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INTRODUCTION |
The serine/threonine kinase protein kinase B, also called Akt, is
the cellular homologue of a viral oncogene, v-Akt (1-3). Akt contains
a pleckstrin homology domain at its N terminus, a catalytic domain, and
a short C-terminal tail, and is closely related to protein kinase A and
protein kinase C in its amino- and C-terminal regions. Akt plays a
critical role in mediating cell proliferation, differentiation, and
survival signals propagated from certain growth factors (4, 5). Akt
activation is dependent on phosphatidylinositol 3-kinase
(PI-3K),1 since wortmannin
and dominant negative mutants of PI-3K block Akt activation (6) and
constitutively active mutants of PI-3K activate Akt (7, 8). Activation
of Akt requires that the products of PI-3K, phosphatidylinositol
3,4,5-trisphosphate (PIP3) and phosphatidylinositol
3,4-bisphosphate interact with the pleckstrin homology domain of Akt
and recruit it to the plasma membrane (4, 9, 10, 11). Subsequently, Akt
undergoes phosphorylation at two sites, Thr308 in the
kinase domain and Ser473 in the C-terminal domain.
Phosphatidylinositol 3,4-bisphosphate and PIP3 activate
3-phosphoinositide-dependent kinase-1 (PDK1), which
phosphorylates Thr308 (4, 12). Phosphorylation of
Ser473 is also dependent on products of PI-3K; however, the
identity of this kinase, termed PDK2, is unknown (11, 12).
Cellular stresses, such as heat shock and hyperosmolarity, stimulate
both p38 kinase and Akt activity (13). p38 kinase, a homologue of the
yeast HOG1, is activated by dual phosphorylation on Thr and Tyr within
a Thr-Gly-Tyr motif by MAP kinase kinases MKK3 and MKK6 (14, 15). The
activation of MKK3 and MKK6 is regulated by phosphorylation on Ser and
Thr residues by one of several MAP kinase kinase kinases.
Chemoattractant stimulation and cross-linking of Fc
receptors
stimulate PI-3K-dependent transient activation of Akt and
p38 kinase in human neutrophils (16-18). MAP kinase-activated protein
kinase-2 (MK2), a direct target of p38, has been reported to
phosphorylate Ser473 of Akt in vitro (19), and
activated Akt associates with a substrate of MK2, heat shock protein 27 (Hsp27) (20). Direct regulation of Akt activity by p38 kinase, however,
has not been demonstrated previously. Additionally, Alessi et
al. (19) suggested that a role for the p38 kinase module in Akt
activation was unlikely in intact cells, since IGF-1 activates Akt, but
not MK2, in HEK 293 cells. The present study examined the hypothesis
that p38 kinase regulates Akt activation in human neutrophils. We show for the first time that Akt activation is regulated by PI-3K-mediated p38 kinase activity in intact cells. We also report that Akt forms a
stable complex with p38 kinase, MK2, and Hsp27 and that, upon stimulation with fMLP, Hsp27 dissociates from this complex.
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EXPERIMENTAL PROCEDURES |
Materials--
PD98059, SB203580, wortmannin, and LY294002 were
obtained from Calbiochem. Final concentrations used were 50 µM PD98059, 10 µM SB203580, 100 nM wortmannin, and 100 µM LY294002, except
where otherwise indicated. fMLP and histone H2B were obtained from
Sigma. GST-Akt agarose beads, active recombinant MK2, anti-Akt2, and anti-Akt3 were obtained from Upstate Biotechnology, Inc. (Lake Placid,
NY). Anti-phospho-p38, anti-p38, anti-phosho-Ser473-Akt,
anti-phospho-Thr308-Akt, and anti-Akt antibodies were
obtained from New England Biolabs, Inc. (Beverly, MA). Recombinant
Hsp27 and anti-mouse Hsp27 were obtained from Stressgen Biotechnologies
Corp. (Victoria, Canada). Anti-MK2 was obtained from Research
Biochemicals International (Natick, MA). Anti-Fc
RIIa Fab monoclonal
antibody (IV.3) and Fc
RIIIb F(ab')2 monoclonal antibody
(3G8) were obtained from Medarex (Annandale, NJ). Goat anti-mouse IgG
and goat anti-rabbit IgG were obtained from Vector (Burlingam, CA).
Goat anti-mouse IgG, specific for F(ab')2, was obtained
from Jackson ImmunoResearch Laboratories (West Grove, PA).
