Title: Protein Phosphatase 2A Regulates Apoptosis in Neutrophils by Dephosphorylating Both p38 MAPK and Its Substrate Caspase 3
Abstract: The induction of apoptosis in neutrophils is an essential event in the resolution of an inflammatory process. We found recently that the reduction of the activity of the neutrophil survival factor p38 MAPK and dephosphorylation and thus activation of caspases must occur to initiate such cell death in these leukocytes. Here, we report a previously undetected early and transient activation of protein phosphatase 2A (PP2A) in neutrophils undergoing apoptosis. The pharmacological inhibition of this phosphatase during Fas-induced apoptosis augmented the levels of phosphorylation of both p38 MAPK and caspase 3, resulting in a decreased activity of caspase 3 and an increased neutrophil survival. The complementary finding of a time-dependent association among PP2A, p38 MAPK, and caspase 3 in intact neutrophils indicated that there is a direct regulatory link among these signaling enzymes during Fas-provoked apoptosis. Moreover, immunoprecipitated active p38 MAPK and recombinant phosphorylated caspase 3 were dephosphorylated by exposure to purified PP2A in vitro. Consequently, the early and temporary activation of PP2A in neutrophils impaired not only the p38 MAPK-mediated inhibition of caspase 3 but also restored the activity to caspase 3 that had already been phosphorylated and thereby inactivated. These findings indicate that PP2A plays a pivotal dual role in the induction of neutrophil apoptosis and therefore also in the resolution of inflammation. The induction of apoptosis in neutrophils is an essential event in the resolution of an inflammatory process. We found recently that the reduction of the activity of the neutrophil survival factor p38 MAPK and dephosphorylation and thus activation of caspases must occur to initiate such cell death in these leukocytes. Here, we report a previously undetected early and transient activation of protein phosphatase 2A (PP2A) in neutrophils undergoing apoptosis. The pharmacological inhibition of this phosphatase during Fas-induced apoptosis augmented the levels of phosphorylation of both p38 MAPK and caspase 3, resulting in a decreased activity of caspase 3 and an increased neutrophil survival. The complementary finding of a time-dependent association among PP2A, p38 MAPK, and caspase 3 in intact neutrophils indicated that there is a direct regulatory link among these signaling enzymes during Fas-provoked apoptosis. Moreover, immunoprecipitated active p38 MAPK and recombinant phosphorylated caspase 3 were dephosphorylated by exposure to purified PP2A in vitro. Consequently, the early and temporary activation of PP2A in neutrophils impaired not only the p38 MAPK-mediated inhibition of caspase 3 but also restored the activity to caspase 3 that had already been phosphorylated and thereby inactivated. These findings indicate that PP2A plays a pivotal dual role in the induction of neutrophil apoptosis and therefore also in the resolution of inflammation. Human neutrophils are short-lived cells that undergo apoptosis soon after they are released from the bone marrow (1Cartwright G.E. Athens G.W. Wintrobe M.M. Blood. 1964; 24: 780-803Crossref PubMed Google Scholar, 2Grigg J.M. Savill J.S. Sarraf C. Haslett C. Silverman M. Lancet. 1991; 338: 720-722Abstract PubMed Scopus (179) Google Scholar, 3Savill J.S. Wyllie A.H. Henson J.E. Walport M.J. Henson P.M. Haslett C. J. Clin. Investig. 1989; 83: 865-875Crossref PubMed Scopus (1353) Google Scholar). The life span of these leukocytes is regulated by endogenous factors and extracellular stimuli, which in turn determine the length of an inflammatory process. Neutrophil apoptosis can be delayed by several factors in the local environment, such as lipopolysaccharides and granulocyte-macrophage colony-stimulating factor, whereas it is accelerated by Fas ligand (4Haslett C. Br. Med. Bull. 1997; 53: 669-683Crossref PubMed Scopus (134) Google Scholar, 5Ward I. Dransfield I. Chilvers E.R. Haslett I. Rossi A.G. Trends Pharmacol. Sci. 1999; 20: 503-509Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Knowledge about the signaling mechanisms involved in the regulation of apoptosis in human neutrophils is still fairly limited, in part because of methodological limitations, that is, the short life span of neutrophils rules out transfection, and microinjection of these cells is not feasible (6Glogauer M. Hartwig J. Stossel T. J. Cell Biol. 2000; 150: 785-796Crossref PubMed Scopus (101) Google Scholar). In previous studies, our research team (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar) and other investigators (8Aoshiba K. Yasui S. Hayashi M. Tamaoki J. Nagai A. J. Immunol. 