From the
Departments of Medicine and
||Biochemistry and Molecular Biology, and the
Core Proteomics Laboratory, University
of Louisville, Louisville, Kentucky 40202-1764 and the
**Veterans Affairs Medical Center, Louisville,
Kentucky 40206
Received for publication, April 2, 2003 , and in revised form, May 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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Characterization of signal pathways that regulate apoptosis have identified phosphoinositide 3-kinase (PI-3K)1 as a transducer of survival signals. The serine/threonine kinase, Akt, is a major target of PI-3K. Akt is present in the cytosol of unstimulated cells in a low activity conformation. Activation of PI-3K generates 3'-phosphorylated phosphoinositides that induce translocation of Akt to the plasma membrane (811). Phosphoinositides also activate phosphoinositide-dependent kinase 1 (PDK1) and 2 (PDK2) that phosphorylate Akt on Thr-308 and Ser-473, respectively (1221). We reported previously that the p38 mitogen-activated protein kinase (MAPK) substrate, MAPK-activated protein kinase-2 (MAPKAPK-2), phosphorylates and activates Akt and that Akt forms a signaling module containing p38 MAPK, MAPKAPK-2, and Hsp27 in human neutrophils (21). Upon cellular stimulation Hsp27 dissociates from this module.
A number of Akt substrates, including BAD, caspase 9, I
kinase, apoptosis signal-regulating kinase 1 (Ask1), and the forkhead
transcription factors FKHR, FKHRL1, and AFX play a role in cell survival
(2229).
Recently, Sheth et al.
(30) showed that introduction
of recombinant Hsp27 into neutrophils delayed apoptosis, but the mechanism of
action was not determined. Hsp27 binds to and inactivates the pro-apoptotic
molecules caspase 3, caspase 9, and cytochrome c
(3134).
Phosphorylated Hsp27 has been shown to bind an adaptor protein Daxx and
inhibit Fas-mediated apoptosis
(35). Based on these data,
Hsp27 was postulated to inhibit apoptosis through its activity as a molecular
chaperone.
Our previous report showing physical association and dissociation of Hsp27 and the Akt signaling module in human neutrophils suggested the hypothesis that Hsp27 modulates neutrophil apoptosis by control of Akt activation. In the present study we identify Hsp27 as an Akt substrate that dissociates from Akt upon phosphorylation. We demonstrate that disruption of the interaction between Hsp27 and Akt impairs Akt activation, leading to an enhanced rate of constitutive neutrophil apoptosis. These data provide evidence for a novel role of Hsp27 in the control of neutrophil apoptosis through regulation of Akt activity.
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MATERIALS AND METHODS |
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Isolation of Neutrophils and Culture ConditionsNeutrophils were isolated from venous blood obtained from healthy volunteers as described previously (5). Neutrophil preparations routinely contained >95% neutrophils, as determined by morphology, and were >97% viable by trypan blue dye exclusion. Neutrophils were suspended in RPMI 1640 supplemented with 10% fetal calf serum, L-glutamine, penicillin, and streptomycin and incubated for the indicated times at 37 °C in 5% CO2.
Western BlottingNeutrophils were lysed 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 as described previously (21). Monoclonal Hsp27 antibody; polyclonal phospho-Ser-15, Ser-78, and Ser-82 Hsp27 antisera; polyclonal Akt antibody; monoclonal Akt antibody; and phospho-Ser-473 Akt antibody were used at a dilution of 1:1000 in 5% bovine serum albumin or 5% milk Tween 20/Tris-buffered saline.
Introduction of Antibodies into NeutrophilsWe used the method of Tezel and Wax (37) to introduce antibodies into human neutrophils. Briefly, 2 x 107 neutrophils/200 µl of RPMI 1640 were prewarmed for 5 min at 37 °C and subjected to anti-Hsp27 antibody (20 µg) or isotype control antibody (20 µg) and incubated at 37 °C for 2 h. The neutrophils were washed three times and then resuspended in 1 ml of Krebs-Ringer phosphate buffer containing 5.5 mM dextrose, pH 7.4 (KRPD).
