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• W1969388212 abstract "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 (IC50 3–5 μm) are only approximately 10-fold higher than those required to inhibit p38 MAP kinase (IC50 0.3–0.5 μm). 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 μm. 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 (IC50 3–5 μm) are only approximately 10-fold higher than those required to inhibit p38 MAP kinase (IC50 0.3–0.5 μm). 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 μm. interleukin mitogen-activated protein protein kinase B 3-phosphoinositide-dependent protein kinase retinoblastoma phosphatidylinositol c-Jun NH2-terminal kinase stress-activated protein kinase 4-morpholinepropanesulfonic acid dithiothreitol phosphatidylinositol 3,4,5-trisphosphate Interleukin-2 (IL-2)1 is a potent T cell growth factor that mediates its effects via a high affinity heterotrimeric receptor comprising α, β, and γc 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 3′ (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 PD098059 (1.Crawley J.B. Willcocks J. Foxwell B.M. Eur. J. Immunol. 1996; 26: 2717-2723Crossref PubMed Scopus (42) Google Scholar) and those of others (2.Minami Y. Oishi I. Liu Z.J. Nakagawa S. Miyazaki T. Taniguchi T. J. Immunol. 1994; 152: 5680-5690PubMed Google Scholar, 3.Evans G.A. Goldsmith M.A. Johnston J.A. Xu W. Weiler S.R. Erwin R. Howard O.M.Z. Abraham R.T. O'Shea J.J. Greene W.C. Farrar W.L. J. Biol. Chem. 1995; 270: 28858-28863Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) 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 IC50 of 3–5 μm, suggesting a possible role for p38 MAP kinase in this process (4.Crawley J.B. Rawlinson L. Lali F.V. Page T.H. Saklatvala J. Foxwell B.M.J. J. Biol. Chem. 1997; 272: 15023-15027Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Recently, we have further investigated the role of p38 MAP kinase in proliferation by mapping the subdomains of the IL-2 receptor β 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 β chain (5.Hunt A.E. Lali F.V. Lord J.D. Nelson B.H. Miyazaki T. Tracey K.J. Foxwell B.M.J. J. Biol. Chem. 1999; 274: 7591-7597Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). However, the A region is not required for proliferation (2.Minami Y. Oishi I. Liu Z.J. Nakagawa S. Miyazaki T. Taniguchi T. J. Immunol. 1994; 152: 5680-5690PubMed Google Scholar, 5.Hunt A.E. Lali F.V. Lord J.D. Nelson B.H. Miyazaki T. Tracey K.J. Foxwell B.M.J. J. Biol. Chem. 1999; 274: 7591-7597Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), indicating that neither p38 MAP kinase nor p54 MAP kinase is essential for this function. Furthermore, CNI-1493 (6.Bianchi M. Bloom O. Raabe T. Cohen P.S. Chesney J. Sherry B. Schmidtmayerova H. Calandra T. Zhang X. Bukrinsky M. Ulrich P. Cerami A. Tracey K.J. J. Exp. Med. 1996; 183: 927-936Crossref PubMed Scopus (123) Google Scholar, 7.Cohen P.S. Nakshatri H. Dennis J. Caragine T. Bianchi M. Cerami A. Tracey K.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3967-3971Crossref PubMed Scopus (102) Google Scholar), an inhibitor of p38 and p54 MAP kinase activation by IL-2 was unable to inhibit proliferation (5.Hunt A.E. Lali F.V. Lord J.D. Nelson B.H. Miyazaki T. Tracey K.J. Foxwell B.M.J. J. Biol. Chem. 1999; 274: 7591-7597Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). 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 MAP kinase activity (8.Whitmarsh A.J. Yang S.H. Su M.S. Sharrocks A.D. Davis R.J. Mol. Cell. Biol. 1997; 17: 2360-2371Crossref PubMed Scopus (438) Google Scholar,9.Clerk A. Sugden P.H. FEBS Lett. 1998; 426: 93-96Crossref PubMed Scopus (207) Google Scholar), but the possibility that the anti-proliferative effects of SB203580 may be mediated by effects on p54 MAP kinase can also be discounted by the studies of Hunt et al. (5.Hunt A.E. Lali F.V. Lord J.D. Nelson B.H. Miyazaki T. Tracey K.J. Foxwell B.M.J. J. Biol. Chem. 1999; 274: 7591-7597Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The above data suggest the existence of a novel target of SB203580 that is critical for mitogenic signaling. To help understand the mechanism of action and possible targets of SB203580, the effect of this inhibitor on cell cycle-regulated proteins was examined. We found that the IL-2-induced hyperphosphorylation of Rb was greatly reduced in SB203580-treated cells, whereas the drug had no effect on cytokine-induced p27kip degradation or Myc expression. Subsequent studies investigated the effects of SB203580 on PI 3-kinase/protein kinase B (PKB), as these kinases have been implicated in the regulation of Rb phosphorylation (10.Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). We observed that although PI 3-kinase activity was unaffected, the phosphorylation of PKB on Thr308 and Ser473 was inhibited, resulting in an inhibition of IL-2-induced kinase activity. The concentrations of SB203580 required were commensurate with antiproliferative effects of the drug and provide evidence that 3-phosphoinositide-dependent protein kinase 1 (PDK1; the Thr308 kinase) is inhibited by SB203580. It has recently been reported that PDK1 also acts as the Ser473 kinase (11.Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). These results could provide a mechanism for the antiproliferative effect of SB203580. Furthermore these data will have important implications for the interpretation of results from numerous published studies in which SB203580 has been used at concentrations in excess of 2 μm. IL-2 was a generous gift from Dr. P. Lomedico (Roche Molecular Biochemicals). Antiphospho-specific Ser473 and Thr308 PKB were from New England Biolabs (Hitchin, Herts, UK), whereas sheep anti-PKB and other reagents for in vitroPKB kinase assay were sold as a kit by Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Rb was from Pharmingen (San Diego, CA), and the monoclonal anti-cyclin D3 and anti-p27kip1 were from Pierce. Rabbit anti-p70S6 kinase was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and was used for both immunoprecipitation and Western blotting. Rabbit anti-c-Myc antibody was from Santa Cruz (Santa Cruz, CA). SB203580 was purchased from Calbiochem-Novabiochem, and U5 anti-p85α monoclonal antibody was a generous gift from Professor D. Cantrell (Imperial Cancer Research Fund, London). Rabbit antisera to p38 MAP kinase was from Professor J. Saklatvala (Kennedy Institute of Rheumatology, London). Second-layer antibodies (horseradish peroxidase-conjugated) were purchased from DAKO (DAKO A/S Denmark). Recombinant PDK1 and recombinant PKB were kindly provided by Dr. L. Stephens (Babraham Institute, Cambridge, UK) and Dr. D. Alessi (Dundee, UK). CN1-1493 was from Dr. K. Tracey (Picower Institute of Medical Research New York). The IL-2-dependent murine T cell line, CT6, was grown and maintained as described previously (12.Willcocks J.L. Hales A. Page T.H. Foxwell B.M. Eur. J. Immunol. 1993; 23: 716-720Crossref PubMed Scopus (9) Google Scholar). These cells were rested by washing three times in RPMI and culturing overnight in RPMI, 5% fetal calf serum in the absence of growth factor, antibiotics, or β-mercaptoethanol supplements. 2–5 × 106 rested CT6 cells were resuspended in 2 ml of RPMI, 5% fetal calf serum and preincubated with inhibitors or vehicle control as indicated in figure legends. Cells were then stimulated with 20 ng/ml recombinant human IL-2 for 5 min at 37 °C and pelleted in a minifuge for 30 s, medium was aspirated, and the pellet was lysed in the appropriate buffer. BA/F3 cells stably expressing deletion mutants of IL-2 receptor β chain (a generous gift from Professor T. Taniguchi, Tokyo, Japan) were maintained in glutamine containing RPMI further supplemented with 5% fetal calf serum and 0.2 μg/ml G418 (Calbiochem-Novabiochem) as described previously (13.Hatakeyama M. Mori H. Doi T. Taniguchi T. Cell. 1989; 59: 837-845Abstract Full Text PDF PubMed Scopus (302) Google Scholar). Human peripheral blood mononuclear cells were prepared from buffy coat leukophoresis residues (North London Blood Transfusion Service, Colindale, London UK) and activated with 50 ng/ml OKT3 for 48 h. The cells were then washed extensively, rested overnight, and washed again before activating with IL-2; such cell preparations were >90% T cells (14.Page T.H. Lali F.V. Groome N. Foxwell B.M. J. Immunol. 1997; 158: 5727-5735PubMed Google Scholar). Cellular proliferation assays were performed by measurement of [3H]thymidine incorporation as described previously (12.Willcocks J.L. Hales A. Page T.H. Foxwell B.M. Eur. J. Immunol. 