Two distinct forms of MAPKAP kinase-2 in adult cardiac ventricular myocytes

Hsp27 kinase activities were studied in adult rat ventricular myocytes following sequential chromatography on Mono Q and Mono S. A basal level of activity was present following cell isolation. FPLC on Mono Q revealed three peaks of activity, peaks 'a', 'b', and 'c'. A fourth peak, 'd', was detected upon subsequent chromatography of the Mono Q flow-through on Mono S. Immunoblotting revealed that peaks 'a', 'b', and 'c' contained predominantly a 49 kDa form of MAPKAP kinase-2. Peak 'd' contained a 43 kDa form. 'In-gel' kinase assays using hsp27 indicated both forms of MAPKAP kinase-2 were active. No other bands of hsp27 kinase activity were detected. Both forms of hsp27 kinase immunoprecipitated with a MAPKAP kinase-2 antibody and have therefore been named MAPKAP kinase-2alpha (p49) and MAPKAP kinase-2beta (p43). MAPKAP kinase-2beta chromatographed on Superose 12 as a 60.7 kDa monomer whereas the behavior of MAPKAP kinase-2alpha suggested both a 65.7 kDa monomer and higher molecular mass complexes. Both activities phosphorylated hsp27 on serine residues, and two-dimensional phosphopeptide mapping indicated the same sites were phosphorylated. A tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated both MAPKAP kinase-2alpha and MAPKAP kinase-2beta activity. Inhibition of MEK activation with PD 98059 or p38alpha/beta MAP kinase activity with SB203580 blocked activation by PMA. However, whereas PD 98059 inhibited only the PMA-stimulated activation, SB203580 inhibited both PMA-stimulated and basal hsp27 phosphorylation. These data demonstrate the presence of two forms of MAPKAP kinase-2 in adult ventricular myocytes. Both forms are activated indirectly by the ERK MAP kinase pathway and directly by p38 MAP kinase but independently regulated.     

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 MAP kinase pathway regulates angiotensin II-induced contraction of rat vascular smooth muscle

Angiotensin II (ANG II) is a multifunctional hormone that exerts potent vasoconstrictor and hypertrophic effects on vascular smooth muscle. Here, we demonstrate that the p38 mitogen-activated protein (MAP) kinase pathway is involved in ANG II-induced vascular contraction. Addition of ANG II to rat aortic smooth muscle cells (SMC) caused a rapid and transient increase of p38 activity through activation of the AT(1) receptor subtype. This response to ANG II was strongly attenuated by pretreating cells with antioxidants and diphenylene iodonium and was mimicked by exposure of cells to H(2)O(2). Stimulation of p38 by ANG II resulted in the enzymatic activation of MAP kinase-activated protein (MAPKAP) kinase-2 and the phosphorylation of heat shock protein 27 (HSP27) in aortic SMC. Pretreatment of cells with the specific p38 MAP kinase inhibitor SB-203580 completely blocked the ANG II-dependent activation of MAPKAP kinase-2 and phosphorylation of HSP27. ANG II also caused a robust activation of MAPKAP kinase-2 in the intact rat aorta. Incubation with SB-203580 significantly decreased the potency of ANG II to induce contraction of rat aortic rings and depressed the maximal hormone response. These results suggest that the p38 MAP kinase pathway selectively modulates the vasoconstrictor action of ANG II in vascular smooth muscle.     

COM crystals activate the p38 mitogen-activated protein kinase signal transduction pathway in renal epithelial cells

Interaction of calcium oxalate monohydrate (COM) crystals with renal cells has been shown to result in altered gene expression, DNA synthesis, and cell death. In the current study the role of a stress-specific p38 MAP kinase-signaling pathway in mediating these effects of COM crystals was investigated. Exposure of cells to COM crystals (20 microg/cm(2)) rapidly stimulated strong phosphorylation and activation of p38 mitogen-activated protein kinase (p38 MAP kinase) and re-initiation of DNA synthesis. Inhibition of COM crystal binding to the cells by heparin blocked the effects of COM crystals on p38 MAPK activation. We also show that specific inhibition of p38 MAPK by 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl) imidazole (SB203580) or by overexpression of a dominant negative mutant of p38 MAP kinase abolishes COM crystal-induced re-initiation of DNA synthesis. The inhibition is dose-dependent and correlates with in situ activity of native p38 MAP kinase, determined as mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) activity in cell extracts. In summary, inhibiting activation of p38 MAPK pathway abrogated the DNA synthesis in response to COM crystals. These data are the first demonstrations of activation of the p38 MAPK signaling pathway by COM crystals and suggest that, in response to COM crystals, this pathway transduces critical signals governing the re-initiation of DNA synthesis in renal epithelial cells.     

Involvement of mitogen-activated protein kinase homologues in the regulation of lipopolysaccharide-mediated induction of cyclo-oxygenase-2 but not nitric oxide synthase in RAW 264.7 macrophages.