Measurement of Akt Kinase ActivityAkt kinase activity was
measured by the ability of the immunoprecipitated enzyme to phosphorylate
histone H2B, as described previously
(21). After incubation with
the indicated reagents, neutrophils were washed with KRPD and resuspended in 1
ml of KRPD and stimulated with 0.3 µM fMLP for 2 min.
Neutrophils were then centrifuged at 2500 x g, and cells were
lysed 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 x g for 15 min at 4 °C, and cleared supernatants
were incubated with 5 µl of anti-Akt antiserum, while rotating continuously
for 1 h at 4 °C, followed by addition of 10 µl of protein A-Sepharose
for an additional 1 h. Protein A-Sepharose beads were then 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 of [-32P]ATP,
1 mM dithiothreitol, 85.7 µg/ml histone H2B, and kinase buffer.
Following incubations at 25 °C for 30 min, the reactions were terminated
by the addition of 6 µlof6x Laemmli buffer. The samples were boiled
for 3 min, proteins separated by 10% SDS-PAGE, and 32P
incorporation visualized by autoradiography.
Recombinant Active Kinase Phosphorylation of Hsp27Active
recombinant Akt (400 ng) or active recombinant MAPKAPK-2 (40 ng) was incubated
in 30 µl of reaction mixture containing 5 µCi of
[-32P]ATP, recombinant Hsp27 (1 µg), and kinase buffer
(20 mM HEPES, 10 mM MgCl2, 10 mM
MnCl2). The reaction was terminated by adding 6 µl of 6x
Laemmli buffer.
For in vitro kinase reactions performed in the absence of radiolabeled ATP, active recombinant Akt (400 ng) was incubated in a 30-µl reaction mixture containing 1 µM ATP, 1 µg of recombinant Hsp27, 20 mM HEPES, 10 mM MgCl2, 10 mM MnCl2. The reaction was terminated by boiling. This in vitro phosphorylated recombinant Hsp27 (25 ng) was added to 20 µl of glutathione-Sepharose-coupled GST or GST-AKT to perform GST pull-down assays.
Subcloning Hsp27 and Akt into GST Fusion VectorsTo create GST fusion proteins, Akt-wt was excised from pUSEAktwt (Upstate Biotechnology, Inc.) with restriction enzymes BamHI/PmeI and ligated into BamHI/SmaI sites of pGEX-4T-2 (Amersham Biosciences). Hsp27 was excised from pcDNA3.1Hsp27wt with restriction enzymes EcoRI/XhoI and ligated into EcoRI/XhoI sites of pGEX-5X-2 (Amersham Biosciences). All positive clones were confirmed by DNA sequencing.
Preparation of GST, GST-Hsp27, and GST-Akt-SepharoseGST-pGEX-4T-2, GST-Hsp27pGEX-4T-2, and GST-AktpGEX-5X-2 cDNAs were transformed into Escherichia coli BL21(DE3)PlysS, and the expression and purification of GST and GST-Hsp27 were performed, as described previously (21).
GST Pull-down AssayNeutrophils (2 x 107) were lysed with 200 µlof lysis 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, 1mM sodium orthovanadate, and 1% (v/v) Triton X-100. GST or GST-Akt-Sepharose (20 µl) was added to the lysates and incubated at 4 °C for 1 h with shaking. The beads were washed three times with Krebs+ buffer. The proteins were eluted from the beads by adding 40 µl of 2x Laemmli buffer. The samples were boiled for 3 min, and proteins were separated by 10% SDS-PAGE. Proteins were transferred onto nitrocellulose and immunoblotted for Hsp27 and Akt, as described above.
