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 characterized an element in the 5'-flanking region of the rat GH-responsive serine protease inhibitor (Spi) 2.1 gene responsible for its induction by GH. This element binds a hepatic nuclear protein(s) in a GH state-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 mechanism of this process, hepatic nuclear extracts were analyzed by electrophoretic mobility shift assays using a DNA fragment (-147 to -103) of the Spi 2.1 gene. Treatment of extracts with phosphatases resulted in a marked reduction of GH state-specific binding. Addition of phosphatase inhibitors antagonized the reduction in binding after phosphatase treatment. The specific nature of the phosphorylation event involved in binding was explored using phosphotyrosine antibodies and a protein tyrosine phosphatase. Treatment of nuclear extracts with either of these reagents ablated binding to the response element. Because the tyrosine-phosphorylated transcription factor protein p91 has recently been implicated in cytokine signal transduction mediated by JAK2, we sought evidence that p91 was part of the GH-responsive binding complex. Analysis of an enriched preparation of GH-inducible binding complexes by Western blots using anti-p91 demonstrated no immunoreactivity. We conclude that tyrosine phosphorylation of a nuclear factor is required for GH state-specific binding to this GH response element in vivo, but that p91 is not present in the binding complex.
Berry
S A
SA
Department of Pediatrics, University of Minnesota, Minneapolis 55455.
Bergad
P L
PL
Whaley
C D
CD
Towle
H C
HC
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015153:Blotting, Western; D002467:Cell Nucleus / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism; D013006:Growth Hormone / Q000494:pharmacology*; D007016:Hypophysectomy; D008099:Liver / Q000648:ultrastructure; D008297:Male; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D051381:Rats; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000276:immunology / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D004268:DNA-Binding Proteins; C091103:LOC299282 protein, rat; D009687:Nuclear Proteins; D050794:STAT1 Transcription Factor; D015534:Trans-Activators; D019000:Phosphotyrosine; D014443:Tyrosine; D013006:Growth Hormone; D004247:DNA; D017027:Protein Tyrosine Phosphatases