p38α phosphorylates STAT4 on serine 721.

(A), IL-12–activated p38 phosphorylates the C-terminus of STAT4 in vitro. NK3.3 cells were left unstimulated (lane 1) or were stimulated with 10 μg/mL anisomycin (lane 2) or with IL-12 (lane 3). Lysates were immunoprecipitated with anti-p38 Ab. A kinase reaction was performed on the immunoprecipitates using GST-STAT4 as a substrate (upper panel). The filter was probed for p38 showing equal loading of the proteins (lower panel). (B), Mutation of serine 721 blunts the enhancement seen by overexpression of p38α/ MKK6. NIH3T3 cells were transiently transfected with the p3xGAS-luc and the pCMV-β-galactosidase reporters, together with expression vectors encoding the human IL-12R subunits. Where indicated, cells were cotransfected with control vector (EV), wild-type STAT4, STAT4 S721A, MKK6, or p38α. The experiment was performed as described in Figure1C. Unstimulated, ■; +IL-12, ▪. (C), Overexpression of MKK6 and p38α is sufficient to mediate STAT4 serine 721 phosphorylation. 293T cells were transiently transfected with control vector (lane 1) or as indicated. Lysates were immunoprecipitated with anti-STAT4 Ab, resolved by SDS/PAGE, transferred to membrane and blotted with antiphosphoserine 727 STAT3 Ab that also detects serine phosphorylated STAT4 (upper panel). The blot was stripped and reprobed with anti-STAT4, demonstrating equal levels of proteins (lower panel).