Phosphorylation is not essential for protection of L929 cells by Hsp25 against H2O2 -mediated disruption actin cytoskeleton, a protection which appears related to the redox change mediated by Hsp25

Small stress proteins protect against the cytotoxicity mediated by oxidative stress. The relationship between Hsp25 expression and the integrity of the actin network was studied in H₂O₂-treated murine L929 fibrosarcoma cells overexpressing endogenous wild-type (wt-) or non-phosphorylatable mutant (mt-) Hsp25. We show here that both proteins prevented actin network disruption induced by a 1 h treatment with 400 μM H₂O₂. In contrast, SB203580, a p38 MAPkinase inhibitor which suppresses Hsp25 phosphorylation, abolished the protective activity conferred by both wt- and mt-Hsp25. Hence, phosphorylation does not appear essential for Hsp25 protective activity against H₂O₂-induced actin disruption, and SB203580-sensitive events other than Hsp25 phosphorylation may be important for actin network regulation. Since, in L929 cells, wt- or mt-Hsp25 expression modulates intracellular glutathione levels, analyses were performed which revealed a direct correlation between glutathione and the integrity of the actin network. Moreover, laser scanning confocal immunofluorescences revealed that only a small fraction of wt- or mt-Hsp25 colocalized with actin microfilaments. Taken together, our results suggest that, in L929 cells, the protection against actin network disruption is probably a consequence of the redox change mediated by Hsp25 rather than a direct effect of this stress protein towards actin.     

Overexpression of HSP25 reduces the level of TNFα-induced oxidative DNA damage biomarker, 8-hydroxy-2′-deoxyguanosine,in L929 cells

Previously we and others have demonstrated that oxidative stress involving generation of reactive oxygen species (ROS) is responsible for the cytotoxic action of TNFα. Protective effect of small heat shock proteins (HSP) against diverse oxidative stress conditions has been suggested. Although overexpression of small HSP was shown to provide an enhanced survival of TNFα-sensitive cells when challenged with TNFα, neither the nature of TNFα-induced cytotoxicity nor the protective mechanism of small HSP has been completely understood. In this study, we have attempted to determine whether TNFα induces oxidative DNA damage in TNFα-sensitive L929 cells. We chose to measure the level of 8-hydroxy-2′-deoxyguanosine (8 ohdG), which has been increasingly recognized as one of the most sensitive markers of oxidative DNA damage. Our results clearly demonstrated that the level of 8 ohdG increased in L929 cells in a TNFα dose-dependent manner. Subsequently, we asked whether small HSP has a protective effect on TNFα-induced oxidative DNA damage. To accomplish this goal, we have stably transfected into L929 cells, which are devoid of endogenous small HSP, with the mouse small hsp cDNA (hsp25). We found that TNFα-induced 8 ohdG was decreased in cells overexpressing exogenous small HSP25. We also found that the cell-killing activity of TNFα was decreased in these cells as measured by clonogenic survival. Taken together, results from the current study show that a cytotoxic mechanism of TNFα involves oxidative damage of DNA, and that overexpression of the small HSP25 reduces this oxidative damage. We suggest that the reduction of oxidative DNA damage is an important protective mechanisms of small HSP against TNFα. J. Cell. Physiol. 174:27–34, 1998. © 1998 Wiley-Liss, Inc.     

Protein kinase inhibitors can suppress stress-induced dissociation of Hsp27

We previously showed that the aggregated form of Hsp27 in cultured cells becomes dissociated as a result of phosphorylation with various types of stress. In order to clarify the signal transduction cascade involved, the effects of various inhibitors of protein kinases and dithiothreitol on the dissociation of Hsp27 were here examined by means of an immunoassay after fractionation of cell extracts by sucrose density gradient centrifugation. The dissociation of Hsp27 induced by exposure of U251 MG human glioma cells to metals (NaAsO₂ and CdCl₂), hypertonic stress (sorbitol and NaCl), or anisomycin, an activator of p38 mitogen–activated protein (MAP) kinase, was completely suppressed by the presence of SB 203580 or PD 169316, inhibitors of p38 MAP kinase, but not by PD 98059 and Uo 126, inhibitors of MAP kinase kinase (MEK), nor by staurosporine, Go 6983, and bisindolylmaleimide I, inhibitors of protein kinase C. Phorbol ester (PMA)–induced dissociation of Hsp27 was completely suppressed by staurosporine, Go 6983, or bisindolylmaleimide I and partially suppressed by SB 203580, or PD 169316 but not by PD 98059 or Uo 126, indicating mediation by 2 cascades. The presence of 1 mM dithiothreitol in the culture medium during exposure to chemicals suppressed the dissociation of Hsp27 induced by arsenite and CdCl₂ but not by other chemicals. These results suggest that the phosphorylation of Hsp27 is catalyzed by 2 protein kinases, p38 MAP kinase–activated protein (MAPKAP) kinase- 2/3 and protein kinase C. In addition, metal-induced signals are sensitive to reducing power.     