1999; 162: 1692-1700PubMed Google Scholar) have observed that p38 mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; PP, protein phosphatase; Ab, antibody; Cal, calyculin; JNK, c-Jun NH2-terminal kinase; ATF, activating transcription factor. exhibits constitutive activity in freshly isolated human neutrophils, and we noted that this activity is transiently decreased and subsequently regained during spontaneous and Fas-induced apoptosis. Furthermore, we proposed that the constitutive activity of p38 MAPK in neutrophils is probably caused by factors present in the blood; therefore, the elimination of such elements during the isolation of these primary cells could explain the subsequent decrease in this activity. In the same study, we also found that the inactivation of the intrinsic p38 MAPK activity increased the number of apoptotic neutrophils. The mechanism underlying Fas-induced inactivation of p38 MAPK has not been identified. However, in a very recent study (9Alvarado-Kristensson M. Melander F. Leandersson K. Ronnstrand L. Wernstedt C. Andersson T. J. Exp. Med. 2004; 199: 449-458Crossref PubMed Scopus (188) Google Scholar), we identified caspase 8 and 3 as downstream targets of p38 MAPK in neutrophils, and we found that p38 MAPK-induced phosphorylation of the serine 150 residue of caspase 3 and the serine 364 residue of caspase 8 impaired the activities of these caspases, thereby favoring survival of the neutrophils. These observations suggest that neutrophil apoptosis is initiated by and dependent on the inactivation of p38 MAPK. The molecular mechanisms whereby extracellular factors modify the activity of p38 MAPK in neutrophils undergoing apoptosis are currently unknown. The serine/threonine kinase p38 MAPK is activated by the concomitant phosphorylation of tyrosine and threonine residues within a conserved threonine-glycine-tyrosine motif in its activation loop (10Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar). Accordingly, p38 MAPK can be down-regulated by various protein phosphatases that dephosphorylate either the threonine or the tyrosine residue or both (11Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (712) Google Scholar). One such enzyme is protein phosphatase type 2A (PP2A). PP2A is a phosphoserine/phosphothreonine phosphatase, which has been reported (12Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (778) Google Scholar, 13Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (242) Google Scholar) to dephosphorylate the threonine residue of p38 MAPK and thereby impair its activity. PP2A is also a highly conserved phosphatase that plays essential roles in several signal transduction pathways, translational control, endosome trafficking, cell cycle regulation, and apoptosis (14Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). The PP2A holoenzyme consists of a 36-kDa catalytic subunit C (Cα or Cβ) and a 65-kDa structural subunit A (Aα or Aβ), that together form the inseparable AC core dimer (PP2AAC). The A subunit appears to function primarily as a scaffolding structure that assembles the different subunits into the holoenzyme complex. PP2A also comprises a third highly diversified regulatory subunit B (B, B′, or B″) that regulates the substrate specificity and subcellular localization of the holoenzyme complex (14Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). Theoretically, the various combinations of the subunits can give rise to a large number of different PP2A holoenzymes. In the present study, we conducted experiments to determine whether PP2A is involved in the regulation of neutrophil apoptosis and thus also in the termination of inflammatory responses. Cells—Human neutrophils were isolated under endotoxin-free conditions from whole blood drawn from healthy volunteers. The neutrophils were isolated by dextran sedimentation followed by a brief hypotonic lysis of contaminating erythrocytes and centrifugation on Ficoll-Paque (Amersham Biosciences) at 4 °C, as described originally (15Bo ̈yum A. Scand. J. Clin. Lab. Investig. Suppl. 1968; 97: 77-89PubMed Google Scholar). The isolated cells were immediately suspended at 4 °C in a complete RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum, 0.2 mml-glutamine, and 100 μg/ml penicillin and streptomycin at a concentration of 5 × 106 cells/ml. Cell aliquots for the time 0 values were taken immediately after the cells were suspended in this medium. The incubations of the cells were then initiated by placing them in multiwell cell culture plates at 37 °C in a humidified 5% CO2 and 95% air environment. Fas receptors were engaged by incubating the cells with an anti-Fas monoclonal Ab (150 ng/ml) (catalog no. CH-11, Beckman) for the indicated periods of time. Immunoprecipitation and Western Blot Analysis—Neutrophils were lysed in a buffer (if not otherwise indicated) containing 1% (v/v) Triton