by Fan Dong, and Andrew C. Larner Activation of Akt kinase by granulocyte colony-stimulating factor (G-CSF): evidence for the role of a tyrosine kinase activity distinct from the janus kinases by Fan Dong, and Andrew C. Larner Blood Volume 95(5):1656-1662 March 1, 2000 ©2000 by American Society of Hematology

Activation of Akt by G-CSF treatment of BAF3 cells expressing the wild-type G-CSF receptor.(A) Induction of Akt phosphorylation. Activation of Akt by G-CSF treatment of BAF3 cells expressing the wild-type G-CSF receptor.(A) Induction of Akt phosphorylation. Cells were incubated in serum-free medium for 6 hours and then stimulated with G-CSF (100 ng/mL) for 10 minutes with or without pretreatment for 15 minutes with wortmannin (WM) or Ly294 002 (LY). Akt phosphorylation was determined using a phospho-specific antibody that recognizes Akt only when phosphorylated on Serine 473 (upper panel). The membrane was reprobed with anti-Akt antibody (lower panel). (B) Activation of Akt kinase activity. Akt was immunoprecipitated from whole-cell extracts prepared from unstimulated or G-CSF–stimulated cells. The kinase activity of Akt was determined by in vitro kinase assay using histone H2B as a substrate (upper panel). The amounts of Akt kinase in each sample were determined by probing the membrane with anti-Akt antibody (lower panel). Fan Dong, and Andrew C. Larner Blood 2000;95:1656-1662 ©2000 by American Society of Hematology

Kinetics of G-CSF–induced Akt phosphorylation in BAF3 cells expressing the different forms of the G-CSF receptor.(A) Schematic diagram of the wild-type (WT) and truncated forms of the G-CSF receptor. Kinetics of G-CSF–induced Akt phosphorylation in BAF3 cells expressing the different forms of the G-CSF receptor.(A) Schematic diagram of the wild-type (WT) and truncated forms of the G-CSF receptor. Boxes B1, B2, and B3 denote subdomains conserved in several members of the cytokine receptor superfamily. The numbers in parentheses indicate amino acid positions; TM, transmembrane domain. (B) Akt phosphorylation induced by G-CSF in BAF3 cells expressing the different G-CSF receptor forms. Cells were left untreated or treated with G-CSF for the indicated times. Whole-cell extracts were immunoblotted with anti-phospho-Akt antibody (upper panel) and reprobed with anti-Akt antibody (lower panel). (C) G-CSF stimulated phosphorylation of Akt in myeloid 32D cells expressing the wild type or the D715 form of the receptor. Fan Dong, and Andrew C. Larner Blood 2000;95:1656-1662 ©2000 by American Society of Hematology

Inhibition of sustained activation of Akt by wortmannin Inhibition of sustained activation of Akt by wortmannin.BAF3 cells expressing the D715 receptor were unstimulated or stimulated with G-CSF for 10 minutes prior to addition of wortmannin (100 nM) to the cultures. Inhibition of sustained activation of Akt by wortmannin.BAF3 cells expressing the D715 receptor were unstimulated or stimulated with G-CSF for 10 minutes prior to addition of wortmannin (100 nM) to the cultures. Whole-cell extracts were prepared at the indicated times and used for analysis of Akt phosphorylation (upper panel). The same membrane was incubated with anti-Akt antibody (lower panel). Fan Dong, and Andrew C. Larner Blood 2000;95:1656-1662 ©2000 by American Society of Hematology

Effects of dominant negative (DN) Jak mutants on the activation of Akt and Stat5.(A) COS-7 cells were transfected with cDNAs encoding the wild-type G-CSF receptor and Stat5a only or were transfected together with cDNAs encoding the kinase inactive Jaks as i... Effects of dominant negative (DN) Jak mutants on the activation of Akt and Stat5.(A) COS-7 cells were transfected with cDNAs encoding the wild-type G-CSF receptor and Stat5a only or were transfected together with cDNAs encoding the kinase inactive Jaks as indicated. Twenty hours after transfection, cells were starved for 4 hours prior to stimulation with G-CSF for 10 minutes. Whole-cell extracts were prepared and used for the analysis of Akt phosphorylation (upper panel). The membrane was reprobed with anti-Akt antibody (lower panel). (B) The same extracts were used for the analysis of Stat5a activation by EMSA using GRR probe. The complex that contains Stat5 is indicated with an arrow and labeled “GRR.” Fan Dong, and Andrew C. Larner Blood 2000;95:1656-1662 ©2000 by American Society of Hematology