Geldanamycin, an inhibitor of Hsp90 increases cytochrome P450 2E1 mediated toxicity in HepG2 cells through sustained activation of the p38MAPK pathway

Cytochrome P450 2E1 (CYP2E1) can mediate reactive oxygen species (ROS) induced cell death through its catalytic processes. Heat shock protein 90 (Hsp90) is an important molecular chaperone which is essential for cellular integrity. We previously showed that inhibition of Hsp90 with Geldanamycin (GA), an inhibitor of Hsp90 increased CYP2E1 mediated toxicity in CYP2E1 over-expressing HepG2 cells (E47 cells) but not in C34-HepG2 cells devoid of CYP2E1 expression. The aim of the present study was to test the hypothesis that the potentiation of CYP2E1 toxicity in E47 cells with GA may involve changes in mitogen activated protein kinase signal transduction pathways. GA was toxic to E47 cells and SB203580, an inhibitor of p38 MAPK prevented this decrease in viability. The protective effects of SB203580 were effective only when SB203580 was added before GA treatment. GA activated p38 MAPK in E47 cells and this activation was an early and a sustained event. GA elevated ROS levels and lipid peroxidation and lowered GSH levels in E47 cells and these changes were blunted or prevented by treatment with SB203580. Apoptosis was increased by GA and prevented by pre-treatment with SB203580. The loss in mitochondrial membrane potential in E47 cells after GA treatment was also decreased significantly with SB203580 treatment. The activity and expression of CYP2E1 and Hsp90 levels were not altered by SB203580. In conclusion, the inhibition of Hsp90 with GA increases the toxicity of CYP2E1 in HepG2 cells through an early and sustained activation of the p38 MAPK pathway.     

The protein kinase inhibitor SB203580 uncouples PMA-induced differentiation of HL-60 cells from phosphorylation of Hsp27

HL-60 cells are an attractive model for studies of human myeloid cell differentiation. Among the well-examined parameters correlated to differentiation of HL-60 cells are the expression and phosphorylation of the small heat shock protein Hsp27. Here we demonstrate that PMA treatment of HL-60 cells stimulates different MAP kinase cascades, leading to significant activation of ERK2 and p38 reactivating kinase (p38RK). Using the protein kinase inhibitor SB 203580, we specifically inhibited p38RK and, thereby, activation of its target MAP kinase-activated protein kinase 2(MAPKAP kinase 2), which is the major enzyme responsible for small Hsp phosphorylation. As a result, PMA-induced Hsp27 phosphorylation is inhibited in SB 203580-treated HL-60 cells indicating that p38RK and MAPKAP kinase 2 are components of the PMA-induced signal transduction pathway leading to Hsp27 phosphorylation. We further demonstrate that, although PMA-induced phosphorylation is inhibited, SB 203580-treated HL-60 cells are still able to differentiate to the macrophage-like phenotype as judged by decrease in cell proliferation, induction of expression of the cell surface antigen CD11b and changes in cell morphology. These results indicate that, although correlated, Hsp27 phosphorylation is not required for HL-60 cell differentiation. However, the results do not exclude that increased Hsp27 expression is involved in HL-60 cell differentiation.     

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, FcgammaR cross-linking, or phosphatidylinositol 3,4,5-trisphosphate. Concentration inhibition studies showed that Ser(473) phosphorylation was inhibited by 0.3 microm SB203580, while inhibition of Thr(308) phosphorylation required 10 microm 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 glutathione S-transferase (GST) pull-down studies showed that Akt was associated with p38 kinase, MK2, and Hsp27 in neutrophils, and Hsp27 dissociated from the complex upon activation. Active recombinant MK2 phosphorylated recombinant Akt and Akt in anti-Akt, anti-MK2, anti-p38, and anti-Hsp27 immunoprecipitates, and this was inhibited by an MK2 inhibitory peptide. We conclude that Akt exists in a signaling complex containing p38 kinase, MK2, and Hsp27 and that p38-dependent MK2 activation functions as PDK2 in human neutrophils.     

