CHARACTERIZATION AND CLONING OF STAT5 FROM IM-9 CELLS AND ITS ACTIVATION BY GROWTH-HORMONE

The interaction of GH with its receptor has been shown to lead to the phosphorylation of the signal transducer and activator of transcriptio n (STAT) family of transcription factors. We demonstrate here that GH activates the tyrosine phosphorylation of STAT5 in the human IM-9 lymp hocyte cell line. Western blotting indicates that GH also activates ST AT5 in human embryonic kidney cells (293), which stably express the ra bbit GH receptor. Although it has been shown previously that GH activa tes both STATs 1 and 3 in the 3T3-F442A mouse preadipocyte cell line, we demonstrate that GH also activates STAT5 in these cells. Using elec trophoretic mobility shift assay, we examined the interaction of prote ins with DNA elements containing consensus STAT-binding sequences. Pro teins prepared from GH-treated 3T3-F442A cells bound to the c-sis indu cible element of the human c-fos gene (m67 SIE), whereas proteins from GH-treated IM-9 cells did not. However, proteins from GH-treated IM-9 cells did interact with oligonucleotides containing either an interfe ron response element or the lactogenic hormone-responsive region. Trea tment of IM-9 cells with interferon-gamma also induced protein interac tions with these elements, although the complexes were distinctly diff erent than those seen with GH treatment. Using STAT-specific antibodie s, we demonstrate that the GH-induced DNA-protein complex formed with the lactogenic hormone-responsive region contained STAT5, while the in terferon-gamma-induced complex contained STAT1. These results implicat e STAT5 as a downstream mediator of GH action in IM-9 cells. We report here the cloning of two forms of STAT5, STAT5A and STAT5B, from an IM -9 cDNA library. Northern blot analysis demonstrated multiple forms of STAT5 mRNA in IM-9 cells.