PIP3 was obtained from Matreya (Pleasant Gap, PA).
Adenovirus vector and adenovirus containing genes for constitutively
active MKK3 and MKK6 were obtained from Dr. Yibin Wang (University of
Maryland). GST-MK2 was kindly provided by Dr Matthias Gaestel (Martin
Luther University, Halle-Wittenberg, Germany). The synthetic MK2
inhibitory peptide (AFHRAFNRQLANGVAEIR-amine) was obtained from the
Macromolecular Structure Analysis Facility at the University of
Kentucky (Lexington, KY). The synthetic EGFR peptide
(NH2-RRELVEPLTPSGEAPNQALLR-COOH) was obtained from Macromolecular Resources, Colorado State University (Fort Collins, CO).
Neutrophil Isolation--
Neutrophils were isolated from healthy
donors using plasma-Percoll gradients, as described previously (21).
After isolation, neutrophils were washed and resuspended with
lipopolysaccharide-free Krebs-Ringer phosphate buffer (pH 7.2)
containing 0.2% dextrose (Krebs). Microscopic evaluation of isolated
cells treated by trypan blue exclusion indicated that 95% of cells
were neutrophils and those were >98% viable.
Fc
R Cross-linking--
Fc
R cross-linking was performed as
described previously (17).
Adenovirus Transfection of 293 Cells--
HEK 293 cells in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
were plated onto 100-mm tissue culture dishes 1 day prior to
transfection. Immediately prior to transfection, the medium was
replaced by 4 ml of complete Dulbecco's modified Eagle's medium
containing 2% fetal bovine serum, and the cells were infected with 500 plaque-forming units of appropriate adenovirus. Following
incubation at 37 °C in a 5% CO2 incubator for 1 h,
6 ml of Dulbecco's modified Eagle's medium containing 2% fetal
bovine serum was added back to each plate. Following 20 h of
incubation, cells were lysed and assayed for p38 kinase or Akt kinase activity.
Delivery of Synthetic Peptide into Human Neutrophils--
The
synthetic MK2 inhibitory peptide or control peptide representing a
portion of epidermal growth factor receptor (EGFR) was introduced into
human neutrophils by incubating cells with peptide for 40 min at
37 °C in a solution containing 20 mM Hepes, 5 mM KCl, 150 mM NaCl, 1 mM
MgCl2, 1 mM CaCl2, and 10 mM glucose before exposing the cells to hypotonic shock for
20 s to stimulate intracellular delivery of the peptide, as
described previously by Zu et al. (22).
Measurement of Akt Kinase Activity--
Akt kinase activity was
measured by the ability of immunoprecipitated enzyme to phosphorylate
histone H2B. Briefly, 1 × 107 neutrophils were
prewarmed for 5 min at 37 °C prior to stimulation with fMLP. The
reaction was terminated by centrifugation at 2500 × g
followed immediately by lysis in buffer containing 1% (v/v) Nonidet
P-40, 10% (v/v) glycerol, 137 mM NaCl, 20 mM
Tris-HCl, pH 7.4, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 5 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium
orthovanadate, and 1% (v/v) Triton X-100. Lysates were centrifuged at
15,000 × g for 15 min at 4 °C, and supernatants
were incubated with 2 µl of anti-Akt antiserum rotating
continuously for 1 h at 4 °C and protein A-Sepharose beads for
an additional 1 h. Beads were washed once each in lysis buffer and
kinase buffer (20 mM HEPES, 10 mM
MgCl2, 10 mM MnCl2) and incubated
in a 30-µl reaction mixture containing 5 µCi
[
-32P]ATP, 1 mM dithiothreitol, 85.7 µg/ml histone H2B, and kinase buffer. Reactions were incubated at
25 °C for 30 min and terminated by the addition of 6 µl of 6×
Laemmli buffer. The samples were boiled for 3 min, the products were
resolved by 10% SDS-PAGE, and products were visualized by autoradiography.
Measurement of p38 Kinase Activity--
p38 MAP kinase activity
was measured by assaying the ability of immunoprecipitated enzyme to
phosphorylate ATF2, as described previously (23).
Measurement of MK2 Activity--
MK2 activity was measured by
assaying the ability of immunoprecipitated enzyme to phosphorylate
recombinant Hsp27, as described previously by Krump et al.
(24).
