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. 2006 Dec 18;175(6):937–946. doi: 10.1083/jcb.200604073

Figure 5.

Figure 5.

The mutant of STAT5A, which lacks MgcRacGAP binding site, was not efficiently tyrosine phosphorylated by ITD-Flt3 stimulation and did not enter the nucleus even after tyrosine phosphorylation. (A) The DB2 region of STAT5 directly interacted with MgcRacGAP in vitro. Full-length MgcRacGAP was expressed in Sf-9 cells using the baculovirus vector and was purified from infected Sf-9 cells. The recombinant MgcRacGAP was pulled down by MBP-DB2 or MBP-bound beads and subjected to Western blot analysis with the anti-MgcRacGAP (top) or anti-MBP antibody for the loading control (bottom). (B) The deletion mutant of DB2 did not bind MgcRacGAP, and the STAT5 phosphorylation was considerably impaired by the deletion of DB2. Expression and tyrosine phosphorylation of Flag-tagged STAT5A-dDB2 (top and middle, respectively) were examined in the MOCK or ITD-Flt3–transfected 293T cells. The interactions of MgcRacGAP with the WT-STAT5A or STAT5A-dDB2 were also examined in the MOCK or ITD-Flt3–transfected 293T cells (bottom). Images of the immunoblots using the MOCK or ITD-Flt3–transfected cells are derived from the same exposure of one gel that was cut to remove intervening lanes. (C) The transcriptional activity of STAT5-dDB2 was impaired. Luciferase activities were examined in the lysates of ITD-Flt3–stimulated 293T cells cotransfected with the STAT5-reporter plasmid together with internal control reporter plasmids and the MOCK vector (pME), the expression vector for the Flag-tagged WT-STAT5, or STAT5-dDB2 mutant. The results shown are the mean ± SD of three independent experiments. (D) MgcRacGAP was coprecipitated with JAK2. The cell lysates of 293T cells transfected with the expression vector (pRK5) for JAK2 were subjected to immunoprecipitation with the anti-MgcRacGAP or control antibody, followed by the immunoblotting with the anti-JAK2 (top) or anti-MgcRacGAP antibody (middle). Levels of transfected JAK2 were assayed by blotting with the anti-JAK2 antibody (bottom). (E) STAT5A-dDB2 did not enter the nucleus even after the phosphorylation. The 293T cells were cotransfected with pMKIT/ITD-Flt3 together with the MOCK (left), the expression vector for the Flag-tagged WT-STAT5A (middle), or STAT5A-dDB2 (right). After 24 h, the cells were fixed and immunostained with the anti–p-STAT5 antibody. Bar, 10 μm.

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