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*
Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Boston, MA 02115;
Boston Biomedical Research Institute, Boston, MA 02114;
Department of Pulmonary Diseases, University Hospital Utrecht, Utrecht, The Netherlands;
§
Arthritis Unit, Massachusetts General Hospital, Boston, MA 02114; and
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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63 and 69 kDa (
-
and 
-Pak). Previous studies have shown that products of
phosphatidylinositol 3-kinase and tyrosine kinases are required for the
activation of Paks. We now report that a variety of structurally
distinct compounds which interrupt different stages in
calcium/calmodulin (CaM) signaling block activation of the 63- and
69-kDa Paks in fMLP-stimulated neutrophils. These antagonists included
selective inhibitors of phospholipase C
(1-[6-((17
-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione),
the intracellular Ca2+ channel
(8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate),
CaM (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide;
N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine),
and CaM-activated protein kinases
(N-[2-(N-(chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide).
This inhibition was dose-dependent with IC50 values very
similar to those that interrupt CaM-dependent reactions in vitro. In
contrast, less active analogues of these compounds
(1-[6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione;
N-(6-aminohexyl)-1-naphthalenesulfonamide;
N-(4-aminobutyl)-1-naphthalenesulfonamide; promethazine;
2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine])
did not affect activation of Paks in these cells. CaM antagonists
(N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide;
trifluoperazine), but not their less-active analogues
(N-(6-aminohexyl)-1-naphthalenesulfonamide;
promethazine), were also found to block activation of the small GTPases
Ras and Rac in stimulated neutrophils along with the extracellular
signal-regulated kinases. These data strongly suggest that the
Ca2+/CaM complex plays a major role in the activation of a
number of enzyme systems in neutrophils that are regulated by small
GTPases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and -Pak; Refs.
1, 2, 3, 4, 5) and certain mitogen-activated protein kinase (MAPK)
cascades (e.g., extracellular signal-regulated kinases (ERK-1/2),
p38-MAPK; Refs. 6, 7, 8, 9, 10). Paks are Ser/Thr protein kinases
that undergo autophosphorylation/activation upon interacting with the
active (GTP-bound) forms of the small GTPases(p21) Rac or Cdc42
(11). Activation of the Paks in neutrophils can be blocked
by inhibitors of heterotrimeric G proteins (pertussis toxin) (2, 5), phosphatidylinositol 3-kinase (PI 3-K; i.e., wortmannin, LY
294002) (12), and tyrosine kinases (13, 14).
Paks or Pak-like kinases contain binding sites for the -subunits
of complex G proteins (15), guanine nucleotide exchange
factors (GEF; i.e., Pak-interacting exchange factors)
(16), and adaptor proteins (Nck) (17, 18).
Paks may directly interact with the cytoskeleton through p95
paxillin-kinase linker, which binds directly to both Pak-interacting
exchange factors and the focal adhesion adaptor protein paxillin
(19). Thus, Paks may be capable of integrating messengers
from a number of signal transduction pathways.
Paks can participate in a broad range of cellular events that include
rapid cytoskeletal responses, activation/potentiation of several
distinct MAPK cascades and apoptosis (for review see Ref.
20 and 21). Recent studies have implicated
Pak in the activation of NF-
B in macrophages (22),
tumor growth (23) and the pathogenesis of HIV (24, 25). Paks can also catalyze the phosphorylation in vitro of both
the 47- and 67-kDa subunits of the superoxide
(O2-)-generating system of
phagocytic leukocytes (NADPH-oxidase) (5, 26). However, it
is not known whether Paks participate in the phosphorylation of these
oxidase subunits in vivo.
Recent studies have shown that heterotrimeric G proteins can activate Ras, Src family tyrosine kinases, and the extracellular signal regulated kinases (ERKs-1/2) through a variety of signal transduction pathways (27, 28). One such pathway contains phosphatidylinositol-specific phospholipase C (PI-PLC) and the Ca2+/calmodulin (CaM) complex as major components in the activation of Src and Ras (28). We have recently demonstrated that D-erythro-sphingosine blocks activation of the 63- and 69-kDa Paks in neutrophils if added to the cells either before or after stimulation with fMLP (29). Interestingly, D-erythro-sphingosine can inhibit a number of enzymes activated by the Ca2+/CaM complex (30).