Preparation of GST and GST-MK2-Sepharose Beads--
GST-pGEX-2T
and MK2-GST-pGEX2T cDNAs were transformed into Escherichia
coli BL21PlysS, and the expression and purification of GST and
GST-MK2 fusion protein was performed as described previously (25).
Western Blot Analysis of Phospho-p38 and
Phospho-Akt--
Tyrosine phosphorylation of ERK and p38 kinase and
phosphorylation of Ser473 or Thr308 on Akt was
determined by Western blotting with phosphospecific antisera. Following
lysis, proteins were separated with 10% SDS-PAGE, transferred onto
nitrocellulose membrane, and blocked with 5% milk in Tween 20 Tris-buffered saline (TTBS) (w/v) for 1 h. Blots were probed with
appropriate phosphospecific antibody in 5% bovine serum albumin/TTBS
overnight, and antibodies were detected using peroxidase-conjugated,
secondary antibody in 5% milk/TTBS for 1 h. Products were
visualized by chemiluminescence. To verify equal loading of protein in
each lane, the blots were stripped and reprobed for total p38, ERK, or Akt.
Immunoprecipitation of p38, MK2, Hsp27, and Akt--
Neutrophils
(2 × 107) were prewarmed at 37 °C for 5 min prior
to stimulation with or without 0.3 µM fMLP. The reactions
were stopped by centrifugation followed immediately by the addition of
200 µl of immunoprecipitation (IP) lysis buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% (v/v)
Triton X-100, 0.5% (v/v) Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 20 mM sodium orthovanadate, 10 µM p-nitrophenol phosphate, 20 mM
NaF, 5 mM phenylmethylsulfonyl fluoride, 21 µg/ml
aprotinin, and 5 µg/ml leupeptin. Following centrifugation at
15,000 × g for 15 min at 4 °C, cleared lysates were
incubated with 5 µl of anti-Akt antiserum, 3 µl of anti-p38, 2 µl of anti-Hsp27, or 2 µl of anti-MK2 overnight with continuous rotation at 4 °C. Protein A-Sepharose beads (15 µl) were then added, and samples were rotated for an additional 2 h at 4 °C. Beads were washed once by centrifugation in Krebs buffer and then resuspended in 50 µl of 2× Laemmli buffer and boiled for 3 min. Proteins were separated by 10% SDS-PAGE, transferred onto
nitrocellulose membrane, and blocked with 5% milk/TTBS for 1 h.
Blots were probed with anti-p38 (1:1000), anti-Hsp27 (1:1000), anti-MK2
(1:2000), or anti-Akt (1:1000) antiserum in 5% BSA/TTBS (w/v) and
peroxidase-conjugated secondary antibody in 5% milk/TTBS (w/v).
Products were visualized by chemiluminescence.
GST Pull-down Assay--
Neutrophils (2 × 107)
were lysed with 200 µl of IP lysis buffer. GST-Akt agarose, GST-MK2
Sepharose, protein A-agarose, protein A-Sepharose, or GST-Sepharose
beads were added to the lysates and incubated at 4 °C for 1 h
with shaking. The beads were washed three times with Krebs buffer, and
15 µl of 2× Laemmli buffer was added to each tube. The samples were
boiled for 3 min and then subjected to 10% SDS-PAGE. Proteins were
transferred onto nitrocellulose and immunoblotted for p38, MK2, Hsp27,
and Akt as described above.
Phosphorylation with Active Recombinant MK2--
Neutrophils
(2 × 107) were lysed with 200 µl of IP lysis
buffer. Lysates were precleared with 15 µl of protein A-Sepharose beads for 1 h at 4 °C with shaking. Anti-Akt (3 µl), anti-MK2 (3 µl), anti-Hsp27 (3 µl), or anti-p38 (3 µl) antiserum was added to the precleared neutrophil lysate and incubated overnight at 4 °C
with shaking. Protein A-Sepharose beads (15 µl) were then added to
lysates and incubated for 1 h at 4 °C with shaking. Beads were
washed once each in lysis buffer and kinase buffer (20 mM HEPES, 10 mM MgCl2, 10 mM
MnCl2) and incubated in a 30-µl reaction mixture
containing 3 µl of [
-32P]ATP (1 mCi/100 µl) or 1 µM ATP, 1 µl of active recombinant MK2 (25 ng/µl),
and 26 µl of kinase buffer. Reactions were incubated at 30 °C for
2 h, and the reaction was terminated by the addition of 30 µl of
2× Laemmli buffer. The samples were boiled for 3 min, and products
were resolved by 10% SDS-PAGE. Phosphorylation was visualized by autoradiography.
