Heat Shock Protein 27 Controls Apoptosis by Regulating Akt Activation*

Madhavi J. Rane {ddagger} § , Yong Pan {ddagger} §, Saurabh Singh {ddagger}, David W. Powell ||, Rui Wu {ddagger}, Timothy Cummins {ddagger}, Qingdan Chen {ddagger}, Kenneth R. McLeish {ddagger} || ** and Jon B. Klein {ddagger} ** {ddagger}{ddagger}

From the Departments of {ddagger}Medicine and ||Biochemistry and Molecular Biology, and the {ddagger}{ddagger}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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of the serine-threonine kinase Akt by cytokines, chemokines, and bacterial products delays constitutive neutrophil apoptosis, resulting in a prolonged inflammatory response. We showed previously that Akt exists in a signaling complex with p38 MAPK, MAPK-activated protein kinase-2 (MAPKAPK-2), and heat shock protein-27 (Hsp27); and Hsp27 dissociates from the complex upon neutrophil activation. To better understand the regulation of this signaling module, the hypothesis that Akt phosphorylation of Hsp27 regulates its interaction with Akt was tested. The present study shows that Akt phosphorylated Hsp27 on Ser-82 in vitro and in intact cells, and phosphorylation of Hsp27 resulted in its dissociation from Akt. Additionally, the interaction between Hsp27 and Akt was necessary for activation of Akt in intact neutrophils. Constitutive neutrophil apoptosis was accelerated by sequestration of Hsp27 from Akt, and this enhanced rate of apoptosis was reversed by introduction of constitutively active recombinant Akt. Our results define a new mechanism by which Hsp27 regulates apoptosis, through control of Akt activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are constitutively committed to apoptosis from the time they enter the circulation (13). The rate at which they undergo apoptosis is delayed by inflammatory products such as lipopolysaccharide, interleukin-8, leukotriene B4, and granulocyte-macrophage colony-stimulating factor (4, 5). This delay of neutrophil apoptosis results in enhanced host defense; however, inappropriate delay of neutrophil apoptosis is associated with generalized inflammation and multiorgan failure of the systemic inflammatory response syndrome (6, 7). The mechanisms by which inflammatory products delay neutrophil apoptosis are incompletely understood.

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{kappa}{alpha} 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
fMLP and histone H2B were obtained from Sigma. Active recombinant Akt, active recombinant MAPKAPK-2, anti-phospho-Ser-15-Hsp27, and anti-phospho-Ser-78-Hsp27 were obtained from Upstate Biotechnology, Inc. (Lake placid, NY). Anti-phospho-Ser-473-Akt, anti-PH domain Akt, anti-Akt, and anti-phospho-Ser-82-Hsp27 antisera were obtained from Cell Signaling Inc. (Beverly, MA). Mouse isotype control antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant Hsp27 and anti-mouse Hsp27 were obtained from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada). Goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Vector Laboratories (Burlingame, CA). The synthetic AKTide-2T inhibitory peptide described previously by Obata et al. (36) (Ala-Arg-Lys-Arg-Glu-Arg-Thr-Tyr-Ser-Phe-Gly-His-His-Ala) and the scrambled peptide (His-Ala-Lys-Glu-Ala-Tyr-Gly-His-Ala-Arg-Arg-Phe-Arg-Ala) were synthesized by the Macromolecular Structure Analysis Facility at the University of Kentucky (Lexington, KY). BioPORTER reagent was obtained from Gene Therapy Systems, Inc. (San Diego, CA). pUse-Akt-wt (wild type) and pUseAktCA (myristoylated constitutively active Akt) constructs were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The Hsp27 constructs pcDNA3.1-Hsp27-wt, pcDNA3.1-Hsp27-3A, and pcDNA3.1-Hsp27-3D were kindly provided by Dr. Rainer Benndorf (University of Michigan, Ann Arbor, MI).

Isolation of Neutrophils and Culture Conditions—Neutrophils 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 Blotting—Neutrophils 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 Neutrophils—We 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 Activity—Akt 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 [{gamma}-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 Hsp27—Active recombinant Akt (400 ng) or active recombinant MAPKAPK-2 (40 ng) was incubated in 30 µl of reaction mixture containing 5 µCi of [{gamma}-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 Vectors—To 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-Sepharose—GST-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 Assay—Neutrophils (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 Proteins—GST, 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-Hsp27—Two 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 Quenching—Neutrophils (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)
where A is the mean fluorescence after trypan blue quenching; B is the mean fluorescence measured in 0.02 M sodium acetate buffer, and bgr is the background fluorescence of control neutrophils (38, 39).

