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Prepublished online as a Blood First Edition Paper on May 13, 2002; DOI 10.1182/blood-2001-12-0193.
PHAGOCYTES
From the Département Réponse et Dynamique
Cellulaires/Biochimie et Biophysique des Systèmes
Intégrés (Unité Mixte de Recherche 5092 Commissariat
à l'Energie Atomique/Centre National de la Recherche
Scientifique/Université Joseph Fourier), Grenoble, France.
A tetracycline-controlled expression system was adapted to the
human promyelocytic HL-60 cell line by placement of the transactivator (tTA-off) sequence under the control of the human EF-1 Transendothelial migration of leukocytes and their
accumulation at sites of inflammation are of particular importance for host defense against invading pathogens. Chemotactic factors, including
the N-formyl peptides, C5a, and interleukin-8 (IL-8), which
bind to specific heterotrimeric G-protein-coupled transmembrane receptors, regulate cell migration and activate microbicidal and cytotoxic functions through the release of proteolytic enzymes from
specific granules and the generation of superoxide anions by the
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Activation
of this enzyme proceeds through a multistep assembly of several
components including the 2 transmembrane subunits of cytochrome
b558 (p22phox and
gp91phox) and 4 cytosolic proteins
(p47phox, p67phox,
p40phox, and the small GTPase
Rac).1,2 The triggering and control of bactericidal
functions require a coordinated activation of several signaling
pathways, namely the activation of tyrosine kinases, phospholipases
(PLC Low-molecular-weight GTPases are key regulators of a wide spectrum of
cellular functions.4-7 The binding of guanine nucleotide regulates all members of the Ras-superfamily. Activation of GTPases through guanosine 5'-diphosphate (GDP)-GTP exchange is promoted by
guanine nucleotide exchange factors (GEFs). In leukocytes, GTPases of
the Rho family (Rho, Rac, and Cdc42) participate in the remodeling of
actin cytoskeleton and in the regulation of cell adhesion. In addition
to its contribution as part of the oxidase enzymatic complex, Rac also
appears to be connected with upstream signaling mechanisms regulating
NADPH oxidase activation. p21-activated kinase (PAK), a direct target
of Rac, has been shown to phosphorylate p47phox in vitro in a
Rac-GTP-dependent manner8 and to regulate the
stress-activated kinase p38 SAPK,9 which has been involved
in the signal transduction pathway leading to superoxide
production.10,11 Like Rho, Rac and Cdc42 appear to
interact with distinct regulators and downstream target proteins that
may allow them to contribute to unique cellular functions. However, the
understanding of their mechanism of signaling in neutrophils remains
limited, mainly because the expression of exogenous proteins in these
cells is particularly difficult to perform.
The human promyelocytic HL-60 cell line provides a model system to
study the regulatory mechanisms involved in triggering and control of
neutrophil bactericidal functions. Appropriate expression vectors and
transfection methods have been developed that allow the successful
expression of exogenous proteins in this cell line.12,13
Yet, the yield of transfection is low, excluding transient
transfection. Because of clonal variations, the effects produced by the
stable expression of dominant-interfering proteins are often difficult
to interpret. Moreover, overexpression of exogenous proteins may
severely upset metabolic pathways, making stable cell lines difficult
to establish. The tetracycline-regulated system for tightly controlled
expression of exogenous gene products described by Gossen and
Bujard14 appears to be ideally suited to circumvent these
general problems.
In the present paper, we describe the adaptation of such a
tetracycline-controlled expression system to HL-60 cells. Using luciferase as a reporter gene, we show that the system allows a strong
expression that can be tightly controlled in a tetracycline-responsive manner. This conditional expression system was used to decipher the
chemoattractant-activated signaling pathways that lead to NADPH oxidase
activation. A dominant-inhibitory form (Cdc42N17) and a constitutively
active form (Cdc42V12) of Cdc42 were conditionally expressed. The
expression of Cdc42V12 had no major increasing effect on
chemoattractant-mediated NADPH activity and calcium mobilization,
whereas Cdc42N17 was found to be an effective inhibitor that appeared
to down-modulate specific parallel signaling pathways far upstream of
NADPH oxidase. It interfered with PLC Plasmid constructions
A new transactivator plasmid, pEF-tTA, was constructed by inserting the
fragment BamHI-EcoRI of pUHD15-1 between the
BamHI and NotI sites of the modified pEF-pgk-neo
plasmid. The luciferase reporter plasmid pUHC13-3 was modified by
inserting a SalI-NruI fragment of the plasmid
pCEP4 (Invitrogen, Groningen, The Netherlands) containing the
hygromycin resistance gene into the HpaI site.