Trypsin Digestion and Mass Spectroscopic Analysis--
Coomassie
Blue-stained regions from one-dimensional PAGE were cut from the gel in
~1-mm3 sections and were taken for tryptic hydrolysis
using a modification of the method of Jensen et al. (26).
Essentially, the gel was washed using NH4HCO3
and CH3CN, and then proteins were reduced using
dithiothreitol and alkyated using iodoacetamide. After washing, proteins were hydrolyzed using modified trypsin (Promega). Differences from the method of Jensen et al. (26) were the use of higher (20 mM) dithiothreitol concentration, larger volume
(0.1-ml) washes following alkylation, and exclusion of calcium from the
trypsin mixture.
Peptides were then taken for thin film spotting for matrix-assisted
laser desorption-ionization using
-cyanohydroxycinnamic acid as
matrix on stainless steel targets with 1-2-µl spots. Mass spectral
data were obtained using a Tof-Spec 2E (Micromass) and a 337-nm
N2 laser at 20-35% power in the reflector mode. Spectral data were obtained by averaging 10 spectra, each of which was the
composite of 10 laser firings. Mass axis calibration was accomplished using peaks from tryptic autohydrolysis. Data were analyzed using MassLynx ProteinProbe software and the Mascot data base.
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RESULTS |
p38 Kinase but Not ERK Regulates Akt Phosphorylation--
Both
formyl peptide receptors and Fc
receptors stimulate Akt
phosphorylation in human neutrophils (16). To determine the optimal
time of stimulation, a time course of Akt Ser473
phosphorylation following the addition of 3 × 10
7 M fMLP or Fc
R
cross-linking was performed. Both agonists stimulated optimal Akt
phosphorylation at 2 min (data not shown).
To investigate the involvement of ERK and p38 in
PI-3K-dependent Akt activation in neutrophils, we measured
fMLP-stimulated Akt Ser473 phosphorylation in the presence
or absence of LY294002, wortmannin, PD98059, or SB203580. Fig.
1a shows that wortmannin,
LY294002, and SB203580 inhibited fMLP-stimulated Akt Ser473
phosphorylation, while PD98059 had no effect. To determine whether p38
regulation of Akt Ser473 phosphorylation was unique to
chemoattractant receptors, we examined the effect of SB203580 on
Fc
R-stimulated Akt Ser473 phosphorylation (Fig.
1b). Pretreatment with SB203580 blocked Akt
Ser473 phosphorylation stimulated by Fc
IIa and Fc
IIIb
receptor cross-linking. Thus, p38 kinase inhibition attenuates both
formyl peptide receptor and Fc
R-stimulated Akt phosphorylation.

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Fig. 1.
Effect of PI-3K, p38 kinase, and ERK
inhibition on fMLP-stimulated Akt phosphorylation. a,
immunoblot of neutrophil lysates probed with phosphospecific Akt
Ser473 (pS473-Akt) antiserum.
Pretreatment with the PI-3K inhibitors wortmannin and LY294002 and the
p38 kinase inhibitor SB203580 inhibited Ser473
phosphorylation following fMLP stimulation, whereas the MAP kinase/ERK
kinase/ERK inhibitor PD98059 did not block fMLP-stimulated Akt
phosphorylation, (n = 4). b, immunoblot of
neutrophil lysates probed with phosphospecific Akt Ser473
(pS473-Akt) and Akt antisera. Pretreatment with the p38
kinase inhibitor SB203580 blocked Fc RIIa- and Fc RIIIb-stimulated
Akt phosphorylation (n = 3).
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Ser473 Is More Sensitive to SB203580 Inhibition than
Thr308--
A previous study found that SB203580 inhibited
PDK1 phosphorylation of Akt Thr308 at concentrations
greater than 3 µM (27). To separate the effects of
SB203580 on PDK1 and p38 kinase, we performed concentration inhibition
experiments on phosphorylation of Thr308 and
Ser473. Neutrophils were pretreated with varying
concentrations of SB203580 for 1 h prior to stimulation with fMLP.