Vascular endothelial growth factor induces heat shock protein (HSP) 27 serine 82 phosphorylation and endothelial tubulogenesis via protein kinase D and independent of p38 kinase

Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.     

Analysis of properties of small heat shock protein Hsp25 in MAPK-activated protein kinase 2 (MK2)-deficient cells: MK2-dependent insolubilization of Hsp25 oligomers correlates with susceptibility to stress

Small heat shock proteins (sHsps) exist in dynamic oligomeric complexes and display diverse biological functions ranging from chaperone properties to modulator of apoptosis. So far, the role of stress-dependent phosphorylation of mammalian sHsps for its structure and function has been analyzed by using various phosphorylation site mutants overexpressed in different cell types as well as by non-exclusive inhibitors of the p38 MAPK cascade. Here we investigate the role of phosphorylation of endogenous sHsp in a genetic model lacking the major Hsp25 kinase, the MAP kinase-activated protein kinase MK2. We demonstrate that in MK2-deficient fibroblasts, where no stress-dependent phosphorylation of Hsp25 at Ser86 and no in vitro binding to 14-3-3 was detectable, stress-dependent disaggregation of endogenous Hsp25 complexes is impared and kinetics of arsenite-dependent, H2O2-dependent, and sublethal heat shock-induced insolubilization of Hsp25 is delayed. Similarly, green fluorescent protein-tagged Hsp25 shows retarded subcellular accumulation into stress granules in MK2-deficient cells after arsenite treatment. Decreased insolubilization of Hsp25 in MK2-deficient cells correlates with increased resistance against arsenite, H2O2, and sublethal heat shock treatment and with decreased apoptosis. In contrast, after severe, lethal heat shock MK2-deficient embryonic fibroblasts cells show fast and complete insolubilization of Hsp25 independent of MK2 and no increased stress resistance. Hence, MK2-dependent formation of insoluble stress granules and irreversible cell damage by oxidative stresses and sublethal heat shock correlate and only upon severe, lethal heat shock MK2-independent processes could determine insolubilization of Hsp25 and are more relevant for cellular stress damage.     

Distribution,phosphorylation, and activities of Hsp25 in heat-stressed H9c2 myoblasts: a functional link to cytoprotection

The behavior of the endogenous heat shock protein 25 (Hsp25) in heat-stressed rat H9c2 myoblasts was studied. After mild or severe heating, this protein became less extractable with Triton X-100 and displayed characteristic immunofluorescence patterns, namely (1) granules in the nucleus, and (2) association with F-actin bundles in the cytoplasm. The intranuclear granulation of Hsp25 and its association with F-actin were sensitive to drugs affecting Hsp25 phosphorylation (cantharidin, sodium orthovanadate, SB203580, SB202190). Isoform analysis of Hsp25 translocated to the nucleus-free cytoskeletal fraction revealed only mono- and biphosphorylated Hsp25 and no unphosphorylated Hsp25. Transfected luciferase with initial localization in the nucleosol became colocalized with the Hsp25-containing granules after a heat shock treatment that denatured the enzyme in the cells. The association of Hsp25 with actin filaments after a mild heat stress conferred protection from subsequent F-actin-damaging treatments with cytochalasins (D and B) or severe heat stress. We hypothesize that (1) the binding of heat-denatured nucleosolic proteins to the Hsp25 contained in specific granular structures may serve for the subsequent chaperoning or degradation of the bound proteins, and (2) the actin cytoskeleton is stabilized by the direct targeting of phosphorylated Hsp25 to microfilament bundles.     