In RAW 264.7 macrophages lipopolysaccharide (LPS) stimulated the activation of p42 and p44 MAP kinases and their upstream activator mitogen-activated protein (MAP) kinase kinase (MAPKK), and induced the 69-kDa isoform of cyclo-oxygenase-2 (COX-2) and the 130-kDa isoform of nitric oxide synthase (iNOS). PD 098059, a specific inhibitor of the activation of MAPKK, prevented LPS-mediated activation of MAPKK (IC50 = 3.0 +/- 0.1 microM, n = 3) and p42/44 MAP kinases and substantially reduced the induction of COX-2 by approximately 40%-70%, but was without effect upon the induction of iNOS. In parallel, LPS also stimulated the activation of p38 MAP kinase and the MAPKAP kinase-2, a downstream target of p38 MAP kinase. SB 203580, a specific inhibitor of p38 MAP kinase prevented the activation of p38 MAP kinase (IC50 = 3.3 +/- 1.4 microM, n = 3) and MAPKAP kinase-2 by LPS and reduced the induction of COX-2 by approximately 50-90%, with no significant effect upon iNOS expression. These studies indicate the involvement of both the classical p42/44 MAP kinases and p38 MAP kinase in the regulation of COX-2 but not iNOS induction following exposure to LPS.     

Survival pathways regulating the apoptosis induced by tumour necrosis factor-alpha in primary cultured bovine endothelial cells

The aim of the present study was to identify biochemical pathways driving the resistance of endothelial cells to apoptosis induced by tumour necrosis factor-alpha (TNF). (1) Although nuclear factor-kappa B (NF-kappaB) was activated by TNF, its inhibition by MG-132 failed to sensitize these cells. (2) The activation of protein kinase C (PKC) by phorbol ester completely abolished the TNF-induced cell death. (3) The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (Wo) triggered apoptosis and enhanced the TNF-induced cell death. (4) The MEK inhibitor PD98059 did not affect the TNF-induced apoptotic process. (5) The p38 is activated by TNF and its inhibition by SB203580 sensitized the cells to TNF. This is correlated with the inhibition of phosphorylation of heat-shock protein of 27 kDa (HSP27).These results indicate that TNF activates NF-kappaB, which does not drive any anti-apoptotic response, and p38, which plays an anti-apoptotic function probably through HSP27 phosphorylation. Moreover, PKC and PI3K are involved in the control of survival pathways.     

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.     

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.     

The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase

Pyridinyl imidazole inhibitors, particularly SB203580, have been widely used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase (p38/HOG/SAPKII) in a wide array of biological systems. Studies by this group and others have shown that SB203580 can have antiproliferative activity on cytokine-activated lymphocytes. However, we recently reported that the antiproliferative effects of SB203580 were unrelated to p38 MAP kinase activity. This present study now shows that SB203580 can inhibit the key cell cycle event of retinoblastoma protein phosphorylation in interleukin-2-stimulated T cells. Studies on the proximal regulator of this event, the phosphatidylinositol 3-kinase/protein kinase B (PKB)(Akt/Rac) kinase pathway, showed that SB203580 blocked the phosphorylation and activation of PKB by inhibiting the PKB kinase, phosphoinositide-dependent protein kinase 1. The concentrations of SB203580 required to block PKB phosphorylation (IC(50) 3-5 microM) are only approximately 10-fold higher than those required to inhibit p38 MAP kinase (IC(50) 0.3-0.5 microM). These data define a new activity for this drug and would suggest that extreme caution should be taken when interpreting data where SB203580 has been used at concentrations above 1-2 microM.     

Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins.

MAP kinase-activated protein kinase-2 (MAPKAP kinase-2) phosphorylates the serine residues in murine heat shock protein 25 (hsp25) and human heat shock protein 27 (hsp27) which are phosphorylated in vivo in response to growth factors and heat shock, namely Ser15 and Ser86 (hsp25) and Ser15, Ser78 and Ser82 (hsp27). Ser86 of hsp25 and the equivalent residue in hsp27 (Ser82) are phosphorylated preferentially in vitro. The small heat shock protein is present in rabbit skeletal muscle and hsp25 kinase activity in skeletal muscle extracts co-purifies with MAPKAP kinase-2 activity throughout the purification of the latter enzyme. These results suggest that MAPKAP kinase-2 is the enzyme responsible for the phosphorylation of these small heat shock proteins in mammalian cells.     

p38 MAPK mediates TNF-induced apoptosis in endothelial cells via phosphorylation and downregulation of Bcl-x(L)

The role of p38 mitogen-activated protein kinase (MAPK) in apoptosis is a matter of debate. Here, we investigated the involvement of p38 MAPK in endothelial apoptosis induced by tumor necrosis factor alpha (TNF). We found that activation of p38 MAPK preceded activation of caspase-3, and the early phase of p38 MAPK stimulation did not depend on caspase activity, as shown by pretreatment with the caspase inhibitors z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and Boc-Asp(OMe)-fluoromethylketone (BAF). The p38 MAPK inhibitor SB203580 significantly attenuated TNF-induced apoptosis in endothelial cells, suggesting that p38 MAPK is essential for apoptotic signaling. Furthermore, we observed a time-dependent increase in active p38 MAPK in the mitochondrial subfraction of cells exposed to TNF. Notably, the level of Bcl-x(L) protein was reduced in cells undergoing TNF-induced apoptosis, and this reduction was prevented by treatment with SB203580. Immunoprecipitation experiments revealed p38 MAPK-dependent serine-threonine phosphorylation of Bcl-x(L) in TNF-treated cells. Exposure to lactacystin prevented both the downregulation of Bcl-x(L) and activation of caspase-3. Taken together, our results suggest that TNF-induced p38 MAPK-mediated phosphorylation of Bcl-x(L) in endothelial cells leads to degradation of Bcl-x(L) in proteasomes and subsequent induction of apoptosis.     

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.