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  • Title: In vitro analysis of STAT5 activation by granulocyte-macrophage colony-stimulating factor.
    Author: Sakurai Y, Arai K, Watanabe S.
    Journal: Genes Cells; 2000 Nov; 5(11):937-947. PubMed ID: 11122381.
    Abstract:
    BACKGROUND: The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor activates multiple and complex signalling pathways in response to GM-CSF stimulation. Biochemical studies suggested that signalling pathways are transmitted through protein/protein interactions, but how these biochemical cascades are initiated and transmitted in response to cytokine stimulation is largely unknown. RESULTS: To investigate these events biochemically, we established an in vitro system leading to the GM-CSF-dependent activation of Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 5 in cell homogenates prepared from BA/F3 cells expressing the GM-CSF receptor. Activation of STAT5 DNA binding ability requires both membrane and cytoplasmic fractions while phosphorylation of JAK2 requires only the membrane fraction. Since the addition of anti-betac or phosphotyrosine antibodies inhibited GM-CSF induced STAT5 DNA binding activity, we examined the role of tyrosine residues of betac for in vitro activation of STAT5. Addition of synthetic tyrosine-phosphorylated peptides derived from betac cytoplasmic tyrosines prior to GM-CSF stimulation inhibited the in vitro activation of STAT5. The association between these tyrosine-phosphorylated peptides and STAT5 was observed by using peptide-coupling beads and BA/F3 lysates. CONCLUSIONS: We established a GM-CSF-dependent in vitro system. In cases of STAT5 activation, each phosphorylated tyrosine residue of betac can act as a docking site and enhance STAT5 activation.
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