Adenosine A1 and A3 receptor agonists reduce hypoxic injury through the involvement of P38 MAPK

Activation of either the A₁ adenosine receptor (A₁R) or the A₃ adenosine receptor (A₃R), by their specific agonists CCPA and Cl-IB-MECA, respectively, protects cardiac cells in culture against ischemic injury. Yet the full protective mechanism remains unclear. In this study, we therefore examined the involvement of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK) phosphorylation in this protective intracellular signaling mechanism. Furthermore, we investigated whether p38 MAPK phosphorylation occurs upstream or downstream from the opening of mitochondrial ATP-sensitive potassium (K_ATP) channels. The role of p38 MAPK activation in the intracellular signaling process was studied in cultured cardiomyocytes subjected to hypoxia, that were pretreated with CCPA or Cl-IB-MECA or diazoxide (a mitochondrial K_ATP channel opener) with and without SB203580 (a specific inhibitor of phosphorylated p38 MAPK). Cardiomyocytes were also pretreated with anisomycin (p38 MAPK activator) with and without 5-hydroxy decanoic acid (5HD) (a mitochondrial K_ATP channel blocker). SB203580 together with the CCPA, Cl-IB-MECA or diazoxide abrogated the protection against hypoxia as shown by the level of ATP, lactate dehydrogenase (LDH) release, and propidium iodide (PI) staining. Anisomycin protected the cardiomyocytes against ischemic injury and this protection was abrogated by SB203580 but not by 5HD. Conclusions Activation of A₁R or A₃R by CCPA or Cl-IB-MECA, respectively, protects cardiomyocytes from hypoxia via phosphorylation of p38 MAPK, which is located downstream from the mitochondrial K_ATP channel opening. Elucidating the signaling pathway by which adenosine receptor agonists protect cardiomyocytes from hypoxic damage, will facilitate the development of anti ischemic drugs.     

Antioxidant effect of quercetin against acute spinal cord injury in rats and its correlation with the p38MAPK/iNOS signaling pathway

AimsThe present study aimed to investigate the correlation between quercetin (Que) and the p38 mitogen-activated protein kinase (p38MAPK)/inducible nitric oxide synthase (iNOS) signaling pathway and to explore its regulating effect on secondary oxidative stress following acute spinal cord injury (SCI), so as to elucidate the protective effects and mechanism associated with Que treatment during acute SCI.Main methodsSprague–Dawley rats were randomly divided into sham-surgery, SCI, Que, methylprednisolone (MP), and specific p38MAPK inhibitor SB203580 treatment groups. Acute SCI models were established in rats by a modified Allen's method. Real-time PCR analysis, western blot assay and immunohistochemistry for molecular changes in the p38MAPK/iNOS signaling pathway, determination of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, reflecting the levels of secondary oxidative stress, and functional or behavioral data, reflecting changes induced by Que and control treatments post-SCI were performed.Key findingsQue significantly increased Basso, Beattie and Bresnahan scores and inclined plane test scores in SCI rats similar to the positive control drug, MP. Que significantly inhibited increases in phosphorylated-p38MAPK (p-p38MAPK) and iNOS expression and reduced the rate of iNOS-positive cells in rats with SCI, similar to the effects of SB203580. In addition, both Que and SB203580 reduced MDA content and enhanced SOD activity in SCI rats, with Que effects being stronger.SignificanceThese experimental findings indicate that in SCI rats, Que has protective effects on the spinal cord by the potential mechanism of inhibiting the activation of p38MAPK/iNOS signaling pathway and thus regulating secondary oxidative stress.     

PMA-induced activation of the p42/44ERK- and p38RK-MAP kinase cascades in HL-60 cells is PKC dependent but not essential for differentiation to the macrophage-like phenotype

The signaling mechanisms leading to phorbol ester myristate (PMA)-induced differentiation of HL-60 cells to the macrophagelike phenotype were investigated by using different protein kinase inhibitors. The protein kinase C inhibitor Ro 31-8220 specifically blocks PMA-induced differentiation, activation of the p42/44^ERK- and p38^RK-MAP kinase cascades and Hsp27-phosphorylation in HL-60 cells. Because Ro 31-8220 does not inhibit activation of the MAP kinase cascades by protein kinase C (PKC)-independent signals such as epidermal growth factor (EGF), heat shock, or anisomycin in these cells, only PMA-induced activation of the MAP kinases can be downstream of PKC. The MEK1 inhibitor PD 098059 and the p38^RK inhibitor SB 203580 also were used to analyze whether the PMA-induced PKC-dependent activation of MAP kinases is involved in the differentiation process. Under certain conditions, PD 098059 can completely block the PMA-induced activation of the p42^ERK as monitored by imunoprecipitation kinase assay by using the substrate myelin basic protein. SB 203580 specifically inhibits activation of p38^RK as judged by MAPKAP kinase 2 activity against the substrate Hsp27 and also blocks Hsp27 phosphorylation in the cells. In contrast, neither PD 098059 nor SB 203580 nor both inhibitors together prevent PMA-induced differentiation of the HL-60 cells to the macrophagelike phenotype. The results suggest the existence of a diversification of PMA-induced signaling in HL-60 cells downstream of PKC, leading to activation of MAP kinases that are not essential for differentiation and to phosphorylation of other, so far unidentified, targets responsible for differentiation. J. Cell. Physiol. 173:310–318, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation.