To construct the response plasmid pTet-Bsd-IRES-EGFP, the
XhoI-XbaI fragment of the pUHD10-3 plasmid was
inserted into the vector pcDNA3.1/hygro(-) (Invitrogen) after cleavage
by NruI and XbaI. The hygromycin resistance gene
was excised with NaeI and replaced by an
EcoRV-SmaI fragment of the plasmid pEF/Bsd
(Invitrogen) encoding the blasticidin resistance gene. A
BamHI site located at the end of the Tet operator was
eliminated using the Quick change site-directed mutagenesis kit
(Stratagene, La Jolla, CA). A BamHI-NotI fragment
of the plasmid pIRES2-enhanced green fluorescence protein (EGFP)
(Clontech, Palo Alto, CA) containing an internal ribosomal entry site
(IRES) sequence and the coding sequence for EGFP were inserted between
the BamHI and PmeI sites in the multicloning site
of the mutated plasmid.
The myc-tagged Cdc42N17 and Cdc42V12 cDNAs provided by Dr P. Chavrier (Paris, France) were subcloned in the multicloning site of the
response plasmid pTet-Bsd-IRES-EGFP, between the NotI and BamHI sites.
Generation of stable cell lines expressing the transactivator
tTA
Expression of tTA-regulated gene products A selected tTA-expressing clone was stably transfected with the myc-Cdc42N17- or myc-Cdc42V12-IRES-EGFP expression plasmids. Selection was performed in the presence of blasticidin (10 µg/mL). Resistant clones were analyzed for EGFP expression by using a FACScalibur flow cytometer (Becton Dickinson) and the Cell Quest analysis program.For detection of myc-tagged proteins, cells lysates were subjected to immunoprecipitation with anti-c-myc antibodies (9E10; Roche Diagnostics, Meylan, France) coupled to protein-G Sepharose beads. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting was performed with specific antibodies to Cdc42 (sc-87; Santa Cruz Biotechnology, Santa Cruz, CA) and the enhanced chemiluminescence detection system (ECL) (Amersham, les Ulis, France). Superoxide production assays HL-60 cell differentiation was induced with N,6O-2'-dibutyryl adenosine 3',5' cyclic monophosphate (Bt2cAMP) or, when specified, with dimethyl sulfoxide (DMSO).16 Superoxide production was assayed by following cytochrome c reduction upon the application of N-formyl Met-Leu-Phe-Lys-OH (fMLFK) or phorbol 12-myristate 13-acetate (PMA) (1 µM and 1 µg/mL, respectively).16A cell-free NADPH activation assay was performed with sonicated membranes, and cytosolic fractions were prepared from bovine neutrophils17 and from HL-60 cells16 differentiated with Bt2cAMP in the presence (50 ng/mL) or the absence of doxycycline. Superoxide generation was measured in 96-well microplates.18 Recombinant myc-Cdc42N17 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. After purification by affinity on glutathione Sepharose beads, the myc-Cdc42N17 protein was enzymatically cleaved from GST.19 [35S]GTP  S binding to Rac was measured by
adding 1 µM [35S]GTPS to the cell-free system
preincubation mixture. GTPS-loaded Rac was pulled down with a
bacterially synthesized GST-fusion of the PAK1 Rac-binding
domain.20
In vivo phosphorylation of p47phox and p40phox HL-60 cells were grown and differentiated in the presence (50 ng/mL) or absence of doxycycline and were subjected to metabolic labeling with [33P] orthophosphate16 Immunoprecipitations of p47phox and p40phox were performed as described.16,21 After SDS-PAGE, 33P-labeled proteins were detected by autoradiography. The presence of p47phox and p67phox was assayed in lysates of unlabeled cells using specific antibodies22 and ECL.IP3 formation, cytosolic calcium mobilization, and kinase assays Inositol-1,4,5-trisphosphate (IP3) level was determined using an [3H] IP3 radioreceptor assay kit (NEN Life Science Products, Boston, MA). Cytosolic calcium measurements were performed as described.23Akt kinase activity was measured in cell lysates using a nonradioactive Akt kinase assay kit (Cell Signaling Technology, Beverly, MA). Extracellular signal-related kinase 1/2 (Erk1/2) phosphorylation was detected by immunoblotting with a phosphospecific antibody, p-ERK (E4) (sc-7383; Santa Cruz Biotechnology). Total Erk2 