Effects of different inhibitors on G-CSF–induced activation of Akt and Stats.(A) BAF3 cells expressing the wild-type G-CSF receptor were unstimulated (lane 1) or stimulated with G-CSF for 10 minutes without (lane 2) or with preincubation with wortmannin (WM... Effects of different inhibitors on G-CSF–induced activation of Akt and Stats.(A) BAF3 cells expressing the wild-type G-CSF receptor were unstimulated (lane 1) or stimulated with G-CSF for 10 minutes without (lane 2) or with preincubation with wortmannin (WM: 100 nM; lane 3), Ly294 002 (LY: 10 μM; lane 4), genistein (GN: 200 μM; lane 5), herbimycin A (HB: 1 μg/mL; lane 6), PP1 (10 μM; lane 7), bisindolylmaleimide (BM: 5 μM; lane 8) or cycloheximide (CHX: 30 μg/mL; lane 9). The preincubation times were 15 minutes except for herbimycin A (180 minutes). Whole-cell extracts were prepared and used for analysis of Akt phosphorylation by Western blotting. (B) The same extracts were used for the analysis of Stat5a activation by EMSA. (C) Peripheral blood neutrophils were left unstimulated or stimulated with G-CSF for 5 minutes following pretreatment with wortmannin or PP1 for 15 minutes as indicated. Whole-cell extracts were examined for Akt phosphorylation. Fan Dong, and Andrew C. Larner Blood 2000;95:1656-1662 ©2000 by American Society of Hematology

PP1 has no effect on the activation of JNK and p42, p44 MAPK PP1 has no effect on the activation of JNK and p42, p44 MAPK.BAF/WT cells were stimulated with G-CSF for 10 minutes with or without pretreatment with wortmannin (WM) or PP1. PP1 has no effect on the activation of JNK and p42, p44 MAPK.BAF/WT cells were stimulated with G-CSF for 10 minutes with or without pretreatment with wortmannin (WM) or PP1. (A) Whole-cell extracts were prepared, and JNK was immunoprecipitated with specific antiserum. The kinase activity of JNK was determined by in vitro kinase assay using ATF2 as a substrate (upper panel). The membrane was subsequently probed for JNK (lower panel). (B) The same cell extracts were also used in Western blot analysis for the determination of p42, p44 MAPK phosphorylation using a phospho-specific antibody (upper panel). Equal loading was confirmed by incubating the membrane with an anti p42, p44 MAPK antibody (lower panel). (C) Whole-cell extracts were also examined for Akt phosphorylation. Fan Dong, and Andrew C. Larner Blood 2000;95:1656-1662 ©2000 by American Society of Hematology

Effect of overexpression of different Jaks, c-Src, or Lyn on the activation of Akt and Stat5.COS-7 cells were transfected with either the Stat5a cDNA only (lanes 1 and 7) or together with cDNAs encoding the different Jaks (lane 2 to 4), c-Src (lane 5), Syk ... Effect of overexpression of different Jaks, c-Src, or Lyn on the activation of Akt and Stat5.COS-7 cells were transfected with either the Stat5a cDNA only (lanes 1 and 7) or together with cDNAs encoding the different Jaks (lane 2 to 4), c-Src (lane 5), Syk (lane 6), or Lyn (lane 8). Twenty hours after transfection, cells were serum-starved for 4 hours prior to preparation of whole-cell extracts. (A) Akt phosphorylation was determined by Western blotting using phospho-specific Akt antibody (upper panel). The blot was reprobed with anti-Akt antibody (lower panel). (B) Activation of Stat5a was measured by EMSA using GRR probe. Fan Dong, and Andrew C. Larner Blood 2000;95:1656-1662 ©2000 by American Society of Hematology