GST Pull-down Assay of Recombinant ProteinsGST, GST-Akt, or GST-Hsp27-Sepharose (20 µl) were added to 25 ng of recombinant protein and 50 µl of Krebs+ in a 0.5-ml Eppendorf tube. The samples were incubated with shaking at 4 °C for 1 h. Sepharose beads were precipitated by centrifugation and washed 6 times with Krebs+ buffer. Forty µl of 2x Laemmli buffer were added to each tube, the samples boiled for 3 min, and proteins separated by 10% SDS-PAGE. Proteins were transferred onto nitrocellulose and immunoblotted for Hsp27 and Akt, as described above.
FITC Conjugation of Anti-Hsp27Two hundred µg of anti-Hsp27 dissolved in 500 µl of phosphate-buffered saline were mixed dropwise with 25 µl of N-hydroxysuccinimide-fluorescein (Pierce) from a 2 mM stock solution in Me2SO. The reaction vials were left on ice for 2 h. Unreacted fluorescein was removed by gel filtration using gel columns equilibrated with phosphate-buffered saline. Protein concentration was determined using the BCA protein assay. The fluorescein-labeled antibody was stored at 4 °C after adding 0.1% NaN3.
Trypan Blue QuenchingNeutrophils (1 x 106)
were suspended in 200 µl of Krebs+. FITC-conjugated anti-monoclonal Hsp27
and mouse isotype control antibodies were incubated with neutrophils at 37
°C for 4 h. The cells were then incubated with or without 200 µl of
0.02% trypan blue (TB) in 0.02 M sodium acetate buffer, pH 5.8, at
room temperature (38).
Following incubation, cells were pelleted by centrifugation. The supernatant
was discarded, and cells were washed twice with phosphate-buffered saline
supplemented with 0.1% sodium azide and 0.2% bovine serum albumin. The cells
were resuspended in 0.02 M sodium acetate buffer, pH 5.8, and mixed
gently by vortexing. Cells were filtered through a cheesecloth, and the cells
were transferred into 12 x 75 mm polypropylene tubes. The percentage of
FITC antibody inside the cell was determined by flow cytometry and calculated
using Equation 1,
![]() | (Eq. 1) |
Introduction of Recombinant Akt and Anti-Hsp27 into Neutrophils Protein transfection reagent BioPORTER was used to transfect constitutively active recombinant Akt and anti-Hsp27 antibody according to the manufacturer's protocol. BioPORTER reagent was dissolved in 250 µl of methanol. Five µl of BioPORTER suspension was aliquoted and allowed to air-dry for 34 h in the laminar flow hood. Anti-Hsp27 antibody alone (14.5 µg) or anti-Hsp27 antibody and constitutively active recombinant Akt (1 µg) were added to the dried BioPORTER. This mixture was incubated for 5 min at room temperature. The tubes were vortexed gently for 35 s at low speed. Neutrophils (1.6 x 106/100 µl in KRPD) were added and incubated for 4 h at 37 °C with shaking. After 4 h, the neutrophils were pelleted and transferred to Eppendorf tubes and washed in cold Krebs+. The cells were washed and resuspended in 300 µl of binding buffer (10 mM HEPES/NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2) and assayed for annexin V binding assay.
Annexin V BindingNeutrophils were incubated with 10 µl of FITC-labeled ApopNexin at room temperature for 15 min in the dark. Cells were washed twice with binding buffer by centrifugation at 400 x g and then resuspended in binding buffer at a concentration of 1 x 106/ml. Two hundred µl of neutrophil cell suspension were placed in an 8-well confocal chamber and viewed by confocal microscopy or were transferred into 12 x 75 mm polypropylene tubes, and fluorescence was determined by flow cytometry.
