Biomolecular interaction analysis of IFN gamma-induced signaling events in whole-cell lysates: prevalence of latent STAT1 in high-molecular weight complexes.
The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA. The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferon gamma (IFN gamma) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immuno-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFN gamma-receptor alpha-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) non-activated STAT1 is present in a higher molecular weight complex(es) and, at least for IFN gamma-primed cells, is available for recruitment to the activated IFN gamma-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.
Growth Regulation Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch), Australia.
M
M
Lackmann
A G
AG
Harpur
A C
AC
Oates
R J
RJ
Mann
A
A
Gabriel
W
W
Meutermans
P F
PF
Alewood
I M
IM
Kerr
G R
GR
Stark
A F
AF
Wilks
D000595:Amino Acid Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D053616:Janus Kinase 3; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017471:Receptors, Interferon / Q000378:metabolism*; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains; D000096743:Interferon gamma Receptor
D004268:DNA-Binding Proteins; D007371:Interferon-gamma; D053616:Janus Kinase 3; D010446:Peptide Fragments; D011505:Protein-Tyrosine Kinases; D017471:Receptors, Interferon; D050794:STAT1 Transcription Factor; D015534:Trans-Activators; D014443:Tyrosine; D000096743:Interferon gamma Receptor; C494079:STAT1 protein, human; C507933:JAK3 protein, human