Figure 1
Figure 1. Flt3-ITD–induced STAT5 activation is independent of Jak or Src kinases. (A) Flt3-ITD phosphorylates STAT5 in AG490-resistant manner. The 32D Flt3-ITD cells were starved for 6 hours in the presence or absence of PKC412 (100 nM) or AG490 (50 μM). Cell lysates were immunoblotted against activation-specific phospho-Flt3 (Y591) or STAT5 (Y694) antibodies. Equal loading of proteins was confirmed by reprobing with antibodies recognizing total proteins. (B) siRNA-mediated down-regulation of Jak2 does not influence Flt3-ITD–mediated STAT5 phosphorylation. 32D Flt3-ITD cells were electroporated with a mixture of 4 siRNAs directed against Jak2, using the Amaxa nucleofection technology (Cologne, Germany). The cells were grown in the absence of IL-3 for the next 30 hours and cell lysates were analyzed for STAT5 activation as described in panel A. Effective down-regulation of Jak2 was verified by staining the membranes with anti-Jak2 antibody. (C) Jak2 phosphorylation is induced by IL-3 but not by Flt3-ITD and can be inhibited by AG490. The 32D cells expressing Flt3-ITD were starved in the absence or presence of AG490 (50 μM) for 6 hours. Subsequently, cells were left unstimulated or were stimulated with IL-3 for 10 minutes at 37°C. Cell lysates were prepared and Jak2 was immunoprecipitated using Jak2 antibodies. Phosphorylation of Jak2 was analyzed using a phosphotyrosine-specific antibody (pY100). The membrane was reprobed with an antibody that recognizes total Jak2. (D) Flt3-ITD–mediated STAT5 phosphorylation is not inhibited by PP2. The 32D Flt3-ITD cells were starved overnight in the presence or absence of PKC412 (100 nM) or PP2 (10 μM). Signaling analyses were performed as described in panel A. (E) PP2 inhibits phosphorylation of Src family kinases. Flt3-ITD–expressing 32D cells were starved overnight in the presence or absence of PP2 (10 μM). Activation of Src kinases was analyzed using a pan-phospho-Src family antibody that recognizes Src family kinases (including the hematopoietic cell type–specific Lyn and Hck kinases) when phosphorylated on the activation loop tyrosine residue analogous to tyrosine 416 (Y416) of c-Src. (F) Src family kinases (SFKs) are dispensable for Flt3-ITD–mediated STAT5 activation. SFK-deficient SYF or control NIH3T3 stably expressing Flt3-ITD were starved and analyzed for signaling activation as described in panel A. (G) No phosphorylation of SFKs is observed in SYF cells. SYF, 32D, or NIH-3T3 cells expressing Flt3-ITD were analyzed for the activation of SFKs as described in panel E. (H-I) Jak2 or Tyk2 is not involved in Flt3-ITD–mediated STAT5 activation. Jak2 (γ2A)– or Tyk2 (U1A)–deficient and control cells (2C4 and 2ftgh) stably expressing Flt3-ITD were serum starved and lysates were analyzed for activation of STAT5 as described in panel A. Deficiency for Jak2 or Tyk2 in γ2A or U1A cells, respectively, was confirmed by probing total cell lysates with Jak2 or Tyk2 antibodies.

Flt3-ITD–induced STAT5 activation is independent of Jak or Src kinases. (A) Flt3-ITD phosphorylates STAT5 in AG490-resistant manner. The 32D Flt3-ITD cells were starved for 6 hours in the presence or absence of PKC412 (100 nM) or AG490 (50 μM). Cell lysates were immunoblotted against activation-specific phospho-Flt3 (Y591) or STAT5 (Y694) antibodies. Equal loading of proteins was confirmed by reprobing with antibodies recognizing total proteins. (B) siRNA-mediated down-regulation of Jak2 does not influence Flt3-ITD–mediated STAT5 phosphorylation. 32D Flt3-ITD cells were electroporated with a mixture of 4 siRNAs directed against Jak2, using the Amaxa nucleofection technology (Cologne, Germany). The cells were grown in the absence of IL-3 for the next 30 hours and cell lysates were analyzed for STAT5 activation as described in panel A. Effective down-regulation of Jak2 was verified by staining the membranes with anti-Jak2 antibody. (C) Jak2 phosphorylation is induced by IL-3 but not by Flt3-ITD and can be inhibited by AG490. The 32D cells expressing Flt3-ITD were starved in the absence or presence of AG490 (50 μM) for 6 hours. Subsequently, cells were left unstimulated or were stimulated with IL-3 for 10 minutes at 37°C. Cell lysates were prepared and Jak2 was immunoprecipitated using Jak2 antibodies. Phosphorylation of Jak2 was analyzed using a phosphotyrosine-specific antibody (pY100). The membrane was reprobed with an antibody that recognizes total Jak2. (D) Flt3-ITD–mediated STAT5 phosphorylation is not inhibited by PP2. The 32D Flt3-ITD cells were starved overnight in the presence or absence of PKC412 (100 nM) or PP2 (10 μM). Signaling analyses were performed as described in panel A. (E) PP2 inhibits phosphorylation of Src family kinases. Flt3-ITD–expressing 32D cells were starved overnight in the presence or absence of PP2 (10 μM). Activation of Src kinases was analyzed using a pan-phospho-Src family antibody that recognizes Src family kinases (including the hematopoietic cell type–specific Lyn and Hck kinases) when phosphorylated on the activation loop tyrosine residue analogous to tyrosine 416 (Y416) of c-Src. (F) Src family kinases (SFKs) are dispensable for Flt3-ITD–mediated STAT5 activation. SFK-deficient SYF or control NIH3T3 stably expressing Flt3-ITD were starved and analyzed for signaling activation as described in panel A. (G) No phosphorylation of SFKs is observed in SYF cells. SYF, 32D, or NIH-3T3 cells expressing Flt3-ITD were analyzed for the activation of SFKs as described in panel E. (H-I) Jak2 or Tyk2 is not involved in Flt3-ITD–mediated STAT5 activation. Jak2 (γ2A)– or Tyk2 (U1A)–deficient and control cells (2C4 and 2ftgh) stably expressing Flt3-ITD were serum starved and lysates were analyzed for activation of STAT5 as described in panel A. Deficiency for Jak2 or Tyk2 in γ2A or U1A cells, respectively, was confirmed by probing total cell lysates with Jak2 or Tyk2 antibodies.

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