Role of the Stat4 N domain in receptor proximal tyrosine phosphorylation

Stat4 is activated by the cytokines interleukin 12 and alpha interferon (IF N-alpha) and plays a significant role in directing development of naive CD3 (+) T cells to the Th1 phenotype. Signal transducers and activators of tran scription (STAT) proteins undergo phosphorylation on a conserved tyrosine r esidue, resulting in homo- and heterodimerization, nuclear translocation, a nd DNA binding. Stat4 can bind to single IFN-gamma-activated sites (GASs) a s a dimer or bind typo tandem GASs as a pair of STAT dimers, or tetramer, s tabilized through N-terminal domain (N domain) interactions between dimers. We uncovered an unexpected effect of the Stat4 N domain in controlling the proximal activation of Stat4 by tyrosine phosphorylation at activated rece ptor complexes. Mutation of the N domain at tryptophan residue W37, predict ed to interrupt N domain dimer formation, unexpectedly prevented IFN-alpha- induced tyrosine phosphorylation of the Stat4 monomer, blocking dimer forma tion and nuclear translocation. Furthermore, N domains appear to exert priv ate STAT functions, since interchanging the N domains between Stat1 and Sta t4 prevented receptor-mediated tyrosine phosphorylation in one case and int errupted STAT-specific gene activation in another. Finally, replacement of the N domain of Stat1 with that of Stat1 abrogated the normal Stat2 depende nce of Stat1 phosphorylation, again suggesting the domains are not equivale nt. Thus, in addition to its role in STAT tetramerization, the conserved ST AT N domain appears to participate in very proximal steps of receptor-media ted ligand-induced tyrosine phosphorylation.