Concentration inhibition studies showed that SB203580 reduced
fMLP-stimulated phosphorylation of Ser473 at 0.3 µM, while at least 10 µM SB203580 was
required to see a diminution of Thr308 phosphorylation
(Fig. 2). The concentrations of SB203580
required to inhibit Akt Thr308 phosphorylation (10 µM) are ~20-fold higher than the IC50 for p38 kinase inhibition (0.3-0.5 µM) (27), while Akt
Ser473 phosphorylation is inhibited by SB203580 at the
IC50 for p38 kinase. These results suggest that inhibition
of p38 kinase attenuates Ser473 phosphorylation, while
Thr308 phosphorylation is independent of p38 kinase.

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Fig. 2.
Effect of p38 inhibition on phosphorylation
of Thr308/Ser473. Immunoblot of neutrophil
lysates probed with phosphospecific Akt Ser473
(pS473-Akt), Thr308 (pT308-Akt), or
Akt antiserum. Pretreatment with SB203580 at concentrations of 0.3 µM or higher inhibited fMLP-stimulated Akt
Ser473 phosphorylation, while inhibition of fMLP-stimulated
Akt Thr308 phosphorylation was not observed at
concentrations below 10 µM (n = 3).
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p38 Kinase Mediates PIP3-dependent Akt
Phosphorylation--
To determine whether p38 kinase is upstream or
downstream of PI-3K in the pathway leading to Akt activation, we
examined the ability of PIP3 to stimulate p38 kinase and
Akt Ser473 phosphorylation in neutrophils. PIP3
stimulated both p38 kinase (Fig.
3a) and Akt Ser473
phosphorylation (Fig. 3b) in a time-dependent
manner with optimal stimulation at 1 min. To determine whether
PIP3-stimulated Akt phosphorylation was mediated by p38
kinase, neutrophils were pretreated with 10 µM SB203580
prior to the addition of PIP3. Fig. 3b shows that SB203580 inhibited PIP3-mediated Akt
Ser473 phosphorylation, indicating that p38 kinase
activation is necessary for PI-3K-mediated activation of Akt in human
neutrophils. PIP3 also stimulated ERK activation with a
time course similar to p38 activation (data not shown).

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Fig. 3.
PIP3-stimulated time course of
p38 and Akt phosphorylation and effect of p38 inhibition on Akt
phosphorylation. a, immunoblot of neutrophil lysates
probed with phosphotyrosine p38 (pp38) and p38 antisera.
PIP3 stimulated optimal p38 phosphorylation by 1 min
(n = 3). b, immunoblot using phosphospecific
Akt Ser473 (pS473-Akt) and Akt antisera.
PIP3-stimulated optimal Akt Ser473
phosphorylation at 1 min. This phosphorylation was inhibited by
pretreatment with 10 µM SB203580 (n = 3).
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Constitutively Active MKK3/6 Stimulates Akt Activation in HEK 293 Cells--
Since neutrophil half-life is not long enough to allow
genetic manipulation, HEK 293 cells were transiently transfected with adenoviruses containing empty vector, MKK3bE (constitutively active MKK3), MKK3A (dominant negative MKK3), MKK6bE (constitutively active
MKK6), or MKK6A (dominant negative MKK6) to directly examine the
ability of p38 kinase to stimulate Akt activation. Fig.
4a shows that MKK3bE and
MKK6bE stimulated increased Akt activity, while the dominant negative
mutants had no effect. Both MKK3bE and MKK6bE stimulated increased p38
kinase activity (Fig. 4b), as measured by an in
vitro kinase assay using ATF2 as substrate.

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Fig. 4.
Expression of constitutively active MKK3 or
MKK6 adenoviruses stimulated p38 and Akt activation in HEK 293 cells. 20 h following adenoviral transfection, HEK 293 cells
were lysed, and lysates were subjected to p38 and Akt in
vitro kinase assay. a, Akt kinase activity was measured
in an in vitro kinase assay by [32P]ATP
phosphorylation of histone H2B. Constitutively active MKK3/6, but not
dominant negative MKK3/6, activated Akt in HEK 293 cells. b,
p38 kinase activity was measured in an in vitro kinase assay
by [32P]ATP phosphorylation of ATF2. These
results indicate that constitutively active MKK3/6 activated p38 kinase
in HEK 293 cells (n = 2).