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RESULTS |
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Akt Phosphorylates Hsp27The ability of Akt to interact directly with Hsp27 suggested the possibility that Akt phosphorylated Hsp27. A manual search of the Hsp27 amino acid sequence failed to identify a consensus RXRXX(S/T) Akt phosphorylation motif. On the other hand, a web-based motif analysis software (SCANSITE) predicted low stringency Akt phosphorylation sites on Hsp27 at Ser-9, Ser-15, Ser-78, and Ser-82 and a high stringency site at Thr-143 (41). Based on these findings, the ability of active recombinant Akt to phosphorylate recombinant Hsp27 in the presence or absence of an Akt inhibitory peptide or a scrambled peptide was examined in an in vitro kinase assay. The Akt inhibitory peptide (Aktide-2T) was described previously by Obata et al. (36) and mimics the optimal phosphorylation sequence of Akt. Fig. 2A shows that Akt phosphorylated Hsp27 (lane 2), and this phosphorylation was inhibited by addition of 20 µM Akt inhibitory peptide (lane 4) but not 20 µM scrambled peptide (lane 3). Equal loading of recombinant Hsp27 was demonstrated by Coomassie Blue staining of the gels.
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MAPKAPK-2 was shown previously to phosphorylate Hsp27 on Ser-15, Ser-78, and Ser-82 (40). To determine whether Akt and MAPKAPK-2 share phosphorylation sites, an in vitro kinase assay with recombinant Hsp27 and active recombinant MAPKAPK-2 or active recombinant Akt was performed. The samples were subjected to immunoblot analysis with phosphospecific Hsp27 antibodies. Separate gels were used to measure anti-Ser(P)-82 (Fig. 2B, gel 1), anti-Ser(P)-78 (gel 2), and anti-Ser(P)-15 (gel 3)-Hsp27 antibody binding. Fig. 2B demonstrates that MAPKAPK-2 phosphorylates Ser-82 (panel 1, lane 4), Ser-78 (panel 3, lane 4), and Ser-15 (panel 5, lane 4), whereas Akt phosphorylates only Ser-82 (panel 1, lane 2). Each gel was stripped and reprobed for total Hsp27 to ensure equal loading of recombinant Hsp27 (Fig. 2B, panels 2, 4, and 6).
To determine whether Akt phosphorylates Hsp27 in intact cells, HEK-293 cells were co-transfected with pUSE and pcDNA3.0 vectors or with c-Myc-tagged constitutively active myristoylated-Akt (pUSEAkt-CA) and wild type Hsp27 (pcDNAHsp27-WT). Post-transfection cells were lysed and subjected to immunoblot analysis with phospho-specific Hsp27 antibodies. Separate gels were used for anti-Ser(P)-82 (gel 1), anti-Ser(P)-78 (gel 2), and anti-Ser(P)-15 (gel 3)-Hsp27 antibodies (Fig. 3). As MAPKAPK-2 phosphorylates Hsp27 on Ser-15, Ser-78, and Ser-82, recombinant Hsp27 phosphorylated in vitro by active recombinant MAPKAPK-2 was included on each gel as a positive control (Fig. 3, lane 3). Cells co-transfected with Akt-CA and Hsp27-wt exhibited phosphorylation of Hsp27 on Ser-82 but not on Ser-78 or Ser-15. No phosphorylation was detected in vector-transfected cells. Active recombinant MAPKAPK-2 phosphorylated recombinant Hsp27 on Ser-82, Ser-78, and Ser-15 as expected (Fig. 3, lane 3). Each gel was stripped and reprobed with anti-Hsp27 and anti-c-Myc antibodies to confirm overexpression of AktCA and Hsp27 (Fig. 3, panels 1 and 2). These findings indicate that Akt phosphorylates Hsp27 on Ser-82 but not on Ser-15 or Ser-78, both in intact cells and in vitro. The absence of phosphorylation of Ser-15 and Ser-78 shows that MAPKAPK-2 was not active in these transfected cells.
