p38 Kinase-dependent MAPKAPK-2 Activation Functions as 3-Phosphoinositide-dependent Kinase-2 for Akt in Human Neutrophils*

Akt activation requires phosphorylation of Thr 308 and Ser 473 by 3-phosphoinositide-dependent kinase-1 and 2 (PDK1 and PDK2), respectively. While PDK1 has been cloned and sequenced, PDK2 has yet to be identified. The present study shows that phosphatidylinositol 3-kinase-dependent p38 kinase activation regulates Akt phosphorylation and activity in human neutrophils. Inhibition of p38 kinase activity with SB203580 inhibited Akt Ser 473 phosphorylation following neutrophil stimulation with formyl-methionyl-leucyl-phenylalanine, Fc g R cross-linking, or phosphatidylinositol 3,4,5-trisphosphate. Concentration inhibition studies showed that Ser 473 phosphorylation was inhibited by 0.3 m M SB203580, while inhibition of Thr 308 phosphorylation required 10 m M SB203580. Transient transfection of HEK293 cells with adenoviruses containing constitutively active MKK3 or MKK6 resulted in activation of both p38 kinase and Akt. Immunoprecipitation and agarose beads, while Akt, p38, and Hsp27 associate with GST-MK2- Sepharose beads. These results confirm the association of Akt, p38, MK2, and Hsp27.

a catalytic domain, and a short C-terminal tail, and is closely related to protein kinase A and protein kinase C in its aminoand 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 (PIP 3 ) 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, Thr 308 in the kinase domain and Ser 473 in the C-terminal domain. Phosphatidylinositol 3,4bisphosphate and PIP 3 activate 3-phosphoinositide-dependent kinase-1 (PDK1), which phosphorylates Thr 308 (4,12). Phosphorylation of Ser 473 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 Ser 473 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.
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 Ͼ98% 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% CO 2 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 MgCl 2 , 1 mM CaCl 2 , 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 ϫ 10 7 neutrophils were prewarmed for 5 min at 37°C prior to stimulation with fMLP. The reaction was terminated by centrifugation at 2500 ϫ 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 g/ml aprotinin, 1 g/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 ϫ g for 15 min at 4°C, and supernatants were incubated with 2 l 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 MgCl 2 , 10 mM MnCl 2 ) and incubated in a 30-l reaction mixture containing 5 Ci [␥-32 P]ATP, 1 mM dithiothreitol, 85.7 g/ml histone H2B, and kinase buffer. Reactions were incubated at 25°C for 30 min and terminated by the addition of 6 l of 6ϫ 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 Ser 473 or Thr 308 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.
GST Pull-down Assay-Neutrophils (2 ϫ 10 7 ) were lysed with 200 l of IP lysis buffer. GST-Akt agarose, GST-MK2 Sepharose, protein Aagarose, 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 l of 2ϫ 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 ϫ 10 7 ) were lysed with 200 l of IP lysis buffer. Lysates were precleared with 15 l of protein A-Sepharose beads for 1 h at 4°C with shaking. Anti-Akt (3 l), anti-MK2 (3 l), anti-Hsp27 (3 l), or anti-p38 (3 l) antiserum was added to the precleared neutrophil lysate and incubated overnight at 4°C with shaking. Protein A-Sepharose beads (15 l) 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 MgCl 2 , 10 mM MnCl 2 ) and incubated in a 30-l reaction mixture containing 3 l of [␥-32 P]ATP (1 mCi/100 l) or 1 M ATP, 1 l of active recombinant MK2 (25 ng/l), and 26 l of kinase buffer. Reactions were incubated at 30°C for 2 h, and the reaction was terminated by the addition of 30 l of 2ϫ 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 ϳ1-mm 3 sections and were taken for tryptic hydrolysis using a modification of the method of Jensen et al. (26). Essentially, the gel was washed using NH 4 HCO 3 and CH 3 CN, and then proteins were reduced p38 Kinase Regulates Akt Activation in Human Neutrophils 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-l spots. Mass spectral data were obtained using a Tof-Spec 2E (Micromass) and a 337-nm N 2 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.