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 3–4 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 3–5 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 Binding—Neutrophils 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Akt Interacts Directly with Hsp27—We reported previously (21) that several members of the p38 MAPK signal transduction pathway, including p38 MAPK, MAPKAPK-2, and heat shock protein 27 (Hsp27), co-immunoprecipitated with Akt from neutrophil lysates. Stimulation by chemoattractants resulted in dissociation of Hsp27 from this complex. Hsp27 is known to associate with and be phosphorylated by MAPKAPK-2 (40). These observations suggested that Hsp27 association with the Akt signaling module resulted from direct interaction with MAPKAPK-2, not with Akt. To define the protein-protein interactions within the module, the ability of recombinant Hsp27 to bind Akt directly was determined by GST pull-down assays. Recombinant Hsp27 was incubated with glutathione-Sepharose coupled to GST or GST-Akt. Fig. 1A shows an immunoblot of this precipitate for Hsp27 demonstrating that recombinant Hsp27 bound to GST-Akt (lane 2) but not to GST alone (lane 1). To confirm this interaction, recombinant Akt was precipitated with glutathione-Sepharose coupled to GST or GST-Hsp27. Fig. 1B shows that recombinant Akt bound to GST-Hsp27 (lane 2) but not GST alone (lane 1). In each case equivalent amounts of recombinant protein were added, as shown in lane 3. These results indicate a direct in vitro interaction between Hsp27 and Akt.



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FIG. 1.
Akt interacts directly with Hsp27. A, the ability of Akt to interact directly with Hsp27 was examined by a GST pull-down assay in which 25 ng of recombinant Hsp27 was incubated with 20 µl of glutathione-Sepharose coupled to GST or GST-Akt/PKB. Precipitated proteins were separated by 10% SDS-PAGE, and Hsp27 was detected by immunoblot analysis. The immunoblot (IB) shows that recombinant Hsp27 precipitated with GST-Akt-Sepharose (lane 2) but not with GST-Sepharose (lane 1). Lane 3 shows an immunoblot of 25 ng of recombinant Hsp27. The results are representative of three separate experiments. B, to confirm the interaction between Akt and Hsp27, a GST pull-down assay was performed in which 25 ng of recombinant Akt was incubated with 20 µl of glutathione-Sepharose coupled to GST or GST-Hsp27. Precipitated proteins were separated by 10% SDS-PAGE, and Akt was detected by immunoblot analysis. Recombinant Akt precipitated with GST-Hsp27-Sepharose (lane 2) but not with GST-Sepharose (lane 1). Lane 3 shows an immunoblot of 25 ng of recombinant Akt. The results are representative of three separate experiments.

 

Akt Phosphorylates Hsp27—The 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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FIG. 2.
In vitro Akt phosphorylation of Hsp27. A, to determine the ability of Akt to phosphorylate Hsp27, 1 µg of recombinant (Recomb) Hsp27, 400 ng of active recombinant Akt, and 5 µCi of [{gamma}-32P]ATP were incubated in the presence or absence of 20 µM scrambled peptide or AKTide-2T. Lane 1 contains a reaction mixture to which recombinant Hsp27 was added in the absence of active recombinant Akt to exclude nonspecific phosphorylation. Proteins were separated by 10% SDS-PAGE, and autoradiography was performed. The autoradiograph shows Hsp27 is phosphorylated by Akt in the presence (lane 3) or absence (lane 2) of scrambled peptide. The Akt inhibitory peptide, AKTide-2T, markedly inhibited Hsp27 phosphorylation (lane 4). Equal loading of recombinant Hsp27 was demonstrated by Coomassie Blue staining. The results are representative of three separate experiments. B, to determine the site of Akt phosphorylation, 1 µg of recombinant Hsp27 and 400 ng of active recombinant Akt (lane 1) or 1 µg of recombinant Hsp27 and 40 ng of active recombinant MAPKAPK-2 (lane 3) were incubated in the presence 1 µM ATP for 30 min at 25 °C. Proteins were separated by 10% SDS-PAGE and subjected to immunoblot (IB) analysis with anti-phospho-Ser-15-, -Ser-78-, and -Ser-82-Hsp27 antibodies. Recombinant Hsp27 in the presence of 1 µM ATP but without any added kinase (lane 2) was included as a negative control. Separate gels were immunoblotted for Ser(P)-82-, Ser(P)-78-, and Ser(P)-15-Hsp27. Each gel was stripped and reprobed for total Hsp27 to demonstrate equal loading of Hsp27. The figure demonstrates that MAPKAPK-2 phosphorylates Ser-82, Ser-78, and Ser-15, whereas Akt phosphorylates only Ser-82. The results are representative of three separate experiments.