In this paper, we describe the effects of selective antagonists of PLC, the intracellular Ca2+ channel, the Ca2+/CaM complex, and CaM-activated protein kinases (CaM-PK) on the activation of Paks in neutrophils. The data indicate that the Ca2+/CaM complex has a major role in regulating the Paks in these cells along with other enzyme systems activated by small GTPases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide
(W-7);
N-(6-aminohexyl)-1-naphthalenesulfonamide
(W-5); N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide
(W-13); N-(4-aminobutyl)-1-naphthalenesulfonamide (W-12);
1-[6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione
(U-73122);
1-[6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione
(U-73343);
8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate
(TMB-8);
N-[2-(N-(chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide
(KN-93); and
2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine]
(KN-92) were purchased from Calbiochem (La Jolla, CA).
Purified mouse mAbs to human Ras was obtained from
PharMingen/Transduction Laboratories (San Diego, CA). A mouse mAb to
full-length human Rac was purchased from Upstate Biotechnology (Lake
Placid, NY). Affinity-purified, rabbit polyclonal Abs that recognized
only the active (doubly phosphorylated) forms of MAP/ERK kinase (MEK)1
and MEK2 (phospho-MEK-1/2
(Ser217/Ser221) Ab) were
obtained from New England Biolabs (Beverly, MA). Affinity-purified,
rabbit polyclonal Abs that recognized only the active (doubly
phosphorylated) forms of ERK (p44/ERK1 and p42/ERK2) were obtained from
Promega (Madison, WI). Affinity-purified rabbit polyclonal Abs that
recognized both the phosphorylated and nonphosphorylated forms of ERK
(p44/42 (ERK-1/2) MAPK Abs) were also purchased from New England
Biolabs. Goat anti-rabbit IgG labeled with HRP, goat anti-mouse
IgG labeled with HRP, a Super Signal substrate Western blotting kit for
luminol-enhanced chemiluminescence, and an ImmunoPure binding/elution
buffer system for stripping and reblotting Western blots were purchased
from Pierce (Rockford, IL). Sources of all other materials are
described elsewhere (1, 2, 3).
Preparation of neutrophils
Guinea pig peritoneal neutrophils were prepared as described previously (31). These preparations contained >90% neutrophils with viabilities always >90%.
Detection of renaturable protein kinases (Paks) in polyacrylamide gels
Paks and certain other protein kinases were detected directly in gels by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate fixed within a gel that corresponds to amino acid residues 297–331 of the 47-kDa protein component of the phagocyte oxidase (p47-phox). This technique was performed as described elsewhere (2) except the amount of cells was reduced to 3 x 106/ml.
Detection of activated Rac and Ras
Activated forms of Rac or Ras were measured in neutrophil lysates by their ability to bind specifically to GST-fusion proteins containing the Cde42/Rac interactive binding (CRIB) domain of Pak1b (crib domain, amino acid residues 56–227) (GST-Pak-CRIB) or the Ras-binding domain of Raf (GST-Raf-RBD) coupled to glutathione agarose beads (32, 33). The beads were subsequently isolated, washed, and subjected to SDS-PAGE as described previously (32, 33). Specific binding of Rac to the GST-Pak crib fusion protein was determined by Western blotting with a specific Ab to this small GTPase. Conditions for Western blotting are described below. Fusion proteins were prepared as described previously (32, 34).
Immunoblotting/detection of activated ERKs and MEK in stimulated neutrophils
Neutrophils (3 x 106/ml) were stimulated and lysed as described (1). Aliquots of these samples were separated by SDS-PAGE (35 µg/lane) on 9.0% (v/v) polyacrylamide slab gels and transferred electrophoretically to Immobilon-P membranes as described (1). Activated ERK and MEK were assayed by Western blotting with Abs that recognized only the activated (doubly phosphorylated) forms of these kinases (35). Activated kinases and the small GTPases were visualized with a luminol-enhanced chemiluminescence detection system (Pierce), which monitored the activity of HRP bound to the secondary Ab (35). Membranes were incubated with the primary Ab against 1.0 µg/ml Rac or Ras (1:500 dilution) for 1 h at room temperature. All other Ab dilutions and conditions for Western blotting are detailed by Huang et al. (14).
