Stat recruitment by tyrosine-phosphorylated cytokine receptors: an ordered reversible affinity-driven process.
Herein, we demonstrate that purified Stat1 binds to its tyrosine-phosphorylated docking site on the IFN gamma receptor alpha chain in a direct, specific, and reversible manner. Using surface plasmon resonance, we determine the affinity (KD = 137 nM) and specificity of the interaction and define the minimum affinity needed for receptor-mediated Stat1 activation. In addition, we quantitate the relative ability of purified Stat1 to interact with tyrosine-phosphorylated binding sites on other Stat proteins. Finally, we describe experiments that imply that the unidirectional release of activated Stat1 from the IFN gamma receptor reflects the preference of free tyrosine-phosphorylated Stat1 monomers to form high avidity reciprocal homodimers rather than reassociating with the receptor binding site. Our results demonstrate that IFN gamma-induced Stat1 activation is an ordered and affinity-driven process and we propose that this process may serve as a paradigm for Stat activation by other cytokine receptors.
Greenlund
A C
AC
Center for Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Morales
M O
MO
Viviano
B L
BL
Yan
H
H
Krolewski
J
J
Schreiber
R D
RD
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D015374:Biosensing Techniques; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000502:physiology; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D018121:Receptors, Cytokine / Q000378:metabolism*; D017471:Receptors, Interferon / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000096743:Interferon gamma Receptor
D004268:DNA-Binding Proteins; D007371:Interferon-gamma; D010748:Phosphopeptides; D011505:Protein-Tyrosine Kinases; D018121:Receptors, Cytokine; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; D015534:Trans-Activators; D000096743:Interferon gamma Receptor; C494079:STAT1 protein, human