 

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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FIG. 3.
Akt phosphorylates Ser-82 on Hsp27 in intact cells. To demonstrate that Akt phosphorylates Hsp27 in intact cells, HEK-293 cells were co-transfected with vectors (pcDNA3.0 and pUse) or c-Myc-tagged constitutively active (myristoylated) Akt (AktCA) and Hsp27-wt. Twenty-four hours later HEK-293 cells were lysed, and the lysates were subjected to immunoblot analysis with phospho-specific Hsp27 antibodies. Separate gels were immunoblotted for Ser(P)-82-, Ser(P)-78-, and Ser(P)-15-Hsp27. The in vitro phosphorylation of 1 µg of Hsp27 by 40 ng of active recombinant MAPKAPK-2 was included as a positive control on each gel (lane 3). The figure shows that co-expression of Akt-CA and Hsp27-WT resulted in phosphorylation of Ser-82 but not Ser-15 or Ser-78. Vector-transfected cells demonstrated no Hsp27 phosphorylation. Blots from each gel were stripped and reprobed with anti-Hsp27 and anti-c-Myc antibodies to confirm overexpression of Hsp27 and Akt. A representative immunoblot for gel 1 demonstrating overexpression of Hsp27 and c-Myc-tagged Akt is shown. Similar results were seen for all gels. These results demonstrate that Akt phosphorylates Hsp27 on Ser-82 but not on Ser-15 or Ser-78 in intact cells. The results are representative of three separate experiments.

 

Phosphorylation of Hsp27 Regulates Association with the Akt Signal Module—We 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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FIG. 4.
Phosphorylation of Hsp27 regulates association with the Akt signal module. A, to determine the effect of phosphorylation on Hsp27 association with Akt, vector (pcDNA3.0), Hsp27 wild type (Hsp27-wt), a phosphorylation-dead mutant of Hsp27 (Hsp27-3A) or a phosphorylation-mimicking mutant of Hsp27 (Hsp27-3D) was overexpressed in HEK-293 cells. Cell lysates were subjected to GST-Akt pull-down assay and immunoblotted for Hsp27 (panel 2). Increased Hsp27 binding to GST-Akt was seen in cell lysates from Hsp27-wt and Hsp27-3A-transfected cells. Hsp27 precipitated by GST-Akt did not differ between Hsp27-3D and vector-transfected cells. Immunoblots (IB) were stripped and reprobed for Akt to ensure equal amounts of GST-Akt were added to all samples (panel 1). Lysates from transfected cells were also subjected to immunoblot analysis for Hsp27 to ensure overexpression of all Hsp27 constructs (panel 3). The results are representative of two separate experiments. B, to confirm that Akt phosphorylation of Hsp27 inhibits association, 25 ng of recombinant wild type Hsp27 or 25 ng of recombinant Hsp27 subjected to in vitro phosphorylation (pHsp27) by active recombinant Akt was incubated for 30 min at 25 °C with 20 µl of glutathione-Sepharose-coupled GST or GST-Akt. Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted for Hsp27. Recombinant Hsp27 (lane 3), but not Akt-phosphorylated Hsp27 (lane 4), precipitated with GST-Akt-Sepharose. Immunoblots of recombinant Hsp27 and pHsp27 added to the GST pull-down assay are shown in lanes 5 and 6. The results are representative of two separate experiments and demonstrate that direct phosphorylation of Hsp27 prevents association with Akt.

 

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 71–74% of FITC-IgG and FITC-anti-Hsp27 were internalized by neutrophils.



