrane plus the recombinant cytosolic proteins p47-phox and p67-phox, wherein superoxide generation was activated by an anionic amphiphile plus guanosine 5'-O-(2-thiotriphosphate) (GTP gamma S) (Uhlinger, D. J., Inge, K. L., Kreck, M. L., Tyagi, S. R., Neckelmann, N., and Lambeth, J. D. (1992) Biochem. Biophys. Res. Commun. 186, 509-516). Based on preincubation with guanine nucleotides, we show that plasma membrane contains G protein(s) that support oxidase activation at submaximal rates. By varying p47-phox and p67-phox concentrations, kinetic parameters (EC50 and Vmax) for each were determined. For both, GTP gamma S increased the Vmax and decreased the EC50, whereas guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) produced the opposite effect, consistent with the participation of a G protein in an activation complex containing p47-phox and p67-phox. Using [35S]methionine-labeled p47-phox and p67-phox, we investigated the association of these components with both normal plasma membranes and chronic granulomatous disease membranes lacking cytochrome b558. p47-phox translocation was stimulated by arachidonate but not GTP gamma S, was about 50% cytochrome-dependent, and occurred independently of p67-phox. Arachidonate-stimulated translocation of p67-phox required both cytochrome and p47-phox and was enhanced by GTP gamma S. The mass of p47-phox and p67-phox which assembled with cytochrome b558 indicated a ternary complex with a 1:1:1 stoichiometry.

Adult↗