Differentiation-regulated serine phosphorylation of STAT1 promotes GAF activation in macrophages.
Gamma interferon (IFN-gamma), a macrophage-activating cytokine, modulates gene expression through the activity of a transcription factor designated IFN-gamma activation factor (GAF). GAF is formed after phosphorylation on tyrosine and dimerization of the 91-kDa protein STAT1. We have recently reported that differentiation of the promonocytic cell line U937 into monocytes increases the amount of cellular GAF after IFN-gamma treatment and at the same time increases the phosphorylation of STAT1. Here we show that activation of the JAK family kinases, which are instrumental in mediating STAT1 phosphorylation on tyrosine, did not increase upon monocytic U937 differentiation. Consistent with this finding, levels of STAT1 tyrosine phosphorylation were virtually identical in promonocytic and monocytic U937 cells. Analysis of STAT1 phosphoamino acids and mapping of phosphopeptides showed an IFN-gamma-dependent increase in Ser phosphorylation in differentiated cells. Analyses of STAT1 isoforms by two-dimensional gel electrophoresis demonstrated a differentiation-induced shift toward more acidic isoforms. All isoforms were equally sensitive to subsequent tyrosine phosphorylation, as indicated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility shift typical for tyrosine-phosphorylated STAT1. Consistent with the importance of Ser phosphorylation for high-affinity binding to the IFN-gamma activation site sequence, phosphatase 2A treatment strongly reduced the formation of IFN-gamma activation site-GAF complexes in an electrophoretic mobility shift assay. Our data indicate that the activity of GAF is modulated by STAT1 serine kinases/phosphatases and suggest that this mechanism is employed in the developmental control of macrophage responsiveness to IFN-gamma.
Eilers
A
A
Vienna Biocenter, Institute of Microbiology and Genetics, Austria.
Georgellis
D
D
Klose
B
B
Schindler
C
C
Ziemiecki
A
A
Harpur
A G
AG
Wilks
A F
AF
Decker
T
T
D000596:Amino Acids / Q000032:analysis; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D050832:Interferon-Stimulated Gene Factor 3; D053613:Janus Kinase 1; D053614:Janus Kinase 2; D008264:Macrophages / Q000502:physiology*; D008954:Models, Biological; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000032:analysis; D011518:Proto-Oncogene Proteins; D050794:STAT1 Transcription Factor; D012694:Serine / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D000596:Amino Acids; D004268:DNA-Binding Proteins; D050832:Interferon-Stimulated Gene Factor 3; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D050794:STAT1 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors; C079868:gamma interferon activation factor; D010768:Phosphoserine; D014443:Tyrosine; D012694:Serine; D011505:Protein-Tyrosine Kinases; D053613:Janus Kinase 1; D053614:Janus Kinase 2; D017346:Protein Serine-Threonine Kinases