19-135 Sigma-Aldrichp38/SAPK2 Inhibitor (SB 203580)

p38 mitogen-activated protein kinase (MAPK) was identified in platelets on the basis of (a) its reactivity with antibodies to C-terminal and N-terminal peptides, and (b) its ability to activate MAPK-activated protein kinase-2, which phosphorylates the small heat shock protein, hsp27. p38 MAPK was activated in platelets by collagen fibers, a collagen-related cross-linked peptide, thrombin, or the thromboxane analogue U46619. A highly specific inhibitor of p38 MAPK, a pyridinyl imidazole known as SB203580, inhibited the platelet enzyme in vitro (IC50 approximately 0.5 microM). At similar concentrations it also inhibited agonist-stimulated phosphorylation of hsp27 in platelets, and platelet aggregation and secretion induced by minimal aggregatory concentrations of collagen or U46619, but not thrombin. Inhibition of aggregation was overcome by increasing agonist dose. SB203580 might act by inhibiting thromboxane generation, but this was only inhibited by 10-20% at low agonist concentrations. p38 MAPK provides a crucial signal, which is necessary for aggregation caused by minimal concentrations of collagen fibers or U46619. Thrombin or high doses of these agonists generate signals that bypass the enzyme, or render the enzyme no longer rate-limiting.

The ERK, JNK/SAPK and p38/RK MAP kinase subtypes (reviewed in [1]) are differentially activated in mammalian cells by various stimuli, which elicit induction of immediate-early (IE) genes, such as c-fos and c-jun (reviewed in [1-3]), as well as phosphorylation of histone H3 [4] and HMG-14 [5]. Anisomycin and UV radiation have been suggested to induce c-fos and c-jun transcription via JNK/SAPK-mediated phosphorylation of TCF (ternary complex factor), for c-fos induction [6-8], and c-Jun and/or ATF-2 for c-jun induction [9-11] [12,13]. We report here that anisomycin and ultraviolet radiation (UV) activate MAP kinase kinase-6 (MKK6) [14,15], p38/RK [16] [17,18] and MAPKAP kinase-2 (MAPKAP K-2) [17-19]. By using the p38/RK inhibitor SB 203580 [20,21], we show that activation of p38/RK and/or its downstream effectors are essential for anisomycin- and UV-stimulated c-fos/c-jun induction and histone H3/HMG-14 phosphorylation, whereas JNK/SAPK activation and phosphorylation of c-Jun and ATF-2 are insufficient for these responses.

The Ca2+-sensitive 85-kDa cytosolic phospholipase A2 (cPLA2) is responsible for thrombin-stimulated mobilization of arachidonic acid for the synthesis of thromboxane A2 in human platelets. We have previously shown that thrombin activates p38 kinase, a recently discovered new member of the mitogen-activated protein kinase family (Kramer, R. M., Roberts, E. F., Strifler, B. A., and Johnstone, E. M. (1995) J. Biol. Chem. 270, 27395-27398) and also induces phosphorylation of cPLA2, thereby increasing its intrinsic catalytic activity. In the present study we have examined the role of p38 kinase in the phosphorylation and activation of cPLA2 in stimulated platelets. We have observed that activation of p38 kinase accompanies receptor-mediated events in platelets and coincides with cPLA2 phosphorylation. Furthermore, in the presence of inhibitors of p38 kinase, the proline-directed phosphorylation of cPLA2 was completely blocked in platelets stimulated with the thrombin receptor agonist peptide SFLLRN and was suppressed during the early (up to 2 min) phase of platelet stimulation caused by thrombin. Unexpectedly, we found that prevention of proline-directed phosphorylation of cPLA2 in stimulated platelets did not attenuate its ability to release arachidonic acid from platelet phospholipids. We conclude that: 1) cPLA2 is a physiological target of p38 kinase; 2) p38 kinase is involved in the early phosphorylation of cPLA2 in stimulated platelets; and 3) proline-directed phosphorylation of cPLA2 is not required for its receptor-mediated activation.

A class of pyridinyl imidazoles inhibit the MAP kinase homologue, termed here reactivating kinase (RK) [Lee et al. (1994) Nature 372, 739-746]. We now show that one of these compounds (SB 203580) inhibits RK in vitro (IC50 = 0.6 microM), suppresses the activation of MAPKAP kinase-2 and prevents the phosphorylation of heat shock protein (HSP) 27 in response to interleukin-1, cellular stresses and bacterial endotoxin in vivo. These results establish that MAPKAP kinase-2 is a physiological RK substrate, and that HSP27 is phosphorylated by MAPKAP kinase-2 in vivo. The specificity of SB 203580 was indicated by its failure to inhibit 12 other protein kinases in vitro, and by its lack of effect on the activation of RK kinase and other MAP kinase cascades in vivo. We suggest that SB 203580 will be useful for identifying other physiological roles and targets of RK and MAPKAP kinase-2.