In certain experiments (Fig. 6
), products of the chemiluminescence
detection system were removed by washing the membranes two times (10
min/wash) with TBST (20 mM Tris-HCl (pH 7.4) containing 150 mM NaCl and
0.01% (v/v) Tween 20). These blots were then reprobed with a different
Ab as described above so that both Ags could be visualized
simultaneously (Ref. 35 ; Fig. 6). At the end of these
experiments, both the immunodetection system and the bound Abs were
removed from the blot by incubating the membranes with ImmunoPure
elution buffer (Pierce) for 30–60 min at room temperature followed by
two washes with TBST. The blots were then stained with an Ab that
recognized both the phosphorylated and nonphosphorylated forms of ERK
to confirm that equal amounts of protein were present in each lane of
the gel.
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O2- release from neutrophils was measured as described previously (2).
Analysis of data
Unless otherwise noted, all of the autoradiographic observations were confirmed in at least three separate experiments performed on different cell preparations. The numbers of observations (n) are also based on different cell preparations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils stimulated with the chemoattractant fMLP exhibit rapid
activation of two Paks with molecular masses of 63 and 69
kDa along with two unidentified renaturable protein kinases with masses
of 49 and 40 kDa (Fig. 1;
Refs. 2 and 4). These kinases can be detected
directly in gels by their ability to undergo renaturation and catalyze
the phosphorylation of a peptide substrate fixed within a gel.
Positions of the protein kinases are visualized by autoradiography
after exposure of the gel to [-32P]ATP
(2). The peptide used corresponds to amino acid residues
297–331 of p47-phox and contains several of the phosphorylation sites
of this protein.
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c). In contrast, 50 µM W-13 was only partially effective
in blocking activation of these kinases and 50 µM W-5 lacked activity
(Fig. 1, d and e). Increasing the concentration
of W-13 to 100 µM markedly reduced activation of the 63- and 69-kDa
Paks, whereas the unchlorinated derivative W-12 was inactive even at
this concentration (Fig. 1, f and g).
Fig. 2 presents dose-response data for
blockade of Pak activation by W-7 and several other structurally
diverse, selective inhibitors that block different aspects of
Ca2+ signaling. The antagonists employed
consisted of inhibitors of phospholipase C (PLC) (U-73122)
(37), the intracellular Ca2+channel
(TMB-8) (38), the Ca2+/CaM complex
(trifluoperazine (TFP)) (39), and
Ca2+/CaM-activated protein kinases (KN-93)
(40). These compounds were chosen, in part, because of the
availability of less active analogues to monitor possible nonspecific
effects of the antagonists on cells. The less-active analogues of TFP,
U-73122, and KN-93 are promethazine (PMZ), U-73343, and KN-92,
respectively (37, 39, 40). All of the antagonists listed
above blocked activation of the 63- and 69-kDa Paks in fMLP-stimulated
neutrophils in a dose-dependent manner at their effective
pharmacological doses (Refs. 37, 38, 39, 40, 41, 42 ; see below).
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and 2. Treatment of neutrophils with 50 µM
W-7, 15 µM TFP, 1.0 µM U-73122, 200 µM TMB-8, or 50 µM KN-93
before stimulation with 1.0 µM fMLP for 15 s reduced the content
of 32P in the 63- and 69-kDa bands by 93 ±
3% and 92 ± 3%, 87 ± 14% and 82 ± 17%, 83 ±
12% and 84 ± 10%, 78 ± 14% and 73 ± 25%, and
72 ± 5% and 73 ± 9% (SD, n = 3–5),
respectively. In contrast, similar treatment of the cells with 50 µM
W-5, 15 µM PMZ, 1.0 µM U-73343, or 50 µM KN-92 did not
significantly reduce the content of 32P in the
63- or 69-kDa bands (data not shown; n = 3–5). Addition of 50 µM W-7, 100 µM W-13, 15 µM TFP, 1.0 µM U-73122, 200 µM TMB-8, or 100 µM KN-93 to the phosphorylation step of the "in gel" renaturation assay with the p47-phox peptide substrate did not affect this reaction (n = 2; data not shown). These data strongly indicate that these antagonists did not interact with the 63- and 69-kDa Paks themselves, but on upstream components involved in the activation of the kinases (see Discussion). The concentrations of inhibitors and conditions used did not affect cell viability, as measured by the exclusion of trypan blue or by the release of lactate dehydrogenase from the cells (data not shown; Ref. 37, 38 , and 41).