in lysates was estimated by immunoblotting with anti-Erk2 antibodies (sc-154; Santa Cruz Biotechnology) and ECL. Rac2-GTP and Ras-GTP pull-down GTP-bound Rac2 was pulled down with a bacterially synthesized GST fusion of the PAK1 Rac-binding domain.20 Similarly, the amount of Ras-GTP was measured by using a GST fusion of the Ras-binding domain of Raf1.24 Cell lysis and pull-down assays were performed as described.20 Rac2 and Ras were detected by ECL after immunoblotting with specific antibodies to human Rac2 (sc-96; Santa Cruz Biotechnology) and to human H-, K- and N-Ras (Ab3; Oncogene Research Products, Cambridge, MA).Ras-GRF cellular distribution Cells, differentiated in the presence (50 ng/mL) or absence of doxycycline, were stimulated for various periods of time with fMLFK (1 µM) at 37°C. After subcellular fractionation,16 solubilized membrane fractions were submitted to SDS-PAGE and immunoblotting was performed with a polyclonal antibody to Ras-GRF (sc-963; Santa Cruz Biotechnology).
Adaptation of a tetracycline-controlled expression system to the human promyelocytic HL-60 cell line The tTA-off sequence14 was placed under the control of the promoter of the human EF-1 chromosomal gene. This
promoter has previously been shown to efficiently stimulate
transcription in myeloid cells.12 Luciferase was used as a
reporter gene to assay for the functional expression of the
transactivator and for the tetracycline control of target gene
expression. Two clones were found to express a functional
transactivator. As reported for other mammalian cells, the tTA-off
transactivator tightly controlled protein expression in a
tetracycline-responsive manner in HL-60 cells. Luciferase activity
dropped by 3 orders of magnitude for tetracycline concentrations
ranging from 20 to 100 ng/mL (Figure 1A).
Luciferase activity could be reinduced after tetracycline removal
(Figure 1B). However, several passages were required for full
reinduction when doses of tetracycline greater than 50 ng/mL had
been applied.
This system offers the advantage of comparing the effect of an exogenous protein by simply turning on and off its expression, thereby avoiding comparison with independent clones that may not have the same genetic background. Furthermore, the expression of potentially toxic proteins can be maintained at a moderate level by the presence of appropriate doses of tetracycline. In the following study, the tetracycline analogue doxycycline, which is more efficient than tetracycline, was used to repress the system. To alleviate the laborious analysis of positive clones and to identify clones with the highest level of expression, a response plasmid was used that allows the bicistronic expression of both the protein of interest and the EGFP. It has been previously shown that the expression of EGFP in neutrophils does not affect NADPH oxidase activity or chemotaxis.25 Conditional expression of dominant-inhibitory and constitutively active forms of the GTPase Cdc42 tTA-expressing HL-60 cells, referred to as parental cells, were used to investigate the effects produced in intact cells by the expression of a dominant-inhibitory (N17) or a constitutively active (V12) mutant of the Rho-GTPase Cdc42, a candidate molecule for the regulation of myeloid cell microbicidal and cytotoxic functions. Preliminary experiments indicated that the expression of the Cdc42 mutants did not affect cell growth. Cells could thus be cultured in the absence of doxycycline during the selection step. FACS was used to analyze resistant clones for fluorescence marker expression. As illustrated in Figure 2A for a Cdc42N17-transfected clone, the fluorescence of expressing cells was much above the background fluorescence of parental cells. When EGFP expression was turned off by the addition of doxycycline, the whole cell population was uniformly shifted to low fluorescence levels. An overall 10-fold induction in the absence of doxycycline could be estimated, with more than 90% of the induced cells expressing levels of EGFP that were never reached in the population of uninduced cells.