X-100, Tris 20 mm (pH 7.5), 150 mm NaCl, 1 mm EGTA, 1 mm EDTA, 1 mm β-glycerolphosphate, 2.5 mm sodium pyrophosphate, 0.1 mm Na3VO4, 1 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride after which the remaining cell debris was removed by centrifugation (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar). The supernatant was precleared with protein G PLUS-agarose (Oncogene Science), after which one of the following was added: agarose-conjugated anti-phospho-p38 MAPK (Thr-180/Tyr-182) IgG1 Ab (New England Biolabs), anti-p38 MAPK IgG Ab (Santa Cruz Biotechnology), or anti-caspase 3 polyclonal IgG Ab (Santa Cruz Biotechnology). As a control, one of the following Abs was added: agarose-conjugated anti-c-Myc IgG1 (Clontech), anti-Fyn IgG1, non-immune anti-rabbit IgG (Santa Cruz Biotechnology), non-agarose-conjugated anti-PP2A IgG2bκ (clone 1D6) (Upstate Biotechnology) or anti-E-cadherin IgG2. The samples were subsequently incubated overnight under rotation at 4 °C. Non-agarose-conjugated immunocomplexes were allowed to absorb to protein G-agarose for 1 h at 4 °C. The immunoprecipitates were washed four times with lysis buffer, and either the precipitates listed above or lysates of intact cells were boiled in a sample buffer (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar), after which the proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. The membranes were analyzed with the following: anti-PP2A IgG2bκ Ab (clone 1D6) or monoclonal Ab 4G10 (mouse anti-phosphotyrosine) (Upstate Biotechnology); anti-caspase 3 IgG Ab (Santa Cruz Biotechnology), anti-p38 MAPKα IgG Ab, or anti-hemagglutinin IgG2a Ab (catalog no. F-7, Santa Cruz Biotechnology); anti-phospho-p38 MAPK (Thr-180/Tyr-182) IgG1 Ab, anti-phospho-ATF2 (Thr-71) IgG Ab (New England Biolabs), or anti-phosphoserine IgMκ Ab (catalog no. 16B4, Biomol). As indicated in the figure legends, certain blots were stripped and reprobed according to the instructions of the manufacturers. PP2A Phosphatase Assay—The PP2A phosphatase assay was performed as stipulated by the manufacturer (serine/threonine assay kit 1, Upstate Biotechnology). Briefly, neutrophils were preincubated on ice for 15 min in the absence or presence of 3 μm PP1 (Biomol) or for 45 min with or without 20 μm DEVD-fluoromethyl ketone (ICN Biomedical, Inc.). Thereafter, the cells were exposed to anti-Fas Ab at 37 °C for different periods of time and were then lysed as indicated above in a lysis buffer (1% Nonidet P-40, Tris 50 mm (pH 7.5), 137 mm NaCl, 2 mm NaF, 10% (v/v) glycerol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 1 mm Na3VO4). Subunit C (catalytic) of PP2A was immunoprecipitated as described above. The precipitates were washed by centrifugation twice in the lysis buffer and twice in a serine/threonine kinase buffer (100 μm CaCl2 and 50 mm Tris-HCl (pH 7.0)). The pellet was resuspended in the kinase buffer, and PP2A activity was measured after the addition of 250 μm phosphopeptide. The phosphatase reactions were carried out for 15 min at 30 °C. The release of phosphate from the added phosphopeptide was quantified using a malachite green reagent. In short, a sample (25 μl) of the reaction medium was transferred to a microtiter assay plate, and 100 μl of malachite green reagent was added to each well, after which the plates were incubated for 15 min at 30 °C. Changes in absorbance were measured at 650 nm in a Fluostar plate reader (BMG Lab Technologies). The phosphatase activity in each well was determined as a percentage of the maximal activity recorded in each experiment. Alternatively, PP2A (10 units/ml) purified from human erythrocytes (Upstate Biotechnology) was added to agarose-conjugated active (phosphorylated) p38 MAPK immunoprecipitates in the presence or absence of 10 nm calyculin A (Cal A). The phosphatase reactions were carried out at 30 °C for 30 min, and the activity remaining in the p38 MAPK immunoprecipitates was measured by running a p38 MAPK phosphorylation assay in the presence of ATF2-(19–96), as described previously (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar). Expression of Human Caspase 3—The pET21b vector containing C-terminally His6-tagged human pro-caspase 3 (16Srinivasula S.M. Fernandes-Alnemri T. Zangrilli J. Robertson N. Armstrong R.C. Wang L. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. J. Biol. Chem. 1996; 271: 27099-27106Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) was generously provided by Dr. E. S. Alnemri. Recombinant human caspase 3 was purified as described previously (9Alvarado-Kristensson M. Melander F. Leandersson K. Ronnstrand L. Wernstedt C. Andersson T. J. Exp. Med. 2004; 199: 449-458Crossref PubMed Scopus (188) Google Scholar). In Vitro Assays for p38 MAPK Phosphorylation and PP2A Dephosphorylation—The p38 MAPK phosphorylation assay was performed as reported previously (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar, 9Alvarado-Kristensson M. Melander F. Leandersson K. Ronnstrand L. Wernstedt C. Andersson T. J. Exp. Med. 2004; 199: 449-458Crossref PubMed Scopus (188) Google Scholar) in the presence of recombinant caspase 3 or active ATF2-(19–96). The assays were run for 60 min at 30 °C. Thereafter, radiolabeled phosphorylated caspase 3 was either incubated in the presence of 10 units/ml PP2A (Upstate Biotechnology) or was immunoprecipitated in active PP2A from neutrophils stimulated with anti-Fas Ab for 30 min, and a PP2A phosphatase assay was run as described above. The degree of phosphorylation of recombinant caspase 3 was analyzed by autoradiography, whereas the phosphorylation of ATF2-(19–96) was assessed by Western blotting (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar). 32P Phosphorylation of Caspase 3 in Vivo—Neutrophils (5 × 107/ml) were preincubated for 1 h and 50 min at 37 °C in a calcium- and phosphate-free medium (136 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 5.0 mm NaHCO3, 5.5 mm glucose, 0.1% human bovine serum albumin, and 2 mm HEPES (pH 7.4)) supplemented with [32P]orthophosphate (2 mCi/ml). Thereafter, the cells were washed and resuspended in RPMI medium supplemented with 5% fetal calf serum, and some cells were also exposed to 40 nm Cal A for 10 min at 37 °C. Next, the cells were stimulated with the anti-Fas monoclonal Ab at 37 °C for 30 min in the absence or presence of 40 nm Cal A. Caspases were subsequently immunoprecipitated as described above using agarose-conjugated anti-caspase 3 polyclonal Ab. The degree of phosphorylation was analyzed by autoradiography performed on a PhosphorImager, and the membranes were subsequently immunoblotted for caspase 3. Fluorometric Assays for Caspase Activities—Neutrophils were first incubated in the absence or presence of 40 nm Cal A for 20 min on ice prior to exposure to anti-Fas Ab (150 ng/ml). The cells were incubated with the anti-Fas Ab for 75 min at 37 °C and then harvested and lysed as described previously (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar). DEVD-7-amino-4-methylcoumarin (Upstate Biotechnology), which is a fluorogenic substrate for caspase 3, was added to each cell lysate, and the activity of caspase 3 (cleavage of 7-amino-4-methylcoumarin) was measured as described previously (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar). Analysis of Nuclear Morphology—Neutrophils were incubated for 15 min at 4 °C in the absence or presence of 40 nm Cal A. Thereafter, the cells were washed with RPMI 1640 medium at 4 °C prior to the engagement of their Fas receptor. The cells were incubated for 3 h at 37 °C in the presence of anti-Fas Ab. Subsequently, 25 μl of each cell suspension was transferred to an Eppendorf tube, and 1 μl of a mixture of acridine orange and ethidium bromide (100 μg/ml each) was added to the cell suspension. The stained cells were examined immediately in a Nikon Eclipse E800 fluorescence microscope equipped with a 4,6-diamidino-2-phenylindole/fluorescein isothiocyanate filter. A minimum of 100 nuclei was counted in each sample, and the percentage of nuclei displaying condensed chromatin was calculated. Statistical Evaluations—All data are expressed as means ± S.D. (n < 6) or S.E. (n ≥ 6), and Student's paired t test was used for statistical analysis of the differences. Activity and Regulation of the Serine/Threonine PP2A in Neutrophils Undergoing Apoptosis—To address the possibility that a protein phosphatase regulates the previously demonstrated transient loss of p38 MAPK activity during Fas-induced apoptosis in human neutrophils (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar), we measured the phosphatase activity in immunoprecipitates of PP2A (the catalytic subunit). Spectrophotometric analysis revealed a rapid increase in PP2A activity that reached a peak after 30–45 min and declined to the starting level after 1 h at 37 °C in the absence or the presence of the anti-Fas Ab (Fig. 1A). Notably, PP2A regained its activity after 2 h of Fas-induced apoptosis (Fig. 1A). This pattern of action correlated well with the increased activity of caspase 3 we recorded previously under the same conditions (9Alvarado-Kristensson M. Melander F. Leandersson K. Ronnstrand L. Wernstedt C. Andersson T. J. Exp. Med. 2004; 199: 449-458Crossref PubMed Scopus (188) Google Scholar). Santoro et al. (17Santoro M.F. Annand R.R. Robertson M.M. Peng Y.W. Brady M.J. Mankovich J.A. Hackett M.C. Ghayur T. Walter G. Wong W.W. Giegel D.A. J. Biol. Chem. 1998; 273: 13119-13128Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) have reported that caspase 3 can cleave and thereby activate PP2A during the execution phase of apoptosis. To determine whether