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FIG. 5.
Anti-Hsp27 antibody disrupts Akt-Hsp27 interaction and results in loss of Akt activation. A, to demonstrate entry and distribution of anti-Hsp27 antibody into neutrophils mouse monoclonal Hsp27 antibody was FITC-conjugated and incubated with neutrophils at37 °Cfor4hbythe method of Tezel and Wax (37) (see "Experimental Procedures"), and cells were viewed by confocal microscope. Confocal image suggests cytoplasmic localization of the anti-Hsp27 antibody. Cells pretreated without antibody failed to demonstrate fluorescence. Pretreatment with FITC-anti-Hsp27 antibody for 4 h demonstrates diffuse staining throughout the cell on confocal images. B, to examine the effect of anti-Hsp27 antibody on association of Hsp27 with Akt, neutrophils were incubated with or without 20 µg of monoclonal anti-Hsp27 or mouse isotype control antibodies for 4 h at 37 °C. Cell lysates were immunoprecipitated with anti-Akt antibody, and precipitated proteins were separated by 10% SDS-PAGE. Following transfer to nitrocellulose, immunoblot (IB) analysis for Hsp27 and Akt was performed. Equivalent amounts of Akt were precipitated under all conditions (panel 2). Hsp27 was detected only in untreated and mouse isotype control antibody-loaded neutrophils, whereas Hsp27 was absent from Akt precipitates from neutrophils in which anti-Hsp27 antibody was introduced (panel 1). The results are representative of three separate experiments.

 

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TABLE I
Trypan blue quenching confirms neutrophils internalize FITC-conjugated antibodies

To determine if anti-Hsp27 and isotype control antibodies were internalized or attached to the surface of neutrophils, antibodies were FITC-conjugated and incubated with neutrophils at 37 °C for 4 h followed by incubation with 0.02% trypan blue at room temperature for 30 min. Trypan blue is a non-penetrating dye for viable cells that quenches fluorescence of extracellular FITC. Following trypan blue quenching neutrophils were washed in PBS and resuspended in 0.02 M sodium acetate buffer, pH 5.8. The percentage of FITC-labeled antibody inside the neutrophils was determined by flow cytometry and calculated using Equation 1. These results are expressed as the mean ± S.E. of mean channel fluorescence for three separate experiments. The final column shows the calculated intracellular fluorescence indicating that 71–74% of the FITC-conjugated antibodies were internalized by neutrophils.

 

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 Activation—The 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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FIG. 6.
Disruption of Akt-Hsp27 interaction results in loss of Akt activation. A, to determine the role of Hsp27 association with Akt on Akt activity, neutrophils were incubated with 20 µg of mouse isotype control or 20 µg of monoclonal anti-Hsp27 antibody (ab) for 2 h at 37 °C prior to stimulation with 1 µM fMLP. Following a 2-min stimulation, neutrophils were lysed, and the lysates were subjected to an in vitro immunoprecipitation kinase assay for Akt using histone H2B as substrate. The phosphorylated protein bands were quantitated by a densitometer. The controls were normalized to one, and the results are expressed as mean arbitrary densitometry units ± S.E. for three separate experiments. Untreated (control) and mouse isotype control antibody-loaded neutrophils showed a significant increase in Akt kinase activity following fMLP stimulation. fMLP failed to stimulate an increase in Akt kinase activity in anti-Hsp27 antibody-loaded neutrophils. The asterisk indicates p < 0.05 by Student's t test. B, to determine the effect of Hsp27 sequestration on Akt phosphorylation, untreated (control), isotype control antibody-loaded, and anti-Hsp27 antibody-loaded neutrophils were incubated with or without 1 µM fMLP for 2 min and lysed, and the lysate proteins were separated by 10% SDS-PAGE. Following transfer to nitrocellulose, immunoblot (IB) analysis for Ser(P)-473-Akt and total Akt was performed. Panel 2 shows that equivalent amounts of total Akt were detected in all lysates. Panel 1 shows that fMLP stimulation resulted in Ser-473 phosphorylation of Akt in control and isotype control antibody-loaded neutrophils. Introduction of Hsp27 antibody into neutrophils blocked fMLP-stimulated Ser-473 phosphorylation of Akt. The results are representative of two separate experiments.