Neutrophils stimulated with 1.0 µM fMLP release large quantities of
O2- (i.e., 46 ± 9 nmol
O2-/min/107
cells) as a result of the activation of the NADPH-oxidase system
(2). Compounds W-7, TFP, U-73122, and TMB-8 are known to
inhibit O2- release from
stimulated neutrophils (37, 38, 41). The concentrations of
these drugs that blocked O2-
release (Fig. 3) were very similar to
those that inhibited activation of the 63- and 69-kDa Paks (Fig. 2). In
contrast, treatment of neutrophils with (50–100 µM) KN-93 for 15–30
min at 37°C before stimulation with 1.0 µM fMLP had no effect on
O2- release (n
= 4; data not shown) even though this antagonist was effective against
Pak (Fig. 2D).
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The fact that a variety of antagonists of
Ca2+ signaling events were equally effective in
blocking activation of the Paks and NADPH-oxidase complex in
neutrophils suggested that these inhibitors might be effecting a common
component of these systems. As noted above, activated Rac (GTP-bound)
can trigger autophosphorylation/activation of Pak (11).
Rac-GTP is also an obligatory subunit of the NADPH-oxidase system
(43). The effects of CaM antagonists on the activation of
Rac in fMLP-stimulated neutrophils were examined with a fusion protein
containing the p21-binding domain of Pak that binds only the GTP-bound
form of Rac (33, 44, 45). Rac exhibited maximal activation
within 15 s of cell stimulation, followed by significant
inactivation at 3.0 min (Fig. 4A). This pattern of
activation was virtually identical with that exhibited by the 63- and
69-kDa Paks in fMLP-stimulated neutrophils (Fig. 4B; Refs.
2 and 4). Incubation of neutrophils with 50
µM W-7 or 15 µM TFP for 5.0 min at 37°C before stimulation with
1.0 µM fMLP for 15 s blocked activation of Rac, whereas 50 µM
W-5 or 15 µM PMZ did not effect this process (Fig. 4C).
This treatment of neutrophils with W-7, W-5, TFP, and PMZ reduced
activation of Rac by 91 ± 5%, 9 ± 10%, 97 ± 5%,
and 36 ± 8% (SD, n = 3), respectively. Treatment
of neutrophils with 200 nM wortmannin for 10 min at 37°C before
stimulation with 1.0 µM fMLP for 15 s reduced the activation of
Rac by 68 ± 9% (range, n = 2) (Fig. 2D) as reported in previous studies (33, 44).
In contrast, treatment of neutrophils with 50 µM KN-93 for 30 min at
37°C before stimulation with 1.0 µM fMLP for 15 s did not
effect the activation of Rac (n = 2; Fig. 2D) .
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Previous studies have shown that CaM antagonists can either block
(27, 28) or prolong (46) the activation of
ERKs in various cell types. Because ERKs are effector proteins for the
small GTPase Ras, we examined the effects of CaM antagonists on the
activation of Ras, MEK, and ERK in neutrophils (Figs. 5 and 6).
Previous studies have shown that neutrophils stimulated with fMLP
exhibit rapid activation of Ras (8) and ERK-1/2
(6, 7, 8, 9, 10, 35). We confirmed the activation of Ras in guinea
pig neutrophils stimulated with fMLP using a fusion protein that
contained the Ras binding domain of Raf coupled to glutathione agarose
beads. This fusion protein binds only the activated, GTP-bound form of
Ras (32). Ras exhibited maximal activation within 15–30 s
followed by significant inactivation at 1.0–3.0 min (Fig. 5A). This activation of Ras was insensitive to 200 nM
wortmannin (n = 3; data not shown). Treatment of
neutrophils with 50 µM W-7 or 15 µM TFP for 10 min before
stimulation with 1.0 µM fMLP for 30 s markedly reduced the
activation of Ras, whereas compounds W-5 (50 µM) and PMZ (15 µM)
had little or no effect on this reaction (Fig. 5B). This
treatment with 50 µM W-7, 50 µM W-5, 15 µM TFP, or 15 µM PMZ
reduced the activation of Ras by 80 ± 18%, 10 ± 9%,
97 ± 5%, and 16 ± 27% (SD, n = 3),
respectively.