the second increase in PP2A activity that we observed might have been caspase 3-dependent, we used DEVD-fluoromethyl ketone to inhibit the activity of this caspase. Such treatment decreased the activity in PP2A immunoprecipitates of Fas-treated cells after 2 h of incubation (Fig. 1B). These findings suggested that the second increase in PP2A activity we noticed (Fig. 1A) was caused by the Fas-induced increase in caspase activity in neutrophils. Therefore, we focused further experiments on elucidating the molecular mechanisms that regulated the transient activity of PP2A seen in Fas-treated cells during the first hour of incubation. The activity of PP2A can be modulated by different mechanisms, some of which involve the various regulatory subunits of this protein. In addition, the core subunit can undergo a number of biochemical modifications that deactivate PP2A; for example, some tyrosine kinases belonging to the Src kinase family can phosphorylate PP2A on tyrosine 307 (18Lechward K. Awotunde O.S. Swiatek W. Muszynska G. Acta Biochim. Pol. 2001; 48: 921-933Crossref PubMed Scopus (174) Google Scholar). We found that in neutrophils the level of tyrosine phosphorylation of the core subunit of PP2A was only modestly affected during spontaneous apoptosis (Fig. 1, C and E), whereas it was increased temporarily during the first 45 min of Fas-induced apoptosis (Fig. 1, D and E). A comparison of PP2A tyrosine phosphorylation (Fig. 1E) with PP2A activity (Fig. 1A) during Fas-induced apoptosis revealed a parallel increase during the first 30 min, after which the phosphatase activity started to decrease before there was a drop in the phosphorylation. Incubating the cells with the Src kinase inhibitor PP1 augmented the activity of PP2A 45 min after the initiation of anti-Fas-induced apoptosis (Fig. 1F). This finding indicated that a Src kinase participated in the decrease in PP2A activity seen after 30 min during anti-Fas-induced apoptosis (Fig. 1A). Furthermore, these data argue against the involvement of a Src kinase in the initiation of PP2A activity during either spontaneous or Fas-induced apoptosis in neutrophils. PP2A Can Directly Regulate the Activity of p38 MAPK—Our data showing corresponding activities of p38 MAPK (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar) and PP2A suggested the existence of direct interactions between these signaling proteins, and that assumption was supported by the time-dependent recovery of PP2A from immunoprecipitates of total p38α MAPK (the most abundant p38 MAPK isoform in neutrophils (19Hale K.K. Trollinger D. Rihanek M. Manthey C.L. J. Immunol. 1999; 162: 4246-4252PubMed Google Scholar)) and active p38 MAPK from anti-Fas-treated cells (Fig. 2A). We observed that PP2A was not associated initially with p38 MAPK in freshly isolated neutrophils; instead, the association was first detected 30 min after the engagement of Fas receptors (Fig. 2A). To determine whether PP2A was involved in the Fas-induced dephosphorylation and inactivation of p38 MAPK, we used a precoupled antibody to immunoprecipitate total p38α MAPK from freshly isolated neutrophils (because such cells exhibit a high level of p38 MAPK activity). These immunocomplexes were incubated in the absence or presence of active PP2A (10 units/ml) purified from human erythrocytes (Fig. 2B) for 30 min at 30 °C and were then washed extensively to remove residual PP2A. The p38 MAPK activity that remained in these immunocomplexes was measured by performing an in vitro kinase assay for 1 h at 30 °C in the absence or presence of 10 nm Cal A with added ATF2. We detected a significant decrease in p38 MAPK-induced phosphorylation of ATF2 when immunoprecipitated p38 MAPK was incubated in the presence of PP2A (Fig. 2B). Interestingly, the PP2A-mediated decrease in p38 MAPK-induced phosphorylation of ATF2 was inhibited in the presence of Cal A (Fig. 2B). These results demonstrate that PP2A can directly dephosphorylate and inactivate p38 MAPK obtained from freshly isolated human neutrophils, which strongly suggests that PP2A is responsible for mediating the transient reduction of p38 MAPK activity seen in intact neutrophils. PP2A Causes the Transient Dephosphorylation of p38 MAPK during Apoptosis in Neutrophils—Notably, the peak in PP2A activity (Fig. 1A) and the transient decline in p38 activity (Fig. 3A) (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar) occurred simultaneously, and it was at the same point in time during an incubation that the activity of caspase 3 started to increase and could be detected (9Alvarado-Kristensson M. Melander F. Leandersson K. Ronnstrand L. Wernstedt C. Andersson T. J. Exp. Med. 2004; 199: 449-458Crossref PubMed Scopus (188) Google Scholar). Considering the findings mentioned above, it is interesting that Cal A, which is