Cdc42 mutants were expressed as myc-tagged proteins. Immunoprecipitation with anti-myc antibodies and Western blot analysis using an antibody to human Cdc42 were used to directly monitor myc-Cdc42N17 or myc-Cdc42V12 expression. As shown in Figure 2B for selected clones, cells grown in the absence of doxycycline expressed a high level of the exogenous proteins. Their expression was almost undetectable in cells grown in the presence of doxycycline, reflecting a relatively tight control of the system. Densitometric quantification of the immunoblots indicated at least a 20-fold increase in exogenous protein expression in induced cells. Phenotype induced by the expression of the Cdc42N17 dominant-inhibitory mutant Promyelocytic human leukemia HL-60 cells differentiate to a granulocytic cell type phenotype and acquire maturation markers when treated by Bt2cAMP or DMSO. Following maturation into granulocytes, HL-60 cells express NADPH oxidase components and produce superoxide upon chemoattractant application. Although p22phox, p40phox, and Rac2 are detected in promyelocytes, gp91phox and the cytosolic proteins p67phox and p47phox appear only after myelocyte stages and can be considered differentiation markers.26,27 Likewise, the N-formyl peptide and C5a receptors are expressed in HL-60 cells only after differentiation to neutrophil-like cells.28 Western blot analysis showed that the differentiation of HL-60 cells was unaffected by the expression of Cdc42N17 or by the presence of doxycycline. Although the maturation markers p67phox and p47phox were hardly detectable in undifferentiated cells, they were expressed at the same levels in cells grown in the presence or absence of doxycycline (Figure 3A). Uninduced and induced differentiated cells displayed roughly the same number of N-formyl peptide or C5a receptors at the cell surface in radioactive ligand-binding assays (data not shown).
Phosphorylation of the cytosolic factor p47phox is thought to be a key step in agonist-mediated NADPH oxidase activation.2 It has been shown that recombinant p47phox prephosphorylated by protein kinase C (PKC) can specifically activate NADPH oxidase in a cell-free system.29 Additionally, p40phox takes up phosphate when NADPH oxidase is activated,22 but the exact role of p40phox in enzyme activation remains controversial. We thus examined whether p47phox and p40phox were differentially phosphorylated in cells expressing Cdc42N17. Immunoprecipitations of p47phox or p40phox from lysates of cells metabolically labeled with [33P] orthophosphoric acid indicated that the chemotactic peptide fMLFK induced a comparable increase in the phosphorylation of p47phox (Figure 3B) and p40phox (Figure 3C), regardless of whether cells expressed Cdc42N17. When assayed for NADPH oxidase activity, differentiated parental
tTA-expressing cells responded equally well to chemoattractants whether
they had been grown in the presence or absence of doxycycline (data not
shown), confirming that doxycycline had no effect on cell
differentiation or cellular function. The response of parental cells to
fMLFK was better observed after differentiation with 1 mM
Bt2cAMP, whereas the response to PMA was more potent when cells were differentiated with DMSO. Transfected cells grown in the
presence of 50 ng/mL doxycycline produced an amount of
superoxide that was similar to that produced by the parental cells,
whether they were differentiated with Bt2cAMP and
stimulated with fMLFK (Figure 4A, left
panel) or differentiated with DMSO and stimulated with PMA (Figure 4B,
left panel). The expression of Cdc42V12 did not markedly potentiate
chemoattractant-mediated NADPH oxidase activity (116% ± 21% of the
response observed in uninduced cells; n = 6). In contrast, when
Cdc42N17-transfected cells were grown and differentiated in the absence
of doxycycline, cells did not respond at all to either agonist. The
same phenotype was observed when cells were stimulated with the C5a
anaphylatoxin or with the synthetic peptide WKYMVM, an agonist for the
N-formyl peptide receptor homologue FPRL130
(data not shown). Dose responses for doxycycline showed that the
oxidative response correlated with the dose-dependent expression of
Cdc42N17 (Figure 4A-B, right panels). The inhibition of fMLFK- and
PMA-stimulated superoxide production occurred roughly at the same
levels of Cdc42N17 expression, indicating that there was no clear
differential sensitivity of the 2 pathways.