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Phosphorylation of Hsp27 Regulates Association with the Akt Signal ModuleWe showed previously that Hsp27 dissociated from the Akt complex following neutrophil stimulation with chemoattractants (21), suggesting that Hsp27 phosphorylation might regulate interaction with Akt. To test this hypothesis, HEK-293 cells were transiently transfected with pcDNA3.1-Hsp27 constructs containing wild type (Hsp27-wt), Hsp27-3A in which Ser-15, -78, and -82 were mutated to alanines, or Hsp27-3D in which all three serine residues were mutated to aspartic acid. Hsp27-3A acts as a phosphorylationdead mutant, whereas Hsp27-3D acts as a phosphorylation-mimicking mutant. At 24 h cells were lysed, and the lysate was subjected to a GST-Akt pull-down assay. The precipitated proteins were subjected to immunoblot analysis with anti-Hsp27 antibody (Fig. 4A, panel 2). Increased Hsp27 binding to GST-Akt was observed in cell lysates from Hsp27-wt and Hsp27-3A-transfected cells. The similar amounts of Hsp27 precipitated by GST-Akt in vector and Hsp27-3D-transfected cells were attributed to endogenous Hsp27. Precipitated proteins were also subjected to immunoblot analysis for Akt to ensure that equal amounts of GST-Akt-Sepharose beads were added to each lysate (Fig. 4A, panel 1). Post-transfection cell lysates were also subjected to immunoblot analysis for Hsp27. Fig. 4A, panel 3, demonstrates that all Hsp27 constructs were equally overexpressed in HEK-293 cells. These results show impaired interaction between the phosphorylation-mimicking mutant of Hsp27 (Hsp27-3D) and Akt in intact cells.
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To determine whether Akt-mediated phosphorylation of Hsp27 was sufficient to induce Hsp27 dissociation from Akt, the ability of glutathione-Sepharose coupled to GST-Akt to precipitate recombinant Hsp27-wt (25 ng) or recombinant Hsp27, which was phosphorylated in vitro by active recombinant Akt (25 ng), was assayed. Fig. 4B shows that GST-Akt-Sepharose beads precipitated recombinant Hsp27-wt (lane 3) but not Akt-phosphorylated Hsp27 (lane 4). These data indicate that Hsp27 binding to Akt is regulated by the phosphorylation state of Hsp27 and that Akt-mediated Hsp27 phosphorylation is sufficient to induce Hsp27 dissociation from Akt.
Sequestration of Cellular Hsp27 by Anti-Hsp27 Antibody Hsp27 was reported previously to regulate apoptosis by binding to cytochrome c, procaspase 9, and caspase 3 (3134). Phosphorylated Hsp27 has also been shown to bind the adaptor protein Daxx and inhibit Fas-mediated apoptosis (35). Recently, Tezel and Wax (37) showed that anti-Hsp27 antibodies entered neuronal cells by endocytosis and enhanced the rate of apoptosis. To determine whether a similar approach could be used in human neutrophils, the ability of FITC-labeled anti-Hsp27 antibodies to enter neutrophils and their distribution was examined by confocal microscopy. Fig. 5A shows that incubation of neutrophils with 20 µg of FITC-labeled anti-Hsp27 for 4 h resulted in diffuse intracellular staining, suggesting distribution throughout the cytosol. Internalization of anti-Hsp27 was confirmed by trypan blue quenching (38, 39). Neutrophils were left untreated, incubated with FITC-conjugated anti-Hsp27, or incubated with FITC-conjugated isotype control antibody for 4 h. Neutrophils were incubated with or without trypan blue to quench extracellular fluorescence, and fluorescence intensity was determined by flow cytometry. Table I shows the fluorescence intensities of control, FITC-IgG treated, and FITC-anti-Hsp27-treated neutrophils before and after trypan blue quenching. Trypan blue reduced fluorescence of FITC-IgG-loaded neutrophils by 26% and FITC-Hsp27-loaded cells by 29%. These data indicate that 7174% of FITC-IgG and FITC-anti-Hsp27 were internalized by neutrophils.
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To determine the effect of introduction of anti-Hsp27 antibodies on Hsp27 association with Akt, neutrophils were incubated in the presence or absence of anti-Hsp27 or isotype control antibodies. After 4 h the cells were lysed, and the lysate was subjected to immunoprecipitation with anti-Akt antibody. The immunoprecipitates were immunoblotted for Hsp27. Fig. 5B shows that Hsp27 co-precipitated with Akt in untreated and isotype antibody-loaded cells, whereas Hsp27 failed to co-precipitate with Akt in anti-Hsp27 antibody-loaded cells. The immunoprecipitates were also immunoblotted for Akt to ensure equivalent immunoprecipitation of Akt in all conditions (Fig. 5B, panel 2). These results suggest that introduction of anti-Hsp27 antibody sequesters Hsp27, making it unavailable for interaction with Akt.