an inhibitor of type 1 and 2A protein phosphatases (20Sheppeck II, J.E. Gauss C.M. Chamberlin A.R. Bioorg. Med. Chem. 1997; 5: 1739-1750Crossref PubMed Scopus (142) Google Scholar), augmented the phosphorylation of p38 MAPK during the first hour of incubation and that the effect abolished the temporary decline in p38 MAPK activity (Fig. 3B). These observations support the idea that the initial, transitory inhibition of p38 MAPK during neutrophil apoptosis is caused by the transient increase in PP2A activity. PP2A Dephosphorylates Caspase 3 at Serine 150—We had observed previously that p38 MAPK plays a role in the survival of human neutrophils by virtue of its ability to phosphorylate (at Ser-150) and thereby inactivates caspase 3 (9Alvarado-Kristensson M. Melander F. Leandersson K. Ronnstrand L. Wernstedt C. Andersson T. J. Exp. Med. 2004; 199: 449-458Crossref PubMed Scopus (188) Google Scholar). That finding, along with the present results showing that PP2A participates in the regulation of p38 MAPK activity in neutrophils, prompted us to determine whether PP2A also affects the activity of caspase 3 in neutrophils undergoing apoptosis. Caspase 3 has been found to cleave PP2A in Jurkat cells (17Santoro M.F. Annand R.R. Robertson M.M. Peng Y.W. Brady M.J. Mankovich J.A. Hackett M.C. Ghayur T. Walter G. Wong W.W. Giegel D.A. J. Biol. Chem. 1998; 273: 13119-13128Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), which suggests that these two enzymes could also interact with each other in human neutrophils. Indeed, immunoprecipitation of caspase 3 and PP2A indicated a similar time-dependent interaction between these proteins in neutrophils (Fig. 4A), which suggests that PP2A can directly dephosphorylate caspase 3 and thereby regain its impaired activity. To test that assumption, we performed an assay in vitro to determine whether p38 MAPK-phosphorylated caspase 3 can serve as a substrate for PP2A. Active p38 MAPK that had been immunoprecipitated from newly isolated neutrophils was incubated with recombinant human caspase 3 in the presence of 10 μCi of [γ-32P]ATP (9Alvarado-Kristensson M. Melander F. Leandersson K. Ronnstrand L. Wernstedt C. Andersson T. J. Exp. Med. 2004; 199: 449-458Crossref PubMed Scopus (188) Google Scholar). After p38 MAPK-dependent phosphorylation of caspase 3 had occurred, the agarose-conjugated active p38 MAPK immunoprecipitates were removed. The phosphorylated caspase 3 was incubated with 10 units/ml PP2A for 30 min at 30 °C and was purified of human erythrocytes and PP2A immunoprecipitates from neutrophils that had been exposed to anti-Fas Ab for 30 min in the presence or absence of 10 nm Cal A (Fig. 4B). Thereafter, we analyzed the level of caspase 3 phosphorylation by autoradiography and found that both purified and immunoprecipitated PP2A caused dephosphorylation of caspase 3 and that this dephosphorylation could be inhibited by Cal A (Fig. 4B). Together, these results indicate that PP2A can mediate Fas-induced dephosphorylation of caspase 3. PP2A Participates in Regulation of Neutrophil Apoptosis— The interaction among p38 MAPK, caspase 3, and PP2A in intact neutrophils and our finding that PP2A can directly dephosphorylate p38 MAPK and caspase 3 in vitro suggest that PP2A plays an important role in the regulation of neutrophil apoptosis by directly inducing biochemical changes in both p38 MAPK and caspase 3, resulting in the reduced activity of p38 MAPK but the increased activity of caspase 3. To ascertain the validity of that assumption, we used two different experimental approaches to detect PP2A-dependent dephosphorylation of caspase 3 in intact neutrophils. First, we employed a general anti-phosphoserine antibody to investigate the effect of the PP2A inhibitor Cal A (40 nm) on the level of phosphorylation of immunoprecipitated caspase 3. We chose that method because it enables analysis of the phosphorylation immediately after isolation of the cells. The results show that the inhibition of PP2A in Fas-engaged neutrophils led to increased phosphorylation of caspase 3 (Fig. 5A). Second, we labeled freshly isolated human neutrophils with 32P for 1 h and 50 min and then incubated them for 10 min with or without 40 nm Cal A. We then engaged the Fas receptors of these cells in the absence or presence of Cal A. The time points indicated in Fig. 5B cannot be compared with those in the other figures because the cells in this experiment had been preincubated for a significant period of time at 37 °C before they were exposed to the anti-Fas Ab. Compared with Fas-treated cells, 32P-labeled cells incubated with anti-Fas Ab and Cal A displayed augmented phosphorylation of both the 32-kDa proform and the 20-kDa active form of caspase 3. To ascertain whether PP2A actually is involved in neutrophil apoptosis, we pretreated