To assess the functionality of the NADPH oxidase complex, superoxide
production was assayed by measuring cytochrome c reduction in a
cell-free reconstitution system after the addition of GTP Thus, although the GTP-bound form of Cdc42 has been shown to be ineffective at directly activating NADPH oxidase in a superoxide-generating cell-free system31 and Cdc42V12 expression was not found to increase NADPH oxidase activity in intact cells, Cdc42N17 expression unexpectedly interfered with the production of superoxide. Cdc42N17 expression does not affect PI3K/Akt signaling pathway Studies performed with knock-out mice lacking the isoform of
phosphatidylinositol-3 kinase (PI3K) have shown that PI3K is the
sole isoform involved in chemoattractant-induced superoxide production
in myeloid cells.32-34 This isoform can be directly activated by G protein  subunits in vitro.35 Taken
together, these results link G protein-coupled receptor stimulation to
the formation of phosphatidylinositol-3,4,5-trisphosphate and the activation of the pathways regulated by phosphoinositides bearing phosphate in the D3 position. In addition, PI3K deficiency results in a lack of chemoattractant-mediated activation of
Akt.34
To examine the impact of Cdc42N17 expression on the PI3K
Cdc42N17 interferes with agonist-induced intracellular calcium mobilization, Ras/MAPK signaling, and Rac2 activation The exposure of neutrophils to chemoattractants generates multiple intracellular second messengers and signaling molecules through the activation of phospholipases and protein kinases.3,36 These include isoforms of phosphatidylinositol-specific phospholipase C (PLC2/3), the aforementioned PI3K, protein kinase C,
mitogen-activated protein kinases p44/42 (Erk1/2),11,37
and p38 SAPK.10,38,39 The strict requirement of PLC in
superoxide generation has recently been established with mice lacking
the 2 PLC2/3 isoforms.33,34 Moreover, recent studies
have shown that both Cdc42 and Rac can stimulate the activity of
PLC2 in vitro40 and of PLC1 in RBL-2H3 mast
cells.41,42 These 2 studies provide evidence that small G
proteins may interact at an early step in receptor-mediated intracellular signaling.
To evaluate a possible effect of Cdc42N17 on the signaling pathway
mediated by PLC
We next investigated whether the expression of Cdc42N17 had an effect
further downstream in the chemoattractant-mediated signaling pathway.
Erk1/2 activation was examined after immunoblotting of lysates from
stimulated cells with antibodies that specifically recognize the
phosphorylated forms of both kinases. As shown in Figure
7, fMLFK triggered rapid and transient
(10 to 30 seconds) Erk1/2 phosphorylation in cells grown and
differentiated in the presence of doxycycline. Upon stimulation with
PMA, transient Erk1/2 phosphorylation was also observed but with a
different time course (1 to 5 minutes). With both agonists, Erk2 was
the predominant phosphorylated species. However, neither fMLFK nor PMA
was able to induce Erk1/2 activation in cells expressing Cdc42N17. This
suggests that the dominant-inhibitory form of Cdc42 interferes upstream
of the MAP kinases.
