Abstract
A growth hormone-inducible nuclear factor complex (GHINF), affinity-purified using the growth hormone response element (GHRE) from the promoter of rat serine protease inhibitor 2.1, was found to contain Stat5a and -5b, as well as additional components. The ubiquitous transcription factor yin-yang 1 (YY1) is present in GHINF. An antibody to YY1 inhibited the formation of the GHINF.GHRE complex in an electrophoretic mobility shift assay. Furthermore, Stat5 was co-immunoprecipitated from rat hepatic nuclear extracts with antibodies to YY1. An examination of the GHRE shows that, in addition to two gamma-activated sites, it contains a putative YY1 binding site between the two gamma-activated sites, overlapping them both. Mutation of this putative YY1 site results in a decrease of GHINF.GHRE complex formation in an electrophoretic mobility shift assay and a corresponding decrease in growth hormone (GH) response in functional assays. The glucocorticoid receptor was also present in GHINF, and Stat5 co-immunoprecipitates with glucocorticoid receptor in hepatic nuclear extracts from rats treated with GH. GH activation of serine protease inhibitor 2.1 requires the unique sequence of the GHRE encompassing the recognition sites of several transcription factors, and the interaction of these factors enhances the assembly of the transcription complex.
Highlights
The liver is a major target organ for growth hormone (GH)1 action
To determine what forms of Stat5 were present in this band and to further examine their phosphorylation states, we used antibodies to Stat5a, Stat5b, phosphoserine, and phosphothreonine in GH response element (GHRE) binding assays
We show that both isoforms of Stat5, Stat5a and Stat5b, are present in growth hormone-inducible nuclear factor complex (GHINF) and that they are, in addition to being tyrosine-phosphorylated, serine-phosphorylated
Summary
We found that treating a normal rat with GH 1 h before sacrifice consistently led to accumulation of activated Stat in liver nuclei. As determined by supershift assays with antibodies to phosphoserine or phosphotyrosine with the GHRE, is identical to that observed when a hypophysectomized rat is treated with GH. We used both GH-treated normal and hypophysectomized rats for these studies. Quantitation of the shifted complexes on the film was determined using the Kodak Digital Science ID Image Analysis. YY1 recognition or mutated sites in all oligonucleotides are underlined
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