those leukocytes with Cal A and then analyzed the effect that this phosphatase inhibitor had on Fas-induced activation of caspase 3. The results revealed that the inhibition of PP2A significantly impeded the Fas-provoked activity of caspase 3 (Fig. 5C). In addition, we also analyzed the effect of Cal A on apoptosis by staining the nuclei of anti-Fas-treated neutrophils with acridine orange (Fig. 5D). These experiments show conclusively that the inhibition of PP2A decreased the number of anti-Fas Ab-treated cells with an apoptotic morphology. Together, these findings show that the inhibition of PP2A in Fas-engaged neutrophils led to an increased phosphorylation of caspase 3, which inhibited the activity of this caspase and thereby delayed the apoptotic process. We have shown previously that the ability of p38 MAPK to phosphorylate caspases constitutes a survival signal in isolated human granulocytes (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar, 9Alvarado-Kristensson M. Melander F. Leandersson K. Ronnstrand L. Wernstedt C. Andersson T. J. Exp. Med. 2004; 199: 449-458Crossref PubMed Scopus (188) Google Scholar). Specifically, such biochemical modifications impair the activities of caspases and thereby weaken the capacity of these proteins to induce apoptosis (9Alvarado-Kristensson M. Melander F. Leandersson K. Ronnstrand L. Wernstedt C. Andersson T. J. Exp. Med. 2004; 199: 449-458Crossref PubMed Scopus (188) Google Scholar). Therefore, inactivation of the constitutively active p38 MAPK facilitates stimulation of the caspase cascade during spontaneous and Fas-induced neutrophil apoptosis (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar). The mechanism of the temporary deactivation of p38 MAPK has not yet been examined in human neutrophils. In the present study, we noted that a transient increase in PP2A activity occurred at the same time as the decrease in p38 MAPK activity, which suggests that PP2A could be involved in regulating the phosphorylation and thus also the activity of p38 MAPK. The finding that both the increase in PP2A activity and the decrease in p38 MAPK activity take place at a point in time when caspase 3 starts to display increased activity (7Alvarado-Kristensson M. Porn-Ares M.I. Grethe S. Smith D. Zheng L. Andersson T. FASEB J. 2001; (in press)PubMed Google Scholar, 9Alvarado-Kristensson M. Melander F. Leandersson K. Ronnstrand L. Wernstedt C. Andersson T. J. Exp. Med. 2004; 199: 449-458Crossref PubMed Scopus (188) Google Scholar) implies that p38 MAPK as well as PP2A is involved in the control of neutrophil apoptosis. The existence of a possible regulatory relationship between p38 MAPK and PP2A is supported by results reported by Keyse (11Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (712) Google Scholar), showing that various protein phosphatases can down-regulate the activity of p38 MAPK by dephosphorylating either a specific threonine or a specific tyrosine residue or both. The author documented that the serine/threonine phosphatases PP2A and PP2Cα can deactivate p38 MAPK by phospho-threonine dephosphorylation (11Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (712) Google Scholar). The present finding of a Fas-dependent association between PP2A and p38 MAPK provides the basis for a signaling interaction between these two enzymes and hence implies that PP2A is involved in regulating the induction of apoptosis in neutrophils. To ascertain whether PP2A does play a role in neutrophil apoptosis, we pretreated such leukocytes with the phosphatase inhibitor Cal A, which is known to efficiently block the effects of PP1 and PP2A but not PP2Cα (20Sheppeck II, J.E. Gauss C.M. Chamberlin A.R. Bioorg. Med. Chem. 1997; 5: 1739-1750Crossref PubMed Scopus (142) Google Scholar). Only PP2A and PP2Cα can dephosphorylate p38 MAPK, and therefore it seems reasonable to assume that any influence of Cal A on Fas-induced neutrophil apoptosis is mediated by its effect on PP2A. We found that the inhibition of PP2A by Cal A abolished the Fas-induced transient decrease in p38 MAPK activity and that it also increased the phosphorylation of caspase 3 and thereby reduced the activity of this apoptotic protease and the morphological alterations characteristic of apoptotic cells. Our data reveal an association between p38 MAPK and caspase 3 and show that PP2A interacts with both of those proteins. Consequently, it could be hypothesized that PP2A may directly dephosphorylate not only p38 MAPK but also caspase 3. In accordance with that idea, the results of our experiments performed in vitro revealed such a direct effect of PP2A on caspase 3 and on p38 MAPK, and therefore we propose that, in addition to its influence on p38 MAPK, PP2A has a direct impact on the phosphorylation statues and activity of caspase 3. These findings