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Akt Is Physically Associated with Components of the p38 Kinase
Pathway--
Previous studies reported that MK2 phosphorylates Akt
Ser473 in vitro and that Hsp27 associates only
with active Akt (19, 20). Coupled with our data showing that
phosphorylation of Ser473 is dependent on p38 kinase, we
postulated that Akt exists in a signaling complex with MK2 and p38
kinase. Therefore, the association of Akt with p38 kinase, MK2, and
Hsp27 was examined in unstimulated and stimulated neutrophils. Lysates
prepared from unstimulated and fMLP-stimulated cells were
immunoprecipitated with anti-Akt antibody. Proteins in the
immunoprecipitate were separated by SDS-PAGE and immunoblotted with
anti-Akt, anti-p38, anti-MK2, and anti-Hsp27. Fig.
5a shows that p38 kinase, MK2,
and Hsp27 were all present in Akt immunoprecipitates from unstimulated
cells. Stimulation with fMLP resulted in a time-dependent
dissociation of Hsp27 from the complex. Additionally, neutrophil
lysates immunoprecipitated with anti-Hsp27, anti-MK2, or anti-p38 were
immunoblotted for Akt. Akt was detected in all three immunoprecipitates
(data not shown).

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Fig. 5.
Association of Akt with components of the p38
signal module. a, immunoblot for Akt, p38, MK2, and
Hsp27 in immunoprecipitates with Akt antibody. All three components of
the p38 kinase module were present. Only Hsp27 dissociated from Akt
upon fMLP stimulation (n = 3). b,
immunoblots for MK2, p38, and Hsp27 indicate that they associate with
GST-Akt agarose beads, while Akt, p38, and Hsp27 associate with
GST-MK2-Sepharose beads. These results confirm the association of Akt,
p38, MK2, and Hsp27.
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Another method for detecting protein-protein interactions is a GST
pull-down assay. GST-fused Akt or MK2 proteins were expressed in
E. coli and were immobilized on glutathione-agarose or
glutathione-Sepharose beads. Neutrophil lysates were incubated with the
protein-immobilized beads or GST-Sepharose beads. The proteins attached
to the beads were separated by SDS-PAGE and immunoblotted for Akt, p38
kinase, MK2, and Hsp27. Fig. 5b shows that GST-Akt was
associated with p38 kinase, MK2, and Hsp27 and that GST-MK2 associated
with Akt, p38 kinase, and Hsp27. GST-Sepharose beads alone did not bind to Akt, p38 kinase, MK2, or Hsp27 from neutrophil lysates (data not shown).
MK2 Phosphorylation of Akt--
A direct target of p38 kinase,
MK2, phosphorylates Akt in vitro (19). A previous report
suggested, however, that MK2 is unlikely to mediate Akt activation
because agonists that activate Akt in HEK 293 cells failed to activate
MK2. We examined the ability of recombinant active MK2 to phosphorylate
recombinant Akt (Fig. 6a) and
Akt present in anti-Akt, anti-MK2, anti-p38, and anti-Hsp27 immunoprecipitates from human neutrophils. Fig. 6, a and
b, shows that MK2 stimulated phosphorylation of a 66-kDa
protein in all conditions. The phosphorylated protein was
trypsin-digested, and resulting peptides were subjected to
matrix-assisted laser desorption mass spectroscopic analysis and
identified by peptide mass fingerprinting to be Akt. Recombinant MK2
also stimulated phosphorylation of Ser473 Akt in anti-Akt,
anti-MK2, and anti-Hsp27 immunoprecipitates from neutrophils (Fig.
6c). To determine whether the differences in the role of MK2
as PDK2 could be due to cell-specific differences in the Akt isoforms,
we immunoblotted neutrophil lysates with anti-Akt1, anti-Akt2, and
anti-Akt3 antibodies. All three isoforms of Akt were present in human
neutrophils (data not shown).

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Fig. 6.
MK2 acts as PDK2 in human
polymorphonuclear leukocytes.
a, autoradiograph of SDS-PAGE following the addition of
active recombinant MK2 to recombinant Akt in the presence of
[32P]ATP. Significant Akt phosphorylation was seen at
2 h. b, autoradiograph of SDS-PAGE following the
addition of active recombinant MK2 to neutrophil lysates
immunoprecipitated with anti-Akt, anti-MK2, anti-p38, and anti-Hsp27
(Immuno-Akt, Immuno-MK2, Immuno-p38,
and Immuno-Hsp2) in the presence of [32P]ATP.
Active recombinant MK2 phosphorylated a 66-kDa protein in all of the
immunoprecipitates. c, active recombinant MK2 was added to
neutrophil lysates immunoprecipitated with anti-Akt, anti-MK2, and
anti-Hsp27 in the presence of 1 µM ATP. The samples were
run on SDS-PAGE and immunoblotted with phosphospecific Akt
Ser473 (pS473-Akt). Active recombinant MK2
phosphorylated Ser473 of Akt in all of the
immunoprecipitates.