Disruption of Akt-Hsp27 Interaction Results in Loss of Akt ActivationThe ability of Hsp27 antibody treatment to disrupt the interaction between Akt and Hsp27 allowed us to determine the role of Hsp27 in activation of Akt kinase. Neutrophils were incubated in the presence or absence of anti-Hsp27 or isotype control antibodies for 2 h prior to stimulation with 1 µM fMLP. Cell lysates were subjected to an in vitro immunoprecipitation kinase assay for Akt activity using histone H2B as substrate. The phosphorylated protein bands were quantitated by a densitometer. The controls were normalized to one, and results were expressed as mean arbitrary densitometry units ± S.E. for 3 separate experiments. Fig. 6A shows that fMLP stimulated a significant increase in Akt activity in untreated and isotype antibody-loaded neutrophils. On the other hand, loading neutrophils with anti-Hsp27 antibody blocked fMLP-stimulated Akt activity. Immunoblot analysis of these lysates demonstrated that loading cells with anti-Hsp27 antibody did not alter Akt expression (Fig. 6B, panel 2). Immunoblot analysis with anti-phospho-Ser-473 Akt antibody showed that Akt was phosphorylated following fMLP treatment in untreated and isotype antibody-loaded cells but not in cells loaded with anti-Hsp27 antibody (Fig. 6B, panel 1). Thus, disruption of Hsp27 binding to Akt in intact neutrophils prevented receptor-mediated Akt phosphorylation and activation. These data suggest that Hsp27 is a necessary component for Akt activation.
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Disruption of Akt-Hsp27 Interaction Enhances Neutrophil ApoptosisThe requirement of Hsp27 for Akt activation suggested a possible mechanism by which Hsp27 regulates apoptosis. Therefore, we examined the effect of disrupting the Hsp27 interaction with Akt on constitutive neutrophil apoptosis. Neutrophils were loaded with anti-Hsp27 or isotype control antibodies for 4 h at 37 °C, after which apoptosis was assessed by annexin V binding and electron microscopy. Fig. 7A shows that introduction of anti-Hsp27 into neutrophils resulted in a significant increase in annexin V binding, whereas no binding was observed in cells loaded with isotype control antibody. By electron microscopy typical features of apoptosis, including membrane blebbing and nuclear condensation, were seen in anti-Hsp27-loaded but not isotype control antibody-loaded cells (Fig. 7B). These results indicate that sequestration of Hsp27 results in an accelerated rate of apoptosis.
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To determine whether sequestration of Hsp27 enhanced apoptosis through impaired Akt activation, BioPORTER lipid vesicles were used to introduce anti-Hsp27 antibodies into neutrophils with or without active recombinant Akt. Fig. 8 shows that this method of anti-Hsp27 antibody introduction also induced the morphologic features of apoptosis (panel 1) and increased annexin V binding (panel 2). Simultaneous addition of active recombinant Akt rescued neutrophils from both morphologic changes and increased annexin V binding produced by anti-Hsp27 antibody. Flow cytometric analysis demonstrated that 13% of neutrophils loaded with isotype control antibody bound annexin V, whereas 60% of neutrophils loaded with anti-Hsp27 showed annexin V binding. Simultaneous introduction of active recombinant Akt and anti-Hsp27 reduced the percent of cells binding annexin V to 30%. These data indicate that Hsp27 regulation of Akt activity is one mechanism by which Hsp27 controls apoptosis in human neutrophils.
