Figure 1
Figure 1. Ser725/779 residues are significantly phosphorylated in transformed cell lines and primary samples from leukemia patients. (A) The C-terminal serine residues 725 and 779 of Stat5a were found to be considerably phosphorylated in human leukemic cell lines. K562 and Ku-812 were used as representative CML samples, and Mv4.11 and MOLM13 for AML. In Western blot analysis, Ser779 was found to be phosphorylated predominantly in CML samples, whereas Ser725 phosphorylation (Abcam antibody) was detected in all leukemia cell lines. S5-expressing gpE+86 fibroblasts served as controls. A representative blot of 3 independent experiments was chosen. (B) Different transformed human T- and B-cell lines, Mac-2A (CTCL), MOLT-4, CCRF-CEM, Jurkat, HPB-ALL (all T-ALL), as well as DAUDI and RAMOS cells (Burkitt-lymphoma), REH (B-ALL), and RL-7 (non-Burkitt lymphoma) cell lines were analyzed for serine phosphorylation by Western blot analysis. Daudi cells as well as CCRF-CEM and Mac-2A cells were found to be strongly positive for Ser725 (Eurogentech and Abcam) and Ser779 phosphorylation. Whereas Mac-2A, Daudi, and to a lesser extent Ramos and REH cells showed Ser779 phosphorylation in addition to Tyr-phosphorylation, Ser779 phosphorylation was largely independent of Tyr-phosphorylation in CCRF-CEM and RL-7 cells. In all B-cell lines except Daudi cells, Ser725 phosphorylation was very low but clearly detectable. A murine T-cell lymphoma line (EL-4) was included in the analysis; it was positive for Ser779 phosphorylation, which was independent of Tyr-phosphorylation. The CML line K562 was used as positive control. Similar results were obtained in 3 individual experiments. (C) Protein analysis of primary samples from AML (n = 4), ALL (n = 2), and CML (n = 2) patients as well as from healthy individuals (n = 2) revealed that Stat5a and Stat5b are strongly phosphorylated not only at the critical tyrosine 694 residue, but also at serine 725, and Stat5a additionally at serine 779. Serine 779 phosphorylation was not detected in healthy individuals, whereas serine 725 was found to be phosphorylated in all samples tested. GpE+86 cells expressing S5 were used as controls for all analyses. The experiments were performed in duplicate. (D) Ba/F3 cells were engineered by retroviral transduction to overexpress the cS5 variant as well as fusion kinases, which are known to activate Stat5a and Stat5b in human patients. Modified Ba/F3 cells were subjected to immunoblot analysis to evaluate Stat5 tyrosine and serine phosphorylation. Ser725 (Abcam antibody) and Ser779 were strongly phosphorylated in all factor-independent lines. Parental Ba/F3 cells starved for 20 hours and cells restimulated with IL-3, as well as S5-expressing gpE+86 fibroblasts, served as controls (exposed on the same blot; interjacent bands were cut). The blots are representatives of 4 individual experiments.

Ser725/779 residues are significantly phosphorylated in transformed cell lines and primary samples from leukemia patients. (A) The C-terminal serine residues 725 and 779 of Stat5a were found to be considerably phosphorylated in human leukemic cell lines. K562 and Ku-812 were used as representative CML samples, and Mv4.11 and MOLM13 for AML. In Western blot analysis, Ser779 was found to be phosphorylated predominantly in CML samples, whereas Ser725 phosphorylation (Abcam antibody) was detected in all leukemia cell lines. S5-expressing gpE+86 fibroblasts served as controls. A representative blot of 3 independent experiments was chosen. (B) Different transformed human T- and B-cell lines, Mac-2A (CTCL), MOLT-4, CCRF-CEM, Jurkat, HPB-ALL (all T-ALL), as well as DAUDI and RAMOS cells (Burkitt-lymphoma), REH (B-ALL), and RL-7 (non-Burkitt lymphoma) cell lines were analyzed for serine phosphorylation by Western blot analysis. Daudi cells as well as CCRF-CEM and Mac-2A cells were found to be strongly positive for Ser725 (Eurogentech and Abcam) and Ser779 phosphorylation. Whereas Mac-2A, Daudi, and to a lesser extent Ramos and REH cells showed Ser779 phosphorylation in addition to Tyr-phosphorylation, Ser779 phosphorylation was largely independent of Tyr-phosphorylation in CCRF-CEM and RL-7 cells. In all B-cell lines except Daudi cells, Ser725 phosphorylation was very low but clearly detectable. A murine T-cell lymphoma line (EL-4) was included in the analysis; it was positive for Ser779 phosphorylation, which was independent of Tyr-phosphorylation. The CML line K562 was used as positive control. Similar results were obtained in 3 individual experiments. (C) Protein analysis of primary samples from AML (n = 4), ALL (n = 2), and CML (n = 2) patients as well as from healthy individuals (n = 2) revealed that Stat5a and Stat5b are strongly phosphorylated not only at the critical tyrosine 694 residue, but also at serine 725, and Stat5a additionally at serine 779. Serine 779 phosphorylation was not detected in healthy individuals, whereas serine 725 was found to be phosphorylated in all samples tested. GpE+86 cells expressing S5 were used as controls for all analyses. The experiments were performed in duplicate. (D) Ba/F3 cells were engineered by retroviral transduction to overexpress the cS5 variant as well as fusion kinases, which are known to activate Stat5a and Stat5b in human patients. Modified Ba/F3 cells were subjected to immunoblot analysis to evaluate Stat5 tyrosine and serine phosphorylation. Ser725 (Abcam antibody) and Ser779 were strongly phosphorylated in all factor-independent lines. Parental Ba/F3 cells starved for 20 hours and cells restimulated with IL-3, as well as S5-expressing gpE+86 fibroblasts, served as controls (exposed on the same blot; interjacent bands were cut). The blots are representatives of 4 individual experiments.

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