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 Ser 473 phosphorylation following the addition of 3 ϫ 10 Ϫ7 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 fMLPstimulated Akt Ser 473 phosphorylation in the presence or absence of LY294002, wortmannin, PD98059, or SB203580. Fig.  1a shows that wortmannin, LY294002, and SB203580 inhibited fMLP-stimulated Akt Ser 473 phosphorylation, while PD98059 had no effect. To determine whether p38 regulation of Akt Ser 473 phosphorylation was unique to chemoattractant receptors, we examined the effect of SB203580 on Fc␥R-stimulated Akt Ser 473 phosphorylation (Fig. 1b). Pretreatment with SB203580 blocked Akt Ser 473 phosphorylation stimulated by Fc␥IIa and Fc␥IIIb receptor cross-linking. Thus, p38 kinase inhibition attenuates both formyl peptide receptor and Fc␥Rstimulated Akt phosphorylation.
Ser 473 Is More Sensitive to SB203580 Inhibition than Thr 308 -A previous study found that SB203580 inhibited PDK1 phosphorylation of Akt Thr 308 at concentrations greater than 3 M (27). To separate the effects of SB203580 on PDK1 and p38 kinase, we performed concentration inhibition experiments on phosphorylation of Thr 308 and Ser 473 . 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 Ser 473 at 0.3 M, while at least 10 M SB203580 was required to see a diminution of Thr 308 phosphorylation (Fig. 2). The concentrations of SB203580 required to inhibit Akt Thr 308 phosphorylation (10 M) are ϳ20-fold higher than the IC 50 for p38 kinase inhibition (0.3-0.5 M) (27), while Akt Ser 473 phosphorylation is inhibited by SB203580 at the IC 50 for p38 kinase. These results suggest that inhibition of p38 kinase attenuates Ser 473 phosphorylation, while Thr 308 phosphorylation is independent of p38 kinase.
p38 Kinase Mediates PIP 3 -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 PIP 3 to stimulate p38 kinase and Akt Ser 473 phosphorylation in neutrophils. PIP 3 stimulated both p38 kinase (Fig. 3a) and Akt Ser 473 phosphorylation (Fig. 3b) in a time-dependent manner with optimal stimulation at 1 min. To determine whether PIP 3 -stimulated Akt phosphorylation was mediated by p38 kinase, neutrophils were pretreated with 10 M SB203580 prior to the addition of PIP 3 . Fig. 3b shows that SB203580 inhibited PIP 3 -mediated Akt Ser 473 phosphorylation, indicating that p38 kinase activation is necessary for PI-3K-mediated activation of Akt in human neutrophils. PIP 3 also stimulated ERK activation with a time course similar to p38 activation (data not shown).
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 ATF 2 as substrate.
Akt Is Physically Associated with Components of the p38 Kinase Pathway-Previous studies reported that MK2 phosphorylates Akt Ser 473 in vitro and that Hsp27 associates only with active Akt (19,20). Coupled with our data showing that phosphorylation of Ser 473 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, 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 Ser 473 (pS473-Akt) antiserum. Pretreatment with the PI-3K inhibitors wortmannin and LY294002 and the p38 kinase inhibitor SB203580 inhibited Ser 473 phosphorylation following fMLP stimulation, whereas the MAP kinase/ ERK kinase/ERK inhibitor PD98059 did not block fMLP-stimulated Akt phosphorylation, (n ϭ 4). b, immunoblot of neutrophil lysates probed with phosphospecific Akt Ser 473 (pS473-Akt) and Akt antisera. Pretreatment with the p38 kinase inhibitor SB203580 blocked Fc␥RIIaand Fc␥RIIIb-stimulated Akt phosphorylation (n ϭ 3).

p38 Kinase Regulates Akt Activation in Human Neutrophils
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).
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 glutathioneagarose 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 Ser 473 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).

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 M 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 M 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 Ser 473 phosphorylation (Fig. 7c) and Akt activity (Fig. 7d), while a nonrelated peptide had no effect. Taken together, our results indicate that MK2 phosphorylates Ser 473 , which leads to activation of Akt in human neutrophils.
ERK Activation Is Independent of Akt-We examined the effect of pretreatment with 50 M PD98059 on PIP 3 -stimulated Akt 473 phosphorylation and found that PD98059 did not alter PIP 3 -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 fMLPstimulated 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 Thr 308 by PDK1, and phosphorylation of Ser 473 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 Ser 473 was the result of autophosphorylation following PDK1-dependent phosphorylation of Akt Thr 308 or was due to sequential phosphorylation of Thr 308 and then Ser 473 by PDK1 (34). Our results indicate that p38 kinase is FIG. 2. Effect of p38 inhibition on phosphorylation of Thr 308 /Ser 473 . Immunoblot of neutrophil lysates probed with phosphospecific Akt Ser 473 (pS473-Akt), Thr 308 (pT308-Akt), or Akt antiserum. Pretreatment with SB203580 at concentrations of 0.3 M or higher inhibited fMLP-stimulated Akt Ser 473 phosphorylation, while inhibition of fMLPstimulated Akt Thr 308 phosphorylation was not observed at concentrations below 10 M (n ϭ 3).
p38 Kinase Regulates Akt Activation in Human Neutrophils required for PIP 3 -stimulated activation of p38 kinase and Akt in human neutrophils, and PIP 3 -dependent phosphorylation of Ser 473 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 IC 50 for p38 kinase. We excluded this explanation for our results by demonstrating different SB203580 concentration-inhibition curves for Thr 308 and Ser 473 phosphorylation. SB203580 at the IC 50 for p38 kinase (0.3 M) inhibited Ser 473 phosphorylation, while concentrations of SB203580 below 10 M failed to inhibit Thr 308 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 kinasemediated Ser 473 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 Ser 473 ; 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 Ser 473 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 Ser 473 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 Ser 473 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 pro- 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 [ 32 P]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 [ 32 P]ATP phosphorylation of ATF 2 . These results indicate that constitutively active MKK3/6 activated p38 kinase in HEK 293 cells (n ϭ 2).
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 ϭ 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.
p38 Kinase Regulates Akt Activation in Human Neutrophils teins, 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 pulldown, 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 as-sociation of Hsp27 with Akt, MK2, and p38 kinase following Akt, p38, or MK2 immunoprecipitation and following GST pulldown 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 Ser 473 on Akt. Hsp27 dissociates 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 [ 32 P]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 [ 32 P]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 M ATP. The samples were run on SDS-PAGE and immunoblotted with phosphospecific Akt Ser 473 (pS473-Akt). Active recombinant MK2 phosphorylated Ser 473 of Akt in all of the immunoprecipitates.
FIG. 7. Effect of MK2 inhibitory peptide on MK2 and Akt activation in human neutrophils. a, concentrationinhibition assay of MK2 inhibitor peptide in neutrophils. Preincubation of human neutrophils with 160 M 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 M MK2 inhibitory peptide or 160 M EGFR peptide and [ 32 P]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 Ser 473 (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.
p38 Kinase Regulates Akt Activation in Human Neutrophils 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.