The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers.     

Short-chain fatty acid mediated phosphorylation of heat shock protein 25: effects on camptothecin-induced apoptosis

Although short-chain fatty acid (SCFA)-induced heat shock protein 25 (Hsp25) is associated with increased cellular resistance to injury, withdrawal of lumenal butyrate in vivo is associated with intestinal epithelial injury and apoptosis. Recognizing that SCFA-dependent posttranslational modification of Hsp25 may involve altered Hsp25 phosphorylation, we hypothesized that butyrate regulates Hsp25 phosphorylation and secondarily affects cellular responses to apoptosis-inducing agents. Intestinal epithelial crypt IEC-18 cells were treated with butyrate, propionate, or the histone deacetylase inhibitor trichostatin A for 6-24 h. Immunolocalization of Hsp25 was examined by confocal laser microscopy. Hsp25 phosphorylation was characterized using two-dimensional isoelectric focusing gel electrophoresis. Hsp25 accumulation in cytoskeletal- and mitochondrial-enriched fractions was examined by immunoblotting. The activation of p38 MAP kinase was determined using phospho-specific antibodies and MAPKAPK 2 kinase assays. The effects of SCFA on apoptosis were studied by ELISA detection of cleaved DNA and using antibodies recognizing cleaved caspase-3. Five-millimolar butyrate induced no significant injury to IEC-18 cells. Hsp25 did not accumulate in Triton X-100-insoluble cytoskeletal fractions with butyrate treatment but did localize to mitochondria in a p38 MAP kinase-dependent manner. Hsp25 phosphorylation was induced by butyrate, propionate, and trichostatin A. Butyrate-mediated changes in Hsp25 phosphorylation coincide with the activation of the p38 MAP kinase and MAPKAPK 2. Butyrate, propionate, and low-dose trichostatin A confer significant protection from camptothecin-induced apoptosis, which was not reversed by the p38 inhibitor SB203580. We conclude that butyrate-mediated phosphorylation of Hsp25 is associated with significant resistance to apoptosis, which appears to be independent of p38-mediated targeting of Hsp25 to mitochondria.     

The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor.

Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF-induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation.     

Phosphorylation of initiation factor 4E is resistant to SB203580 in cells expressing a drug-resistant mutant of stress-activated protein kinase 2a/p38

Previous work has shown that increased phosphorylation of eukaryotic initiation factor (eIF) 4E at Ser209 in the C-terminal loop of the protein is observed in response to cellular stress. SB203580, a cell permeable inhibitor of stress-activated protein kinase 2a (SAPK2a/p38), suppresses this response in a number of cell types. To validate the in vivo specificity of this inhibitor for the investigation of signalling pathways, which modulate the phosphorylation of eIF4E, we have used 293 cells which inducibly express either a wild-type form (WT-SAPK2a) or a drug-resistant mutant of SAPK2a (DR-SAPK2a). These data show that while the arsenite-induced increase in the phosphorylation of eIF4E and hsp25 was sensitive to SB203580 in cells expressing WT-SAPK2a, these responses to SB203580 were abrogated in cells expressing DR-SAPK2a. In addition, the phosphorylation of the eIF4E kinase, MAP kinase integrating kinase-1 (Mnk1), which is activated in response to growth factors or stress, was insensitive to SB203580 in DR-SAPK2a-expressing cells. However, a cell-permeable, specific inhibitor of Mnk1, CGP57380 and the phosphatidylinositol-3-kinase (PI3-K) inhibitor, LY294002, prevented eIF4E phosphorylation in 293 cells irrespective of SAPK2a expression. Therefore, this study validates the use of SB203580 for investigating signalling pathways modulating the phosphorylation of eIF4E in cultured cells.