DIFFERENTIATION-REGULATED SERINE PHOSPHORYLATION OF STAT1 PROMOTES GAF ACTIVATION IN MACROPHAGES

Gamma interferon (IFN-gamma), a macrophage-activating cytokine, modula tes gene expression through the activity of a transcription factor des ignated IFN-gamma activation factor (GAF). GAF is formed after phospho rylation on tyrosine and dimerization of the 91-kDa protein STAT1. We have recently reported that differentiation of the promonocytic cell l ine 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 n ot increase upon monocytic U937 differentiation. Consistent with this finding, levels of STAT1 tyrosine phosphorylation were virtually ident ical in promonocytic and monocytic U937 cells. Analysis of STAT1 phosp hoamino acids and mapping of phosphopeptides showed an IFN-gamma-depen dent 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 isof orms were equally sensitive to subsequent tyrosine phosphorylation, as indicated by a sodium dodecyl sulfate-polyacrylamide gel electrophore sis mobility shift typical for tyrosine-phosphorylated STAT1. Consiste nt with the importance of Ser phosphorylation for high-affinity bindin g to the IFN-gamma activation site sequence, phosphatase 2A treatment strongly reduced the formation of IFN-gamma activation site-GAF comple xes 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 cont rol of macrophage responsiveness to IFN-gamma.