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1.0–3.0 min
after stimulation of these cells with fMLP (35). Previous
studies have shown that MEK-2 is the predominant isoform of this kinase
in human neutrophils and undergoes activation in fMLP-stimulated cells
(47). Data presented in Fig. 6 demonstrate the effects of
CaM antagonists on the activation of ERK-1/2 and MEK in neutrophils.
The cells were incubated with the antagonists for 10 min at 37°C and
then stimulated with 1.0 µM fMLP for 3.0 min. A single blot was first
stained for activated ERKs (Fig. 6A) and then stained again
for activated MEK (Fig. 6B). As was the case with Ras, 50
µM W-7 and 15 µM TFP markedly reduced the activation of ERKs and
MEK in these cells whereas 50 µM W-5 and 15 µM PMZ had little or no
effect. Visualization of MEK by the chemiluminescence detection system
required a longer reaction time than that needed for p42-ERK (Fig. 6A), which resulted in the p42-ERK band being over-exposed
in Fig. 6B. Treatment of the cells with 50 µM W-7, 50 µM
W-5, 15 µM TFP, and 15 µM PMZ reduced the activation of p42-ERK by
96 ± 4%, 21 ± 10%, 100 ± 0%, and 19 ± 26%
(n = 2–3), respectively. These data were calculated by
densitometry from blots stained for p42-ERK alone as shown in Fig. 6A. Thus, the Ca2/CaM complex plays a
major role in activating the small GTPases Ras and Rac in neutrophils
along with their downstream effectors (e.g., Paks, NAPDH-oxidase,
ERKs). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is not possible to employ the techniques of molecular biology to
investigate the involvement of the Ca2+/CaM
complex in Pak activation in primary neutrophils because these cells
are short-lived. Therefore, particular care was taken to employ
structurally distinct antagonists, which blocked different stages in
triggering the increase in cytosolic Ca2 and the
subsequent target/effector proteins. The choice was also restricted to
those compounds for which less active analogues were available. The
order of effectiveness of the CaM antagonists in blocking activation of
Paks (i.e., TFP > W-7 > W-13 > PMZ, W-5, and W-12)
(Figs. 1 and 2) was identical with that reported for CaM-dependent
enzymes in vitro (39). Although TFP and W-7 can also
inhibit protein kinase C (PKC) at high concentrations
(52), PKC is not involved in activating the Paks in
neutrophils (1, 4, 5). Moreover, the concentrations of TFP
and W-7 required for blockade of Pak activation (i.e., 10
and 25 µM, respectively) (Fig. 2) were very similar to that observed
for CaM-dependent processes (39) and severalfold less than
that required to inhibit PKC (52). The
IC50 values for TFP, W-7, W-5, and PMZ blocking
the CaM mediated activation of cAMP phosphodiesterase in vitro are
2–10, 28, 240, and 200–340 µM, respectively (39). The
concentrations of TFP, W-7, U-73122, KN-93, and TMB-8 routinely used to
block CaM, CaM, PLC, CaM-PK, and the intracellular
Ca2a channel in vivo are 10, 35,
1.0, 20, and 100–300 µM, respectively (37, 38, 39, 42).
Each of these antagonists were effective at blocking activation of the
Paks in neutrophils at these doses (Fig. 2). In contrast, the
antagonists W-7 (50 µM) and TFP (15 µM) did not effect the
increases in Ca2+ permeability or cytosolic
Ca2+ that occur in fMLP-stimulated neutrophils
(Refs. 38 and 53 ; data not shown), which
indicates that these inhibitors are selective for certain pathways and
do not have a general disruptive effect on the fMLP-receptor.
The GEF that promotes activation of Rac (and hence Pak) in
fMLP-stimulated neutrophils is blocked by antagonists of PI 3-K and
tyrosine kinases (33, 44). Only the -isoform of PI 3-K
is activated when neutrophils are stimulated with fMLP
(49, 50, 51). Unlike other isoforms of PI 3-K, the -isoform
is directly activated by -subunits of complex G proteins and is
not activated by binding to tyrosine phosphorylated proteins
(49, 50, 51). Thus, the tyrosine kinase involved in the
activation of Rac is not likely to be "upstream" of PI 3-K. One
possible explanation for these data is that the relevant GEF requires
both products of PI 3-K and tyrosine phosphorylation for activation
(Fig. 7). Interestingly, Vav, a GEF for
Rac, undergoes enhanced phosphorylation/activation by a Src-related
kinase when bound to PIP3 (54).