1993; 23: 716-720Crossref PubMed Scopus (9) Google Scholar). Cells were lysed in Buffer A (see below) for Western blotting and PKB kinase assays. Kinase assays were performed according to the manufacturer's instructions. Briefly, 4 μg of sheep anti-PKBα was immobilized on 25 μl of protein G-Sepharose overnight (or 1.5 h) and washed in Buffer A (50 mm Tris, pH 7.5, 1 mm EDTA, 1 mmEGTA, 0.5 mm Na3VO4, 0.1% β-mercaptoethanol, 1% Triton X-100, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 0.1 mmphenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, pepstatin, leupeptin, and 1 μm microcystin). The immobilized anti-PKB was then incubated with 0.5 ml of lysate (from 5 × 106 cells) for 1.5 h and washed three times in 0.5 ml of Buffer A supplemented with 0.5 m NaCl, two times in 0.5 ml of Buffer B (50 mm Tris-HCl, pH 7.5, 0.03% (w/v) Brij-35, 0.1 mm EGTA, and 0.1% β-mercaptoethanol), and twice with 100 μl of assay dilution buffer; 5× assay dilution buffer is 100 mm MOPS, pH 7.2, 125 mmβ-glycerophosphate, 25 mm EGTA, 5 mm sodium orthovanadate, 5 mm DTT. To the PKB enzyme immune complex was added 10 μl of assay dilution buffer, 40 μm protein kinase A inhibitor peptide, 100 μm PKB-specific substrate peptide, and 10 μCi of [γ-32P]ATP, all made up in assay dilution buffer. The reaction was incubated for 20 min at room temperature with shaking, then samples were pulse spun, and 40 μl of reaction volume were removed into another tube to which was added 20 μl of 40% trichloroacetic acid to stop the reaction. This was mixed and incubated for 5 min at room temperature, and 40 μl was transferred onto P81 phosphocellulose paper and allowed to bind for 30 s. The P81 pieces were washed three times in 0.75% phosphoric acid then in acetone at room temperature. γ-32P incorporation was then measured by scintillation counting. Cells were lysed in PI 3-kinase lysis buffer (40 mm Tris-HCl, pH 7.5, 200 mm NaCl, 1 mm EGTA supplemented with 1 mm DTT, 1 mm Na3VO4, and 10 μg/ml each of aprotinin, pepstatin, leupeptin) at 10 × 106cells/ml, and the post-nuclear lysate was precleared with 25 μl of protein G-Sepharose for 1 h then preincubated with 5 μg of monoclonal anti-p85α (U5) and further with 25 μl of protein G-Sepharose for the final 1 h. The pellets were washed three times in 0.5 ml of PI 3-kinase assay buffer. The pellet was then resuspended in 25 μl of kinase assay buffer. To this, 10 μl of a 1 mg/ml mixture of phosphatidylinositol and phosphatidylserine (made up in 100 mm HEPES, pH 7.5, and sonicated just before use) was added. The mixture was then preincubated at room temperature for 10 min, and the reaction was started by the addition of 15 μl of ATP mixture (340 μl of water, 4.2 μl of 1 mMgCl2, 16 μl of 100 mm ATP) supplemented with 5 μCi of [γ-32P]ATP. The reaction proceeded for 15 min and was stopped by the addition of 100 μl of 1 m HCl and vortexing, adding a further 200 μl of a 1:1 chloroform:methanol and vortexing again, and microfuge-spinning the tubes for 5 min. The lower layer was removed and dried in vacuo (or at 60 °C on dry block) then redissolved in 10 μl of 4:1 chloroform:methanol before spotting onto silica plates. The plate was developed in a preequilibrated vertical tank with chloroform, methanol, 28% ammonium hydroxide, water (180:140:10.8:27.5) for 3 h (or overnight) followed by phosphorimaging analysis (Fuji FLA-2000). Cells were lysed in 0.5 ml of p70S6 kinase lysis buffer (10 mm potassium phosphate, pH 7.05, 0.5% Triton X-100, 1 mm EDTA, 5 mm EGTA, 1 mm Na3VO4, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin), and the postnuclear lysate was precleared with 20 μl of protein A-agarose for 30 min. The precleared supernatant was then preincubated with 5 μl of rabbit antiserum to p70S6 kinase for 1 h and additionally with 25 μl of protein A-agarose with mixing for a further 1 h, all at 4 °C. The final immune complex was washed twice in 0.5 ml of lysis buffer and twice in 0.5 ml of kinase assay buffer (50 mm MOPS, pH 7.2, 1 mm DTT, 30 mm ATP, 5 mmMgCl2, 10 mm p-nitrophenylphosphate). To washed immune complex pellet was added 45 μl of assay mixture (made up of 35 μl of kinase assay buffer, 5 μl of 125 mm substrate peptide (KKRNRTLTK), 5 μl of 50 