In vitro analysis of STAT5 activation by granulocyte-macrophage colony-stimulating factor.
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.
Sakurai
Y
Y
Department of Molecular and Developmental Biology, Institute of Medical Science, Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, University of Tokyo.
Arai
K
K
Watanabe
S
S
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D053614:Janus Kinase 2; D051379:Mice; D008894:Milk Proteins; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D010748:Phosphopeptides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000037:antagonists & inhibitors; D017434:Protein Structure, Tertiary / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D000906:Antibodies; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D019000:Phosphotyrosine; D014443:Tyrosine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2