Location of Vav or a similar GEF upstream of Rac would account for the
sensitivity of the Pak stimulatory pathway to pertussis toxin,
wortmannin, and herbimycin.
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presents a
modified portion of that model (28). The ability of CaM
antagonists to block activation of Ras and ERK in neutrophils (Figs. 5 and 6) is consistent with a similar stimulatory pathway being present
in these cells. Addition of a GEF to this pathway, which is
synergistically activated by PI 3-K and tyrosine phosphorylation as
described above, will account for all of the data presented herein and
in earlier publications (2, 3, 4, 5, 12, 13, 14). This modified
model predicts that the Ca2+/CaM complex is
required for activation of a tyrosine kinase that lies upstream of the
GEF for Rac. Thus, most (see below) Ca2+/CaM
antagonists block activation of Pak (Figs. 1 and 2) by preventing
activation of Rac (Fig. 4). Activation of the Src-related kinase Lyn by
a mechanism dependent upon Ca2/CaM has been
described in macrophages (55). This scheme can also
explain why agonists that only increase intracellular
Ca2 in neutrophils (thapsigargin, ionophore
A23187) do not trigger activation of Pak (1, 2, 4) because
-subunits of complex G proteins are also required to effectively
activate PI 3-K and thus Rac in these cells. Activation of Tiam1, a GEF
for Rac, is catalyzed by CaM-PK and blocked by KN-93 in fibroblasts
(42). The inability of KN-93 to block activation of Rac in
fMLP-stimulated neutrophils (Fig. 4D) suggests that Tiam1 is
either not involved in this reaction in neutrophils or its activation
under these circumstances does not require CaM-PK. In contrast to studies reported in this paper and elsewhere (33, 44), a previous paper has reported that activation of Rac in human neutrophils is insensitive to an antagonist of PI 3-K (10 µM LY294002) and genistein (45). The "pull-down" assay used to monitor activated Rac only measures the fraction of this GTPase that is accessible to the fusion protein and may not detect membrane associated Rac or Rac that forms high affinity complexes with other effector proteins (44). Differences in assay conditions (e.g., detergents, incubation times) can alter the amount of "protected Rac" and could account for some of the discrepancies in the literature. A corollary of this situation is that the pull-down assay for Rac and the renaturation assay for Pak may monitor different populations of Rac.
It is noteworthy that KN-93, but not KN-92, blocked activation of the
Paks in neutrophils (Fig. 2D) but did not effect the
activation of Rac (Fig. 4D) or
O2- release (see
Results). Thus, KN-93 blocks activation of Pak by a
mechanism different from that of W-7 and TFP. KN-93 does not bind to
CaM but competes for the CaM binding site on CaM-PK (40).
The concentrations of KN-93 that blocked activation of Paks (Fig. 2)
were similar to those that inhibit CaM-PK in other cell types
(40, 42). The question as to whether a CaM-PK may be
involved in the activation of Pak (e.g., by direct phosphorylation?) is
currently under investigation.
In summary, we provide evidence that the Ca2/CaM complex plays a major role in activation of the Paks and ERKs in neutrophils through stimulation of their upstream effectors Rac and Ras. Identifying the direct target(s) of CaM in these pathways may markedly increase our knowledge of the GEFs in neutrophils that control a variety of cellular responses.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John A. Badwey, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Thorn Building, Room 703, 75 Francis Street, Boston, MA 02115.