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Inhibition of Akt Phoshorylation and Activation by MK2 Inhibitory
Peptide--
Zu et al. (22) reported that a peptide
representing the phosphorylation site of Hsp27 inhibited MK2
phosphorylation of substrates. A concentration inhibition study showed
that a 160 µM concentration of the MK2 inhibitory peptide
was required to reduce fMLP-stimulated MK2 phosphorylation of Hsp27
(Fig. 7a). A nonrelated
peptide at the same concentration did not reduce MK2-mediated Hsp27
phosphorylation. The concentration of the MK2 peptide required was
significantly greater than the 30 µM concentration
reported by Zu et al. (22). A similar concentration of the
MK2 peptide was required to inhibit the ability of active recombinant
MK2 to phosphorylate Akt from neutrophil anti-Akt immunoprecipitates
(Fig. 7b). To determine the role of MK2 in Akt
phosphorylation and activation, we preincubated neutrophils with the
MK2 inhibitory peptide prior to stimulation with fMLP. Intracellular
delivery of the inhibitory peptide reduced both fMLP-stimulated Akt
Ser473 phosphorylation (Fig. 7c) and Akt
activity (Fig. 7d), while a nonrelated peptide had no
effect. Taken together, our results indicate that MK2 phosphorylates
Ser473, which leads to activation of Akt in human
neutrophils.

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Fig. 7.
Effect of MK2 inhibitory peptide on MK2 and
Akt activation in human neutrophils. a,
concentration-inhibition assay of MK2 inhibitor peptide in neutrophils.
Preincubation of human neutrophils with 160 µM MK2
inhibitory peptide (MK2 peptide), but not EGFR peptide, inhibited
fMLP-stimulated MK2 kinase activity as measured by phosphorylation of
recombinant Hsp27 by immunoprecipitated MK2 in an in vitro
kinase assay. b, autoradiograph of SDS-PAGE following the
addition of active recombinant MK2 in the presence of 80 or 160 µM MK2 inhibitory peptide or 160 µM EGFR
peptide and [32P]ATP to neutrophil lysates
immunoprecipitated with anti-Akt. MK2 inhibitory peptide, but not EGFR
peptide, inhibited the ability of active recombinant MK2 to
phosphorylate a 66-kDa protein in a concentration-dependent
manner. c, immunoblot using phosphospecific Akt
Ser473 (pS473-Akt). Preincubation of human
neutrophils with MK2 inhibitory peptide, but not EGFR peptide,
inhibited fMLP-stimulated Akt (S473) phosphorylation.
d, preincubation of human neutrophils with MK2 inhibitory
peptide, but not EGFR peptide, inhibited fMLP-stimulated Akt kinase
activity measured by phosphorylation of histone H2B in an in
vitro kinase assay.
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ERK Activation Is Independent of Akt--
We examined the effect
of pretreatment with 50 µM PD98059 on
PIP3-stimulated Akt 473 phosphorylation and found that
PD98059 did not alter PIP3-stimulated Akt phosphorylation
(data not shown), suggesting that ERK is not upstream of Akt. To
determine whether ERK is downstream of Akt, we pretreated neutrophils
with SB203580 prior to stimulation with fMLP and measured ERK activity
by an in vitro kinase assay and by immunoblot for
phospho-ERK. Inhibition of p38 kinase did not alter fMLP-stimulated ERK
activity by either of the two methods (data not shown). These data
suggest that ERK does not participate in the Akt signaling pathway,
despite the requirement of PI-3K for ERK activation.