μm protein kinase A inhibitor, 5 μCi of [γ-32P]ATP), and the reaction was allowed to continue for 30 min at room temperature. The reaction was stopped by the addition of reducing sample buffer and boiling for 5 min. After separation on a peptide gel as described before (15.Crawley J.B. Williams L.M. Mander T. Brennan F.M. Foxwell B.M.J. J. Biol. Chem. 1996; 271: 16357-16362Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), radioactivity incorporated into peptide was quantitated by phosphorimaging. Lipid vesicles were made by drying down a mixture of phosphatidylcholine and phosphatidylserinein vacuo and reconstituting with lipid buffer (0.2m NaCl, 20 mm HEPES, 2 mm EGTA) to a final 5 times working stock (500 μmphosphatidylcholine, 500 μm phosphatidylserine, and 100 μm phosphatidylinositol 3,4,5-trisphosphate (PIP3) and sonicated before use. EE-tagged recombinant PDK1 and PKBα (both >98% pure) were prediluted in enzyme dilution buffer (1 mm DTT, 0.1 m NaCl, 1 mm EGTA, 20 mm HEPES). PDK1 assays were performed with 1 μm EE-PKB and 50 nm EE-PDK1 in the presence of appropriately diluted lipid vesicles, 0.5 μm ATP, and 1 μCi of [γ-32P]ATP in assay buffer (8 mmMgCl2, 0.12 m NaCl, 1.2 mm DTT, 1.2 mm EGTA, 0.01% azide) supplemented with protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin) in a final volume of 5 μl. The reaction was allowed to continue for 5 min at 30 °C and stopped by boiling with 10 μl of 1.5 times SDS sample buffer (with 5 mm EDTA). The PKB autokinase assays were performed as above for PDK1 but in the absence of PDK1 and PIP3. Samples were then resolved on a 10% SDS-polyacrylamide electrophoresis gel and quantitated by phosphorimaging (Fuji FLA-2000). After separation by SDS-polyacrylamide gel electrophoresis, proteins were transferred onto nitrocellulose or polyvinylidene difluoride membranes that were then blocked with 5% (w/v) milk powder in Tris-buffered saline/Tween 20 (0.1%) for 1 h at room temperature. The blocked membranes were then incubated in 1:1000 dilution of first-stage antibody (made up in Tris-buffered saline Tween, 2% bovine serum albumin supplemented with 0.1% azide) overnight, then washed and incubated with 1:1000 horseradish peroxidase-conjugated secondary antibody (made up in Tris-buffered saline Tween, 5% (w/v) milk powder) for 1 h at room temperature before developing with ECL. As we have shown previously (4.Crawley J.B. Rawlinson L. Lali F.V. Page T.H. Saklatvala J. Foxwell B.M.J. J. Biol. Chem. 1997; 272: 15023-15027Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 5.Hunt A.E. Lali F.V. Lord J.D. Nelson B.H. Miyazaki T. Tracey K.J. Foxwell B.M.J. J. Biol. Chem. 1999; 274: 7591-7597Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), the IL-2-induced proliferation of primary human T cells, murine CT6 T cells, or BAF F7 B cells is prevented by the p38 MAP kinase inhibitor with an IC50 of 3–5 μm (Fig. 1). However, as our recent studies (5.Hunt A.E. Lali F.V. Lord J.D. Nelson B.H. Miyazaki T. Tracey K.J. Foxwell B.M.J. J. Biol. Chem. 1999; 274: 7591-7597Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) showed that IL-2-induced proliferation and the inhibitory effects of SB203580 on this event were independent of p38 MAP kinase or even p54 MAP kinase activity in both T cells and B cells, we endeavored to identify other possible targets involved in mediating the anti-proliferative effects. To do this, we investigated events associated with cell cycle progression. SB203580 had no effect on Myc expression, except for a small reduction at 30 μm only (Fig. 2 a). Furthermore, nuclear staining of SB203580-treated CT6 cells with propidium iodide showed no evidence of apoptosis after stimulation with IL-2 for 20 h (data not shown). The expression of hyperphosphorylated Rb and degradation of p27kip1 were also measured as markers of S-phase entry. The addition of IL-2 to resting CT6 cells caused the hyperphosphorylation of Rb as detected by Western blotting (Fig.2 b). The presence of SB203580 in the antiproliferative (0–30 μm) range resulted in a dose-dependent reduction in the hyperphosphorylated form (Fig. 2 b). There also appeared to be some reduction in the total levels of Rb protein. Similar inhibitory effects on Rb hyperphosphorylation and protein levels were obtained with wortmannin and LY294002, both inhibitors of PI 3-kinase. The decrease in Rb protein is likely to be due to the IL-1-converting enzyme (ICE)-mediated proteolysis of the hypophosphorylated form, which has been previously reported (16.Janicke R.U. Walker P.A. Lin X.Y. Porter A.G. EMBO J. 1996; 15: 6969-6978Crossref PubMed Scopus (222) Google Scholar, 17.Dou Q.P. An B. Antoku K. Johnson D.E. J. Cell. Biochem. 