3 Abbreviations used in this paper: Pak, p21-activated protein kinase; CRIB, Cdc42/Rac interactive binding domain; PI 3-K, phosphatidylinositol 3-kinase; CaM, calmodulin; PI-PLC, phosphatidylinositol-specific phospholipase C; CaM-PK, CaM-activated protein kinases; ERK; extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAP/ERK kinase; p47-phox, the 47-kDa protein component of the phagocyte oxidase; O2-, superoxide; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; W-5, N-(6-aminohexyl)-1-naphthalenesulfonamide; TFP, trifluoperazine; PMZ, promethazine; PKC, protein kinase C; GEF, guanine nucleotide exchange factor; W-13, N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide; W-12, N-(4-aminobutyl)-1-naphthalenesulfonamide; U-73122, 1-[6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione; U-73343, 1-[6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione; TMB-8, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate; KN-93, N-[2-(N-(chlorocinnam-yl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide; KN-92, 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine].
Received for publication April 14, 2000. Accepted for publication November 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit with a conserved sequence in Ste20/PAK family protein kinases. Nature 391:191.[Medline]
B activity by multiple signaling pathways requires Pak 1. J. Biol. Chem. 275:19693. in thymocyte development, T cell activation and neutrophil migration. Science 287:1040.2 and -3 and PI3K in chemoattractant-mediated signal transduction. Science 287:1046. in inflammation. Science 287:1049.This article has been cited by other articles:
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  Y.-P. Ching, V. Y.L. Leong, M.-F. Lee, H.-T. Xu, D.-Y. Jin, and I. O.-L. Ng P21-Activated Protein Kinase Is Overexpressed in Hepatocellular Carcinoma and Enhances Cancer Metastasis Involving c-Jun NH2-Terminal Kinase Activation and Paxillin Phosphorylation Cancer Res., April 15, 2007; 67(8): 3601 - 3608. [Abstract] [Full Text] [PDF]
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 E. A. Woolfolk, S. Eguchi, H. Ohtsu, H. Nakashima, H. Ueno, W. T. Gerthoffer, and E. D. Motley Angiotensin II-induced activation of p21-activated kinase 1 requires Ca2+ and protein kinase C{delta} in vascular smooth muscle cells Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1286 - C1294. [Abstract] [Full Text] [PDF]
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 U. K. Misra, T. Sharma, and S. V. Pizzo Ligation of Cell Surface-Associated Glucose-Regulated Protein 78 by Receptor-Recognized Forms of {alpha}2-Macroglobulin: Activation of p21-Activated Protein Kinase-2-Dependent Signaling in Murine Peritoneal Macrophages J. Immunol., August 15, 2005; 175(4): 2525 - 2533. [Abstract] [Full Text] [PDF]
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P. C. Chu, J. Wu, X. C. Liao, J. Pardo, H. Zhao, C. Li, M. K. Mendenhall, E. Pali, M. Shen, S. Yu, et al. A Novel Role for p21-Activated Protein Kinase 2 in T Cell Activation J. Immunol., June 15, 2004; 172(12): 7324 - 7334. [Abstract] [Full Text] [PDF]
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Q. Zhan, Q. Ge, T. Ohira, T. Van Dyke, and J. A. Badwey p21-Activated Kinase 2 in Neutrophils Can Be Regulated by Phosphorylation at Multiple Sites and by a Variety of Protein Phosphatases J. Immunol., October 1, 2003; 171(7): 3785 - 3793. [Abstract] [Full Text] [PDF]
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), TFP (
), W-7 (
) and
TMB-8 (
) for 5.0 min at 37°C and then stimulated with 1.0 µM
fMLP. O2- was assayed as referenced in
Materials and Methods. Data points represent mean values
of two to three different experiments.
1.0 min (dotted arrow) is p90RSK2 .
C, Effects of various antagonists on the activation of
Rac. Cells were treated with the inhibitors for 5.0 min at 37°C and
then stimulated with 1.0 µM fMLP for 15 s. Lane a
is for unstimulated neutrophils. Lane b is for
stimulated cells. Lanes c–f are for stimulated
neutrophils treated with the following: 50 µM W-7 (c),
50 µM W-5 (d), 15 µM TFP (e), and 15
µM PMZ (f). D, Effects of various
antagonists on the activation of Rac. Cells were incubated at 37°C
with or without inhibitors for 10 min (lanes a–c) or 30
min (lanes d–g) and then stimulated with 1.0 µM fMLP
for 15 s. Lanes a and d are for
unstimulated cells. Lanes b and e are for stimulated cells.
Lanes c, f, and g are for stimulated
cells treated with the following: 200 nM wortmannin (c),
50 µM KN-93 (f), and 50 µM KN-92
(g).