An activation pathway has emerged in which G
Similarly, we used the Rac-binding domain of PAK1, which associates
exclusively with the GTP-bound state of Rac, to isolate the active form
of Rac2 from stimulated cells.20 Rac2 is the major isoform
present in differentiated HL-60 cells.27 As shown in
Figure 9, in cells grown and
differentiated in the presence of doxycycline, fMLFK induced a rapid
and transient conversion of Rac2 to the GTP-bound active state, which
culminated at 10 to 20 seconds. In the absence of doxycycline, the
basal level of Rac2 in its GTP-bound form (no fMLFK) was consistently
lower, and fMLFK-mediated Rac2 activation was markedly delayed. The
application of PMA to uninduced cells also stimulated GTP loading of
Rac2, but the kinetics of activation was much slower and more
sustained. The expression of Cdc42N17 led to a drastic inhibition of
PMA-mediated Rac2 activation.
Expression of Cdc42N17 prevents Ras-GRF recruitment to the membrane fraction A consensus has been reached that small GTPase dominant-inhibitory mutants work in cells by competing with their normal counterparts for binding to their specific GEFs. Because many GEFs for the Rho family GTPases (Rho, Rac, and Cdc42) activate more than one Rho family member, Cdc42N17 is expected to block the activation of Rac by directly interfering with and sequestering common GEFs. Impaired GTP-loading on Ras may also result from the sequestration of a GEF common to Cdc42/Rac and Ras. However, in a recent study, Cdc42N17 has been shown to inhibit translocation to the membrane of Ras-GRF1, a brain-restricted GEF for Ras but not for Cdc42.46,47 The activity of Ras-GRF1 is inhibited by the GDP-bound form of Cdc42 but is unaffected by its GTP-bound form,47 suggesting an indirect regulation of this GEF. Similarly, in differentiated HL-60 cells, the activation of Ras-GRF2, a more ubiquitous isoform that has been shown to be a GEF for Ras and Rac but not for Cdc42,48 could be indirectly regulated by Cdc42. When activated, Ras-GRFs translocate to the membrane.The subcellular localization of Ras-GRF2 was therefore examined in
differentiated HL-60 cells when the expression of Cdc42N17 was either
induced or repressed. Induced and uninduced cells expressed the same
amount of Ras-GRF2 (not shown). On stimulation with fMLFK, Ras-GRF2
rapidly and transiently translocated to the membrane of uninduced cells
(Figure 10). In cells expressing
Cdc42N17, translocation did not occur.
In the present study, we show that the conditional expression of Cdc42N17 in neutrophil-like differentiated HL-60 cells has a dramatic inhibitory effect on chemoattractant-induced superoxide production. This inhibitory effect is not attributable to a loss of function of any of the components that form the active NADPH oxidase complex. On cell activation, p47phox is phosphorylated
and translocates to the plasma membrane. It functions as an adapter
protein that promotes the binding of p67phox,
Rac, or both to the flavocytochrome b558. PKC is at least
one of the enzymes able to convert p47phox to a
functional molecule.29,49,50 The respective contribution of each PKC isoform has not been completely elucidated. Although an
antisense approach has shown that PKC Rac binds to p67phox, to the flavocytochrome b558, and to the membrane through distinct regions.55-60 The recent crystal structure of a fragment encompassing amino acids 1 to 203 of p67phox in complex with Rac-GTP60 reveals significant differences in the way p67phox interacts with Rac, in comparison with other structures of Rho family effector complexes. Although Cdc42 does not normally activate NADPH oxidase,31 it becomes active by replacing amino acids 27 and 30 within the effector loop by the corresponding residues of Rac.61 The crystal structure clearly shows that Gly-30 in Rac is indeed part of the protein-protein interface. The replacement in Rac of Ala-27 by the lysine present in Cdc42 leads to a steric clash of the lysine side chain with the p67phox structure, thereby explaining the specific interaction of Rac and p67phox. In addition, p67phox fragment 1-192 inhibits Rac-mediated membrane ruffling but does not inhibit the Cdc42-mediated development of filopodia and RhoA-mediated stress fiber formation.56 The Cdc42 binding to p67phox observed initially62 has been proved to be an artifact caused by the presence in the assay of a Cdc42-binding PAK-like protein.56 Purified recombinant Cdc42N17 did not inhibit the cell-free NADPH oxidase system, indicating that the inhibition of NADPH oxidase activity when Cdc42N17 is expressed did not result from a direct competition between Cdc42N17 and Rac for the formation of an active complex. Tight temporal control of Rac activation is apparently required for its
contribution to NADPH oxidase activation. In cells expressing Cdc42N17,
there is no N-formyl peptide-mediated superoxide production
in spite of the GTP-loading on Rac2 at later time points (Figure 9).