1997; 64: 586-594Crossref PubMed Scopus (49) Google Scholar, 18.Gottlieb E. Oren M. EmBO J. 1998; 17: 3587-3596Crossref PubMed Scopus (74) Google Scholar). These results would also agree with previous studies on the role of PI 3-kinase in IL-2-induced Rb hyperphosphorylation and protein by Brennanet al. (10.Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar) and would support previous indications that PI 3-kinase is a proximal regulator of Rb (10.Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 19.Bacqueville D. Casagrande F. Perret B. Chap H. Darbon J.M. Breton-Douillon M. Biochem. Biophys. Res. Commun. 1998; 244: 630-636Crossref PubMed Scopus (40) Google Scholar, 20.Belham C.M. Scott P.H. Twomey D.P. Gould G.W. Wadsworth R.M. Plevin R. Cell. Signal. 1997; 9: 109-116Crossref PubMed Scopus (16) Google Scholar). The effects of SB203580 on Rb hyperphosphorylation were confirmed in similar studies on activated primary human T cells (Fig. 2 d).Figure 2Effect of SB203580 on cell cycle proteins. Resting CT6 cells (a, b,c) and PBMC (d and e) were preincubated with the indicated doses of SB203580, wortmannin, LY294002, or vehicle control for 1 h before stimulating with 20 ng/ml IL-2 or vehicle control for 3 h (c-Myc) or 20 h (Rb and p27kip). Cells were then lysed and prepared for total cellular c-Myc (a) Rb (b and d) or p27kip (c and e) measurement by Western blotting as described under “Experimental Procedures.” The data are representative of three separate experiments. pRb, hypophosphorylated; ppRb, hyperphosphorylated Rb.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also investigated a second cell cycle-regulated protein, p27kip1. The addition of IL-2 to resting CT6 cells induces the degradation of p27kip1(Fig. 2 c). This degradation was unaffected by SB203580, which if anything, further reduced levels of the protein. Wortmannin and LY294002 similarly had no inhibitory effect on p27kip1 degradation. Again, these studies on p27kip1 degradation were repeated in activated primary human T cells, with no significant inhibition observed with SB203580 or wortmannin, although LY294002 had some inhibitory effect (Fig. 2 e). The characteristic, S-phase hyperphosphorylation of Rb induced by IL-2 has been reported to be mediated by the PI 3-kinase pathway via the distal effector PKB (10.Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Furthermore, both the mitogenic and survival functions of the PI 3-kinase pathway have, in several reports, been attributed to PKB (10.Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 15.Crawley J.B. Williams L.M. Mander T. Brennan F.M. Foxwell B.M.J. J. Biol. Chem. 1996; 271: 16357-16362Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 19.Bacqueville D. Casagrande F. Perret B. Chap H. Darbon J.M. Breton-Douillon M. Biochem. Biophys. Res. Commun. 1998; 244: 630-636Crossref PubMed Scopus (40) Google Scholar, 20.Belham C.M. Scott P.H. Twomey D.P. Gould G.W. Wadsworth R.M. Plevin R. Cell. Signal. 1997; 9: 109-116Crossref PubMed Scopus (16) Google Scholar, 21.Ahmed N.N. Grimes H.L. Bellacosa A. Chan T.O. Tsichlis P.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3627-3632Crossref PubMed Scopus (487) Google Scholar, 22.Haas-Kogan D. Shalev N. Wong M. Mills G. Yount G. Stokoe D. Curr. Biol. 1998; 8: 1195-1198Abstract Full Text Full Text PDF PubMed Google Scholar). We were therefore interested in investigating the possibility that SB203580 mediates its effects on Rb by inhibiting these kinases, especially as wortmannin and LY294002 displayed similar effects. The activation of PKB requires the PI 3-kinase-generated second messenger PIP3 as well as phosphorylation on Thr308 and Ser473 mediated by the PIP3-dependent kinases, PDK1 and PDK2, respectively (23.Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 24.Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). We investigated the effect of SB203580 on PKB activation by looking at IL-2-induced phosphorylation of residue Ser473 of PKB in whole cell lysates using a phospho-specific antibody. In both CT6 and activated human T cells, SB203580 inhibited the phosphorylation of Ser473 in a dose-dependent manner (Fig.3, a and b). Similar studies on the IL-2-responsive BA/F3 F7 B cells gave the same result (Fig. 3 c). The approximate IC50 for the effect of SB203580 on this parameter is ∼5 μm, similar to the concentration required for the inhibitory effects on proliferation (Fig. 1). As expected, wortmannin (Fig. 3) and LY294002 (not shown) also inhibited Ser473 phosphorylation, whereas rapamycin (not shown) had no effect. The phosphorylation of PKB on Thr308 was similarly investigated. As the antibody was not so effective, PKB was first immunoprecipitated, and phospho-Thr308 was detected by Western blotting. SB203580 inhibited Thr308 phosphorylation in CT6 cells with similar efficacy to the Ser473 phosphorylation (Fig.4). Wortmannin, as expected, also inhibited this Thr308 phosphorylation. To confirm that the effects of SB203580 on PKB phosphorylation correlated with kinase activity, assays were performed on immunoprecipitated PKB from IL-2-stimulated CT6 cells (Fig. 5). The drug inhibited PKB activation with an IC50 of 3–10 μm, in agreement with its effects on phosphorylation of the kinase and cell proliferation.Figure 4Effect of SB203580 on phosphorylation of threonine-308 on PKB. Rested CT6 were preincubated with the indicated doses of SB203580 or vehicle control for 1 h or 100 nm wortmannin for 15 min before stimulating with 20 ng/ml IL-2 or vehicle control for 5 min. Cells were then lysed, and PKB was immunoprecipitated (IP) with anti-PKBα antiserum or irrelevant control antibody. Threonine 308 phosphorylation was assayed by Western blotting (upper panel). Membranes were stripped and reprobed for total PKB to confirm equal loading (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5SB203580 inhibits IL-2-induced PKB activity. Rested CT6 cells per sample were preincubated with indicated concentrations of SB203580, wortmannin, or vehicle control before stimulating with 20 ng/ml IL-2 or vehicle control for 5 min. Immunokinase assays were performed as described under “Experimental Procedures.” Panel a is representative of three separate experiments. b, the accumulated data from the three experiments is represented graphically showing inhibition of PKB activity as a percentage of uninhibited, IL-2-activated controls.Error bars equal ± S.D. (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although the above results showed that PKB activation is inhibited, it is still possible that PKB is one of several mitogenic effector molecules downstream of the actual SB203580 target. Therefore the effect of SB203580 on IL-2-induced activation of the PI 3-kinase/PKB pathway was examined. Exposure of CT6 cells to IL-2 leads to a reproducible 2-fold increase in anti-p85-precipitable PI 3-kinase activity. This was unaffected by preincubating the cells with SB203580. In contrast, wortmannin totally inhibited this activity (Fig.6). Furthermore, direct addition of SB203580 to PI 3-kinase assays did not have any effect (results not shown), indicating that PI 3-kinase is not the target of the drug. The effect of SB203580 on PI 3-kinase/PKB pathway was also examined indirectly. Several studies have shown that p70S6 kinase is a distal mediator of PI 3-kinase activity in several systems (15.Crawley J.B. Williams L.M. Mander T. Brennan F.M. Foxwell B.M.J. J. Biol. Chem. 1996; 271: 16357-16362Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 25.Calvo V. Crews C.M. Vik T.A. Bierer B.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7571-7575Crossref PubMed Scopus (167) Google Scholar). As expected, wortmannin (Fig. 6 b) and LY294002 (not shown) inhibited the activation of p70S6 kinase by IL-2, as measured in immunokinase assays. We observed that SB203580 could also inhibit IL-2-induced p70S6 kinase activation, althou" @default.
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• W1969388212 title "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" @default.
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