A functional DNA binding domain is required for growth hormone-induced nuclear accumulation of Stat5B

The mechanisms regulating the cellular distribution of STAT family transcri ption factors remain poorly understood. To identify regions of Stat5B requi red for ligand-induced nuclear accumulation, we constructed a cDNA encoding green fluorescent protein (GFP) fused to the N terminus of Stat5B and perf ormed site-directed mutagenesis. When co-expressed with growth hormone (GH) receptor in COS-7 cells, GFP-Stat5B is tyrosylphosphorylated, forms dimers , and binds DNA in response to GH in a manner indistinguishable from untagg ed Stat5B. In multiple cell types, laser scanning confocal imaging of GFP-S tat5B co-expressed with GH receptor shows that GFP-Stat5B undergoes a rapid , dramatic accumulation in the nucleus upon GH stimulation. We introduced a lanine substitutions in several regions of Stat5B and assayed for GH-depend ent nuclear localization. Only the mutation that prevented binding to DNA ( (VVVI469)-V-466) abrogated GH-stimulated nuclear localization. This mutant fusion protein is tyrosyl-phosphorylated and dimerizes in response to GH. T hese results suggest that either high affinity binding to DNA contributes t o nuclear accumulation of Stat5B or that this region is crucial for two fun ctions, namely accumulation of Stat5B in the nucleus and DNA binding. Thus, we have identified a mutant Stat5 defective in nuclear localization despit e its ability to be tyrosyl-phosphorylated and to dimerize.