suggest that PP2A promotes neutrophil apoptosis through dual actions, more precisely, by dephosphorylating p38 MAPK and thus inhibiting subsequent phosphorylation of caspase 3 and by dephosphorylating caspase 3 that has already been phosphorylated and thereby restoring its activity. The signaling events that occur during the first hour of Fas-induced apoptosis in neutrophils are relatively straightforward to interpret, whereas the subsequent alterations in intracellular signaling are more difficult to understand because of the increased activity of caspases in apoptotic cells. The stimulated caspases are known to degrade and thereby abolish the activities of a number of signaling molecules (21Degterev A. Boyce M. Yuan J. Oncogene. 2003; 22: 8543-8567Crossref PubMed Scopus (986) Google Scholar), or, as in the case of PP2A, they increase its activity (17Santoro M.F. Annand R.R. Robertson M.M. Peng Y.W. Brady M.J. Mankovich J.A. Hackett M.C. Ghayur T. Walter G. Wong W.W. Giegel D.A. J. Biol. Chem. 1998; 273: 13119-13128Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). We found that the inhibition of caspase 3 in neutrophils exposed to anti-Fas Ab for 90 min led to reduced activity of PP2A. Accordingly, it is logical to conclude that the secondary increase in PP2A activity observed in neutrophils with engaged Fas receptors is mediated indirectly by the Fas-induced increase in caspase activities. Based on this knowledge, we focused our present efforts on understanding how PP2A participates in the initial induction phase of apoptosis in neutrophils. PP2A has been implicated primarily as a proapoptotic signal in several human and murine cell lines (22Chiang C.W. Kanies C. Kim K.W. Fang W.B. Parkhurst C. Xie M. Henry T. Yang E. Mol. Cell. Biol. 2003; 23: 6350-6362Crossref PubMed Scopus (128) Google Scholar, 23Deng X. Ito T. Carr B. Mumby M. May Jr., W.S. J. Biol. Chem. 1998; 273: 34157-34163Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 24Ha ̈rma ̈la ̈-Braske ́n A.S. Mikhailov A. So ̈derstro ̈m T.S. Meinander A. Holmstro ̈m T.H. Damuni Z. Eriksson J.E. Oncogene. 2003; 22: 7677-7686Crossref PubMed Scopus (36) Google Scholar), and such an effect is mediated probably by the ability of this protein to dephosphorylate Bcl-2 and Bad (22Chiang C.W. Kanies C. Kim K.W. Fang W.B. Parkhurst C. Xie M. Henry T. Yang E. Mol. Cell. Biol. 2003; 23: 6350-6362Crossref PubMed Scopus (128) Google Scholar, 23Deng X. Ito T. Carr B. Mumby M. May Jr., W.S. J. Biol. Chem. 1998; 273: 34157-34163Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). However, PP2A can also indirectly exert a proapoptotic effect on cells by down-regulating signal transduction molecules such as extracellular signal-regulated kinase (24Ha ̈rma ̈la ̈-Braske ́n A.S. Mikhailov A. So ̈derstro ̈m T.S. Meinander A. Holmstro ̈m T.H. Damuni Z. Eriksson J.E. Oncogene. 2003; 22: 7677-7686Crossref PubMed Scopus (36) Google Scholar). In tumor necrosis factor-α-stimulated human neutrophils, Avdi et al. (25Avdi N.J. Malcolm K.C. Nick J.A. Worthen G.S. J. Biol. Chem. 2002; 277: 40687-40696Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) have demonstrated a functional cross-talk between p38 MAPK and JNK, whereby p38 MAPK acts to limit the activation of a proapoptotic signal from JNK. These authors suggest that this cross-talk is mediated via the p38 MAPK-mediated activation of PP2A and the subsequent inhibition of MAPK kinase 4 and JNK (25Avdi N.J. Malcolm K.C. Nick J.A. Worthen G.S. J. Biol. Chem. 2002; 277: 40687-40696Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In contrast, results from the present study suggest that the proapoptotic impact of PP2A in primary human neutrophils occurs through direct regulation of another member of the MAPK family, namely p38 MAPK, and also caspase 3, which is a downstream substrate of p38 MAPK. These effects are possible because of the interactions of PP2A with p38 MAPK and caspase 3, which enable a transient PP2A-mediated deactivation of p38 MAPK and the dephosphorylation and reconstitution of previously impaired caspase 3 activity. These molecular modifications, in turn, increase the activity of caspase 3 and thus initiate apoptosis in human neutrophils. Taken together, these data could of course be readily explained by the ability of Fas- and tumor necrosis factor-α receptors to activate distinctly different downstream signaling events that result in an increased neutrophil survival. However, the data convincingly reveal the capacity of both PP2A and p38 MAPK to regulate each other and show that such signaling interactions are important in regulating neutrophil survival. We thank E. S. Alnemri for kindly providing the pET21b vector, Lena Axelsson for excellent technical assistance, and Patricia Ödman for linguistic revision of the manuscript.