 |
DISCUSSION |
Both Akt and p38 kinase are rapidly activated in neutrophils by a
number of inflammatory mediators, and one or both kinases participate
in respiratory burst activity, chemotaxis, priming, and apoptosis (17,
18, 28, 29, 31, 32). Previous studies indicated that activation of both
kinases is mediated by products of PI-3K (18, 30). Our study provides
evidence for the first time that p38 kinase participates in the signal
transduction pathway leading to Akt activation. Akt activation requires
its translocation from a cytosolic location to the plasma membrane,
phosphorylation of Thr308 by PDK1, and phosphorylation of
Ser473 by an unknown kinase heretofore called PDK2 (12,
33). All three of these activation steps are dependent on products of
PI-3K (30, 33). Previous reports suggested that phosphorylation of
Ser473 was the result of autophosphorylation following
PDK1-dependent phosphorylation of Akt Thr308 or
was due to sequential phosphorylation of Thr308 and then
Ser473 by PDK1 (34). Our results indicate that p38 kinase
is required for PIP3-stimulated activation of p38 kinase
and Akt in human neutrophils, and
PIP3-dependent phosphorylation of
Ser473 on Akt is inhibited by SB203580. The pyridinyl
imidazole SB203580 is a relatively specific inhibitor of the
and
isoforms of p38 kinase (35). Recently, Lali et al. (27)
reported that SB203580 inhibited PDK1 at concentrations significantly
greater than the IC50 for p38 kinase. We excluded this
explanation for our results by demonstrating different SB203580
concentration-inhibition curves for Thr308 and
Ser473 phosphorylation. SB203580 at the IC50
for p38 kinase (0.3 µM) inhibited Ser473
phosphorylation, while concentrations of SB203580 below 10 µM failed to inhibit Thr308 phosphorylation.
Further evidence that p38 kinase participates in Akt activation was
provided by the ability of constitutively active MKK3 and MKK6 to
stimulate Akt activation following transfection into HEK 293 cells.
These studies also suggest that p38 kinase-mediated Ser473
phosphorylation is sufficient to induce Akt activation independent of
PI-3K. Neutrophils were inadequate for these genetic studies, since
they undergo constitutive apoptosis in culture resulting in survival of
less than 40% of cells at 48 h (36).
Alessi et al. (19) previously showed that MK2 phosphorylates
recombinant Akt on Ser473; however, they discounted a role
for MK2 in intact cells, since fibroblasts showed
IGF-1-dependent Akt activation in the absence of MK2
activation. The present study demonstrates that p38 kinase-stimulated Akt Ser473 phosphorylation is mediated by MK2. Not only did
active recombinant MK2 phosphorylate recombinant Akt in
vitro, but Akt immunoprecipitated from neutrophil lysates was
phosphorylated by active recombinant MK2 as well. Direct evidence for
MK2-mediated phosphorylation of Akt Ser473 in intact
neutrophils was obtained using an MK2 inhibitory peptide described by
Zu et al. (22). Introduction of the inhibitory peptide into
freshly isolated neutrophils inhibited phosphorylation of Hsp27
following immunoprecipitation of MK2 from fMLP-stimulated cells.
Similarly, the MK2 inhibitory peptide reduced fMLP-stimulated Akt
activation and Ser473 phosphorylation, while the control
EGFR peptide had no affect. Taken together, these data indicate that
MK2 acts as PDK2 in human neutrophils.
The concept that signal transduction pathway components form multimeric
complexes held together by scaffolding proteins, rather than existing
free in the cytosol, has received significant experimental support
recently. Scaffolding proteins have been described for two other MAP
kinase modules, ERK and JNK (37, 38). Therefore, the possibility that
p38 kinase, MK2, and Akt form a signaling complex was examined. Using
two separate methods, immunoprecipitation and GST pull-down, the
present study shows for the first time that p38 kinase, MK2, and Akt
exist as a complex that does not dissociate upon activation. Hsp27 was
previously reported to associate with activated Akt (20). Therefore, we
examined the association of Hsp27 with Akt, MK2, and p38 kinase
following Akt, p38, or MK2 immunoprecipitation and following GST
pull-down with MK2 or Akt. Hsp27 was present in these complexes in
unstimulated neutrophils. As opposed to MK2 and p38 kinase, Hsp27
dissociated from Akt immunoprecipitates following neutrophil
stimulation with fMLP. Taken together, our data indicate that three
components of the p38 kinase module, p38 kinase, MK2, and Hsp27, form a
signaling complex with Akt. MK2, which has been shown to phosphorylate
Hsp27 (39, 40), directly phosphorylates Ser473 on Akt.
Hsp27 dissociates from the complex during stimulation, suggesting that
Hsp27 performs a regulatory function. Our data do not indicate whether
Hsp27 dissociates from the complex before or after translocation or
phosphorylation of Akt; therefore, no conclusion as to whether Hsp27
acts as a positive or negative regulator is possible. The presence of
other components in the signaling complex and the scaffolding
protein that binds the complex together remain to be determined.
Identity of scaffolding protein is unknown.