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DISCUSSION |
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To establish the effect of phosphorylation on Hsp27 association with Akt, we used Hsp27 mutants in which Ser-15, Ser-78, and Ser-82 were converted to alanine or aspartic acid. Expression of these mutants showed that wild type and Hsp27-3A (Ser-15, -78, and -82 mutated to alanine) interacted with Akt, whereas Hsp27-3D (Ser-15, -78, and -82 mutated to aspartic acid) failed to interact. The ability of Akt-mediated phosphorylation of Hsp27 to regulate the interaction between Hsp27 and Akt was demonstrated in vitro, suggesting that phosphorylation of Ser-82 controls Hsp27 binding to Akt. These findings indicate that phosphorylation of Hsp27 by either MAPKAPK-2 or Akt could be responsible for dissociation of Hsp27 from Akt following neutrophil stimulation.
Both Akt and Hsp27 are reported to promote cell survival by inhibiting
apoptosis. The mechanisms by which Akt inhibit apoptosis are proposed to
include phosphorylation of the pro-apoptotic Bcl-2 family member BAD,
phosphorylation and inhibition of Forkhead transcription factors,
phosphorylation and inhibition of caspase 9, phosphorylation of
I kinase, and activation of apoptosis signal-regulated kinase 1
(Ask1)
(2229).
It has been postulated that Hsp27 inhibits apoptosis through inactivation of
caspase 3, caspase 9, and cytochrome c
(3134).
Hsp27 has also been shown bind to an adaptor protein Daxx, preventing
association of Daxx with Fas and Ask-1
(35). We reported previously
that Akt plays a significant role in the inhibition of constitutive neutrophil
apoptosis by cytokines, chemokines, and chemoattractants
(4,
5). The present study suggests
that one mechanism by which Hsp27 regulates neutrophil apoptosis is through
control of Akt activation. Introduction of anti-Hsp27 antibodies into
neutrophils blocked Hsp27 interaction with Akt and inhibited Akt activation.
These data indicate that Hsp27 association with the Akt signaling complex is
necessary for Akt activation in human neutrophils. Introduction of these
antibodies also resulted in a marked increase in neutrophil apoptosis that was
rescued by introduction of constitutively active Akt.
Based on our data and previous reports, we propose the following model for Akt activation. Inactive Akt exists in the cytosol complexed with p38 MAPK, MAPKAPK-2, and Hsp27. Through the interaction with both Akt and MAPKAPK-2, Hsp27 may act as a scaffolding protein. PI-3K-generated phosphoinositides induce translocation of the Akt complex to the plasma membrane, activate PDK1, and activate p38 MAPK activity (21). Binding of Akt to phosphoinositides may produce conformational changes, bringing MAPKAPK-2 into close proximity with Ser-473. As we have described previously (21), MAPKAPK-2 activation by p38 MAPK results in Ser-473 phosphorylation of Akt in human neutrophils, forming a docking site for PDK1 (43, 44). Active PDK1 binds to this docking site and phosphorylates Thr-308, resulting in full activation of Akt. Active Akt provides survival signals. Phosphorylation of Hsp27 by MAPKAPK-2 or Akt leads to dissociation of Hsp27 from the complex, which may promote independent survival signals. Determination if Hsp27 regulation of Akt activation represents a general pathway or is unique to neutrophils awaits further experimentation.
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FOOTNOTES |
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Both authors contributed equally to this work.
¶ To whom correspondence should be addressed: Molecular Signaling Group, Donald E. Baxter Biomedical Research Bldg., 570 S. Preston St., Louisville, KY 40202-1764. Tel.: 502-852-0014; Fax: 502-852-4384; E-mail: mrane{at}louisville.edu.
1 The abbreviations used are: PI-3K, phosphatidylinositol 3-kinase; Akt,
protein kinase B; PDK1/PDK2, 3-phosphoinositide-dependent kinase-1/2; fMLP,
fMet-Leu-Phe; MAPK, mitogen-activated protein kinase; MAPKAPK-2,
MAPK-activated protein kinase-2; Hsp27, heat shock protein 27; GST,
glutathione S-transferase; FITC, fluorescein isothiocyanate; wt, wild
type.
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REFERENCES |
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