BINDING OF A GROWTH HORMONE-INDUCIBLE NUCLEAR FACTOR IS MEDIATED BY TYROSINE PHOSPHORYLATION

The nuclear mechanism by which GH acts to induce gene expression after binding to its receptor on the cell surface is not defined. We have c haracterized an element in the 5'-flanking region of the rat GH-respon sive serine protease inhibitor (Spi) 2.1 gene responsible for its indu ction by GH. This element binds a hepatic nuclear protein(s) in a GH s tate-specific manner. Activation of binding by GH does not require de novo protein synthesis, suggesting that a reversible posttranslational process is required for binding to the element. To define the mechani sm of this process, hepatic nuclear extracts were analyzed by electrop horetic mobility shift assays using a DNA fragment (-147 to -103) of t he Spi 2.1 gene. Treatment of extracts with phosphatases resulted in a marked reduction of GH state-specific binding. Addition of phosphatas e inhibitors antagonized the reduction in binding after phosphatase tr eatment. The specific nature of the phosphorylation event involved in binding was explored using phosphotyrosine antibodies and a protein ty rosine phosphatase. Treatment of nuclear extracts with either of these reagents ablated binding to the response element. Because the tyrosin e-phosphorylated transcription factor protein p91 has recently been im plicated in cytokine signal transduction mediated by JAK2, we sought e vidence that p91 was part of the OR-responsive binding complex. Analys is of an enriched preparation of OR-inducible binding complexes by Wes tern blots using anti-p91 demonstrated no immunoreactivity. We conclud e that tyrosine phosphorylation of a nuclear factor is required for OH state-specific binding to this OH response element in vivo, but that p91 is not present in the binding complex.