Specific DNA binding and transactivation potential of recombinant, purified Stat5

Beisenherz-Huss, Christian; Mundt, Maren; Herrala, Annakaisa; Vihko, Pirkko; Schubert, Alexis; Groner, Bernd

doi:10.1016/s0303-7207(01)00588-3

Cited by 8 publications

(7 citation statements)

References 34 publications

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“…As shown in Figure 2G, Flt3 kinase directly phosphorylated STAT5 at similar levels as the PDGFRB, which we used as a positive control, since it is known to directly phosphorylate STAT5. 14 Taken together, our data provide the mechanistic basis of STAT5 activation by oncogenic Flt3-ITD mutations and show that Flt3-ITD directly activates STAT5 in a SFK-and Jakindependent manner. …”

supporting

confidence: 59%

Activation mechanisms of STAT5 by oncogenic Flt3-ITD

Choudhary

Brandts

Schwäble

et al. 2007

Blood

175

129

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IntroductionPreviously, we and others have reported aberrant activation of STAT5 by Flt3-ITD mutations. [1][2][3][4][5][6][7] Activation of STAT5 is increasingly recognized as important for self-renewal of hematopoietic stem cells. 5,8 Constitutive activation of STAT5 has also been observed in human leukemias. 9 In animal models, activation of STAT5 has been shown to be essential for induction of leukemias by Tel-Jak2 and recently by Bcr-Abl and Flt3-ITD. 10,11 Therefore, elucidation of mechanisms of STAT5 activation by Flt3-ITD may help to selectively target oncogenic signals of Flt3-ITD. Materials and methods Cell linesSYF (Src, Yes, and Fyn) cells are derived from Src, Yes, and Fyn knockout mice and express no Src family kinases. 12 ␥2A and U1A cell lines are deficient for Jak2 and Tyk2 kinases, respectively. 13 All cell lines, except 32D, were cultured in DMEM medium supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. The 32D cells were cultured and maintained as described earlier. 1,3,4 Preparation of retroviruses, generation of cell lines, and sources of antibodies and reagents have been described in the supplementary materials and methods, available on the Blood website; see the Supplemental Materials link at the top of the online article. Purification of recombinant proteins and in vitro kinase assaysHis-tagged murine STAT5 and GST fusion proteins with Flt3 or PDGFRB kinase were expressed in Sf9 cells and purified by standard procedures. In vitro kinase assays were performed using about 0.5 g STAT5 and 0.5 g GST-Flt3 or GST-PDGFRB in 20 mM Hepes, 5 mM MnCl 2 , 0.2 mM Na-vanadate, in the absence or presence of 1 mM ATP at 30°C for 20 minutes.Lysate preparation, Western blotting, and 3H thymidine incorporation assays were performed as described earlier. 1,3,4 Results and discussion Flt3-ITD-induced STAT5 activation is independent of Src or Jak kinases STAT5 is generally activated by one of the 3 mechanisms: (1) by Jak kinases, (2) by Src family kinases (SFK), or (3) directly by RTKs such as the EGFR, PDGFRB, or insulin receptors. 14,15 Although phosphorylation of Jak2 or Tyk2 by Flt3-ITD has been shown in previous studies, the role of Jak kinases in Flt3-ITD signaling remains controversial and inconclusive. 16,17 To systematically explore the mechanisms of STAT5 activation by Flt3-ITD, we first tested chemical inhibitors to inhibit SFKs or JAKs. 18,19 Treatment of 32D-Flt3-ITD with AG490 resulted in a decrease of STAT5 phosphorylation, but also in proportional inhibition of Flt3 autophosphorylation, making the interpretation of this result difficult ( Figure 1A). Therefore, we analyzed the effects of Jak2 knockdown using siRNA, which did not have any effect on Flt3-ITD-mediated STAT5 activation ( Figure 1B). Failure of Flt3-ITD to induce phosphorylation of any of the 4 Jak kinases ( Figure 1C and data not shown) also indicated minor, if any, involvement of JAKs in Flt3-ITD-induced STAT5 activation. The SFK inhibitors PP-2 and PP-1 had marginal effect on STAT5 phosphorylation by Flt3-I...

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supporting

confidence: 59%

Activation mechanisms of STAT5 by oncogenic Flt3-ITD

Choudhary

Brandts

Schwäble

et al. 2007

Blood

175

129

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“…Tyrosine phosphorylation is critical for STAT5b activation (32). To determine the effects of the constitutively active Lck kinase on STAT5b phosphorylation, a STAT5b expression construct was transiently transfected into both T-REx-293/Lck(Y505F) and the vector control cells.…”

Section: Resultsmentioning

confidence: 99%

A Constitutively Active Lck Kinase Promotes Cell Proliferation and Resistance to Apoptosis through Signal Transducer and Activator of Transcription 5b Activation

Shi

Cooper

2006

Molecular Cancer Research

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“…We have previously shown that tyrosine phosphorylated, activated Stat5a can be obtained from extracts of Sf9 cells simultaneously infected with baculoviruses encoding Stat5 and Jak2 (39). Sf9 cells properly carry out post-translational modifications of recombinant proteins such as the tyrosine phosphorylation of Stat5 or the O-GlcNAcylation of human cytomegalovirus basic protein 1 and keratins 8, 13, and 18 (40).…”

Section: Stat5a Is Modified With O-linked N-acetylglucosaminementioning

confidence: 99%