The expression of Cdc42N17 seems to have adverse effects on superoxide
generation through interference at multiple levels in the signaling
pathways downstream of chemoattractant receptors. The second
messenger-generating enzyme PLC GTPase N17 mutants produce their inhibitory effects through several mechanisms. They either interfere with the endogenous counterpart for the activation of a downstream effector or sequester an upstream positive regulator, such as a GEF. In the latter case, endogenous GTPase is maintained in a GDP-bound inactive conformation. In fact, the Rho family GTPases are switched to their active conformation by a large number of GEFs that are not always selective for a particular GTPase.4,65,66 Thus, a given dominant-negative GTPase can sequester more than one GEF and produce pleiotropic effects.67 The Cdc42N17 mutant may also produce an inhibitory effect by interfering with GEF activation through a reduced DAG formation and calcium release. For instance, FGD1, a GEF specific for Cdc42, and Vav, which activates multiple Rho family proteins, share a potential DAG-binding domain.66 Because they may thus be regulated by phorbol esters, the inhibition of the PMA-induced NADPH oxidase and the Rac activation we observed are likely to be related to the interference of Cdc42N17 at the GEF level. Phosphorylation and membrane localization of Tiam1, a GEF for Rac1 and Cdc42, are induced by the activation of calcium-calmodulin kinase II and PKC either by ionomycine or by stimulation of lysophosphatidic acid, bombesin, or bradykinin receptors.68,69 Likewise, calcium signaling regulates Ras-GRF1, a brain-restricted GEF for Ras, linking G protein-coupled receptors to the activation of the Ras/MAPK cascade.70 Ras-GRF1 activation is mediated by calmodulin that binds to a calmodulin-binding motif. The widely distributed homologue Ras-GRF2, which activates both Ras and Rac but not Cdc42, seems to be regulated by similar mechanisms.48 Thus, if one assumes that Ras-GRF2 is involved in chemoattractant-mediated signaling to Ras or Rac, a reduced calcium increase may participate in the impairment of Rac and Ras GTP-loading and MAPK cascade activation in Cdc42N17-expressing cells. This view is consistent with the observation that an ionomycin-induced calcium influx restores superoxide production in these cells. In addition, recent studies47 have shown that Cdc42 controls the translocation of Ras-GRF1 to the plasma membrane, but a direct interaction between Ras-GRF1 and Cdc42 has not been observed. In differentiated HL-60 cells expressing Cdc42N17, the inhibition of Ras GTP-loading and Ras-GRF2 translocation | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
), 10 ng/mL (
), 20 ng/mL (
), 50 ng/mL
(
), and 100 ng/mL (
). At indicated times, aliquots containing the
same amounts of cells were withdrawn and assayed for luciferase
activity. No change in growth behavior or cell morphology occurred for
any concentration of antibiotic. (B) Cells maintained in the presence
of doses of tetracycline that repress luciferase expression were washed
twice with phosphate-buffered saline and recultured in the absence of
tetracycline. At indicated times, aliquots containing the same amount
of cells were withdrawn and assayed for luciferase activity. A
substantial increase in luciferase activity could be detected only
after several passages when cells had been treated with 100 ng/mL
tetracycline (
including adapter proteins, a guanine exchange factor, and the small GTPase Ras
phosphorylates specific residues in
p47phox and participates in the signaling
cascade between the N-formyl peptide receptor and NADPH
oxidase activation.
1 or PLC
-RI) signaling to the exocytosis of
secretory granules by interacting with the PLC