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

Abstract

Pyridinyl imidazole inhibitors, particularly SB203580, have been widely used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase (p38/HOG/SAP-KII) 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, phosphoinositidedependent protein kinase 1. The concentrations of SB203580 required to block PKB phosphorylation (IC 50 3-5 M) are only approximately 10-fold higher than those required to inhibit p38 MAP kinase (IC 50 0.3-0.5 M). 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 M. Interleukin-2 (IL-2)1 is a potent T cell growth factor that mediates its effects via a high affinity heterotrimeric receptor comprising ␣, ␤, and ␥ c subunits. Several intracellular signaling pathways are known to be activated by IL-2, including the p42/44 mitogen-activated protein kinase (MAP kinase, also known as ERK2/1), the p38 and p54 MAP kinases (also called stress kinases, or HOG and JNK, respectively), the phosphatidyl inositol 3Ј (PI) 3-kinase pathway and the Jak/STAT (signal transducer and activator of transcription) pathways. Our earlier studies using the MEK (mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase) inhibitor PD-098059 (1) and those of others (2, 3) have indicated that the p42/44 MAP kinase pathway is not required for IL-2-driven proliferation. In contrast, a pyridinyl imidazole inhibitor of p38 MAP kinase, SB203580, inhibited IL-2-driven T cell proliferation with an IC 50 of 3-5 M, suggesting a possible role for p38 MAP kinase in this process (4). Recently, we have further investigated the role of p38 MAP kinase in proliferation by mapping the subdomains of the IL-2 receptor ␤ chain involved in the activation of the kinase. As previously shown for p42/44 MAP kinase, activation of p38 and p54 MAP kinases required the acidic rich A region of the IL-2 receptor ␤ chain (5). However, the A region is not required for proliferation (2, 5), indicating that neither p38 MAP kinase nor p54 MAP kinase is essential for this function. Furthermore, CNI-1493 (6, 7), an inhibitor of p38 and p54 MAP kinase activation by IL-2 was unable to inhibit proliferation (5). Surprisingly, SB203580 was still able to inhibit proliferation in the absence of IL-2 stimulated p38 MAP kinase activation. It has already been reported that SB203580 does inhibit p54...

Myeloid-Related Protein-14 Is a p38 MAPK Substrate in Human Neutrophils

“…fMLP stimulation before permeabilization resulted in a significant increase in MRP-14 staining that localized to the base of the actin cytoskeleton forming lamellipodia. The increased staining for MRP-14 and its localization at the base of lamellipodia were dependent on p38 MAPK-mediated phosphorylation, as both were inhibited by SB203580 at concentrations specific for p38 MAPK inhibition (55). These data suggest that phosphorylation by p38 MAPK results in MRP-14 association with long, unbranched actin filaments that form at the base of lamellipodia.…”

Insulin restores differentiation of Ras-transformed C2C12 myoblasts by inducing NF-κB through an AKT/P70S6K/p38-MAPK pathway

“…C2Ras transformed myoblasts failed to restore di erentiation when rapamycin or PD* were added in the presence of insulin+PD, indicating that the activation of both P70S6K and p38-MAPK/MAP-KAPK-2 was necessary to reach a fully di erentiated phenotype. The pyrinidyl imidazole compounds (such as PD*) have been described as partial inhibitors of activation of AKT and/or P70S6K under di erent acute stimulus in cardiac myocytes and T cells (Lali et al, 2000;Mockridge et al, 2000) but we did not observe this inhibitory e ect upon insulin stimulation in C2Ras myoblasts. However, under rapamycin treatment a partial inhibition of p38-MAPK/MAP-KAPK-2 was observed in C2Ras cells, indicating a crosstalk between P70S6K and p38MAPK/MAP-KAPK-2 pathways.…”

Selective Regulation of ICAM-1 and RANTES Gene Expression after ICAM-1 Ligation on Human Renal Fibroblasts

“…The increase in ICAM-1 mRNA levels seen at 3 h following ICAM-1 cross-linking was inhibited by the p38 inhibitor SB203580 (Fig 7), at a concentration, which maximally inhibits p38 MAP kinase activity without affecting other kinases (50). There was, however, no effect of the MEKK inhibitor, PD 98059 up to a maximum concentration of 50 M (a concentration 25 times its IC 50 ).…”

“…There was, however, no effect of the MEKK inhibitor, PD 98059 up to a maximum concentration of 50 M (a concentration 25 times its IC 50 ). This suggests that it is specifically the p38 MAP kinase that is linked to the induction of ICAM-1.…”