Gamma interferon (IFN-␥), a macrophage-activating cytokine, modulates gene expression through the activity of a transcription factor designated IFN-␥ 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-␥ 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-␥-dependent increase in Ser phosphorylation in differentiated cells. Analyses of STAT1 isoforms by two-dimensional gel electrophoresis demonstrated a differentiationinduced 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-␥ activation site sequence, phosphatase 2A treatment strongly reduced the formation of IFN-␥ 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-␥.The cytokine gamma interferon (IFN-␥) is produced by activated natural killers or T H1 cells in the course of the immune response. It exerts a broad range of biological functions, including antiviral activity and an antiproliferative effect leading to growth inhibition and cellular differentiation (reviewed in references 8 and 36). Studies in mice with targeted disruption of either the IFN-␥ gene or the IFN-␥ receptor gene support the conclusion that one major role of IFN-␥ is that as a macrophage-activating factor (5, 13). Activated macrophages acquire physiological properties relevant to their antimicrobial or antitumor cytotoxicity as well as the potential to present antigen (1). This activation response to IFN-␥ is found only in differentiated cells and is acquired during macrophage development.De novo gene expression is an essential part of the response to IFN-␥, and several IFN-␥-inducible genes have been characterized (reviewed in reference 23). An important mechanism for the transcriptional activation of such genes operates through a promoter element designated the IFN-␥ activation site (GAS) (18,23). This sequence binds an IFN-␥-activated transcription factor (GAF) containing a dimer of tyrosinephosphorylated STAT1 protein (p91) (6; reviewed in references 7 and 28). Dime...

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