Figure 2
Figure 2. Flt3-ITD directly phosphorylates STAT5 in a SOCS1- and SOCS3-resistant manner. (A-B) Flt3-ITD cells are resistant to SOCS1- or SOCS3-induced growth inhibition. Parental 32D or 32D cells expressing Flt3-WT or Flt3-ITD were transduced with retroviruses encoding SOCS1 or SOCS3 and EGFP or vector control (pMY-IRES-EGFP). Thirty hours after transduction, cells were washed and cultured with or without the indicated cytokines for up to one week. The proportion of EGFP-positive cells was analyzed by flow cytometry. (C) 32D Flt3-ITD or Flt3-ITD-SOCS1 cells were starved for 12 hours. Subsequently, cells were exposed to IL-3 or left unstimulated in the presence or absence of the indicated PKC412 concentrations. Each data point represents the mean of 3H thymidine incorporation of 3 samples ± standard deviation. (D-E) SOCS1 or SOCS3 does not inhibit Flt3-ITD–mediated STAT5 activation. The 32D Flt3-ITD, Flt3-ITD-SOCS1, or Flt3-ITD-SOCS3 cells were starved overnight in the presence or absence of IL-3, supplemented with or without PKC412. On the next day, cell lysates were prepared and signaling analyses were performed as mentioned in the legend to Figure 1A. Activation of STAT5 was further confirmed in Flt3-ITD-SOCS1–expressing cells by analyzing the expression of the STAT5 target genes Pim-1 and Pim-2. (F) SFKs are dispensable for Flt3-ITD–mediated STAT5 phosphorylation in the absence of Jak activity. The 32D-Flt3-ITD-SOCS1 cells (with Jak kinase inhibition by SOCS1) were treated with PP2 to inhibit SFKs. Cell lysates were analyzed for STAT5 phosphorylation as described in the legend to Figure 1A. (G) Flt3 directly phosphorylates STAT5. In vitro kinase assays were performed using purified recombinant STAT5 alone, or together with GST-Flt3 or GST-PDGFRB kinase. The phosphorylation of STAT5 was measured with a phospho-STAT5 antibody and loading of STAT5 and Flt3 or PDGFRB was confirmed by reprobing the membrane with a STAT5 or GST antibody. (H) SOCS1 inhibits IL-3 but not Flt3-ITD–mediated STAT5 activation. Activation of STAT5 by Flt3-ITD or IL-3 leads to induction of STAT5 target genes, including SOCS proteins. Up-regulation of SOCS proteins results in inhibition of Jak kinases of STAT5 activation by IL-3. In contrast, Jak-independent STAT5 activation by Flt3-ITD is not inhibited by SOCS proteins such as SOCS1 and SOCS3.

Flt3-ITD directly phosphorylates STAT5 in a SOCS1- and SOCS3-resistant manner. (A-B) Flt3-ITD cells are resistant to SOCS1- or SOCS3-induced growth inhibition. Parental 32D or 32D cells expressing Flt3-WT or Flt3-ITD were transduced with retroviruses encoding SOCS1 or SOCS3 and EGFP or vector control (pMY-IRES-EGFP). Thirty hours after transduction, cells were washed and cultured with or without the indicated cytokines for up to one week. The proportion of EGFP-positive cells was analyzed by flow cytometry. (C) 32D Flt3-ITD or Flt3-ITD-SOCS1 cells were starved for 12 hours. Subsequently, cells were exposed to IL-3 or left unstimulated in the presence or absence of the indicated PKC412 concentrations. Each data point represents the mean of 3H thymidine incorporation of 3 samples ± standard deviation. (D-E) SOCS1 or SOCS3 does not inhibit Flt3-ITD–mediated STAT5 activation. The 32D Flt3-ITD, Flt3-ITD-SOCS1, or Flt3-ITD-SOCS3 cells were starved overnight in the presence or absence of IL-3, supplemented with or without PKC412. On the next day, cell lysates were prepared and signaling analyses were performed as mentioned in the legend to Figure 1A. Activation of STAT5 was further confirmed in Flt3-ITD-SOCS1–expressing cells by analyzing the expression of the STAT5 target genes Pim-1 and Pim-2. (F) SFKs are dispensable for Flt3-ITD–mediated STAT5 phosphorylation in the absence of Jak activity. The 32D-Flt3-ITD-SOCS1 cells (with Jak kinase inhibition by SOCS1) were treated with PP2 to inhibit SFKs. Cell lysates were analyzed for STAT5 phosphorylation as described in the legend to Figure 1A. (G) Flt3 directly phosphorylates STAT5. In vitro kinase assays were performed using purified recombinant STAT5 alone, or together with GST-Flt3 or GST-PDGFRB kinase. The phosphorylation of STAT5 was measured with a phospho-STAT5 antibody and loading of STAT5 and Flt3 or PDGFRB was confirmed by reprobing the membrane with a STAT5 or GST antibody. (H) SOCS1 inhibits IL-3 but not Flt3-ITD–mediated STAT5 activation. Activation of STAT5 by Flt3-ITD or IL-3 leads to induction of STAT5 target genes, including SOCS proteins. Up-regulation of SOCS proteins results in inhibition of Jak kinases of STAT5 activation by IL-3. In contrast, Jak-independent STAT5 activation by Flt3-ITD is not inhibited by SOCS proteins such as SOCS1 and SOCS3.

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