 

Disruption of Akt-Hsp27 Interaction Enhances Neutrophil Apoptosis—The 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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FIG. 7.
Treatment with anti-Hsp27 increases neutrophil apoptosis. To determine the role of Hsp27 in constitutive apoptosis, neutrophils were incubated with 20 µg of mouse isotype control antibody or 20 µg of monoclonal anti-Hsp27 antibody for 4 h at 37 °C. Apoptosis was assessed by morphologic changes determined by electron microscopy and by annexin V binding determined by confocal microscopy. A shows the confocal images of FITC-conjugated annexin V in the two groups of cells. Annexin V binding to a majority of neutrophils was observed following loading with anti-Hsp27 antibody, whereas no annexin V binding was seen in cells loaded with isotype control antibody. B shows the electron microscope images of isotype control and anti-Hsp27 antibody-loaded cells. Features typical of apoptosis, including membrane blebbing and nuclear condensation, were observed in anti-Hsp27 antibody-loaded neutrophils but not in isotype control antibody-loaded cells.

 

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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FIG. 8.
Active recombinant Akt rescues anti-Hsp27-induced neutrophil apoptosis. To determine whether the increased rate of constitutive apoptosis following Hsp27 sequestration was related to inhibition of Akt activity, mouse isotype control antibody, anti-Hsp27 antibody, or both anti-Hsp27 antibody and active recombinant Akt were incorporated into BioPORTER vesicles and introduced into neutrophils by incubation for 4 h at 37 °C. Apoptosis was determined by confocal microscopic imaging of cells following staining with FITC-conjugated annexin V. Panel 1 represents light microscopic images of isotype control antibody (lane 1), anti-Hsp27 antibody (lane 2), and anti-Hsp27 antibody and active recombinant Akt (lane 3)-loaded neutrophils. Isotype control antibody-loaded neutrophils failed to demonstrate morphologic features of apoptosis, whereas neutrophils loaded with anti-Hsp27 antibody demonstrate significant membrane blebbing. Simultaneous introduction of active recombinant Akt and anti-Hsp27 antibody preserved normal morphology. Panel 2 shows that annexin V binding was observed only in anti-Hsp27 antibody loaded neutrophils. No annexin V binding was detected following introduction of isotype control antibody or simultaneous introduction of anti-Hsp27 and recombinant active Akt. These data indicate that simultaneous addition of active recombinant Akt and anti-Hsp27 antibody rescued neutrophils from apoptosis. Results are representative of four separate experiments.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study describes a novel mechanism for Hsp27 regulation of neutrophil apoptosis and provides the first description of Hsp27 regulation of a kinase-signaling module. We reported previously that Akt formed a stable complex with components of the p38 MAPK cascade, including p38 MAPK, MAPKAPK-2, and Hsp27, in human neutrophils (21). Stimulation by chemoattractants or immune complexes resulted in Akt activation, which was accompanied by dissociation of Hsp27 from the complex. Based on the identification of Hsp27 as a substrate of MAPKAPK-2 (40), we expected that Hsp27 would be bound to the complex through a direct interaction with MAPKAPK-2. Contrary to this expectation, the present study shows that Hsp27 directly interacts with Akt. These results are supported by Konishi et al. (42), who reported that Hsp27 associated with Akt in COS-7 cells following heat shock, hyperosmolarity, or chemical stress. The differences in the effect of cell stimulation on Hsp27 association with Akt between the two studies are likely related to different stimuli. Consistent with the results of Konishi et al. (42), we found that heat shock enhanced association of Hsp27 with Akt in human neutrophils (data not shown). The association of Hsp27 with Akt suggested the hypothesis that Akt might phosphorylate Hsp27. Konishi et al. (42) showed that phosphorylated Hsp27 associated with Akt, but the site of phosphorylation and the responsible kinase were not identified. We identified putative Akt phosphorylation sites on Hsp27 using the web-based Scansite motif analysis software (41). Three of the putative sites, Ser-82, Ser-78, and Ser-15, were identified previously as sites of MAPKAPK-2 phosphorylation (39). The ability of Akt and MAPKAPK-2 to phosphorylate Ser-82, Ser-78, and Ser-15 was compared using phosphorylation site-specific antibodies. Our data indicate that Akt phosphorylates Hsp27 on Ser-82, but not on Ser-78 or Ser-15, both in vitro and in intact cells, whereas MAPKAPK-2 phosphorylated all three sites. The ability of Akt to phosphorylate 2 other putative sites, Thr-143 and Ser-9, was not examined.

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{kappa}{alpha} 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.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HL66358-02 (to J. B. K.), Department of Veterans Affairs Merit Review (to K. R. M. and J. B. K.), and an American Heart Association Beginning Grant-in-aid (to M. J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

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. Back



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