Abstract
Previous work has demonstrated that estrogen administration to immature chickens results in a rapid but transient increase in nuclear estrogen receptor content, a large portion of which is associated with the nuclear matrix. The present studies were undertaken to determine whether estrogen produced a more generalized change in the protein composition of the nuclear matrix. High-resolution two-dimensional gel analysis of the matrix revealed a very complex protein pattern, but several major qualitative differences were observed after estrogen treatment. To simplify the number of proteins evaluated, we examined the effects of estrogen on a subset of matrix proteins, namely, calmodulin and its binding proteins. Calmodulin was measured by radioimmunoassay and the binding proteins were detected by interaction of 125I-calmodulin with matrix proteins distributed on one-dimensional polyacrylamide gels. Calmodulin and two specific Ca2+-dependent calmodulin-binding proteins were found to be associated with matrix preparations. The two binding proteins exhibited apparent Mr of 200,000 and 130,000. The Mr 130,000 protein was identified as myosin light chain kinase on the basis of enzymatic activity and immunoreactivity with a specific antibody to this enzyme. Estrogen treatment of immature chickens did not alter the hepatic content of calmodulin. However, the steroid did result in an enrichment of the proportion of calmodulin and its two binding proteins associated with the nuclear matrix within 4 h after injection. The time course of these changes paralleled those previously documented for estrogen receptor. Taken together, these data are compatible with a role for calmodulin and myosin light chain kinase in the response of chicken liver cells to steroid hormones.
Highlights
The liver of the immature chicken constitutes a useful system for the study of estrogen-regulated gene expression in the absence of cell growth and differentiation
One of the binding proteins has been identified as myosin light chain kinase. These changes parallel those previously reported to occur for the estrogen receptor (15). These results suggest that calmodulin and myosin light chain kinase may play a role in the response of chicken liver cells to estrogen
A number of proteins shown here exhibited increases in staining intensities after estrogen induction (Nos. 5, 6, and 11, for example). These changes suggest that the protein composition of the nuclear matrix is hormonally altered
Summary
The liver of the immature chicken constitutes a useful system for the study of estrogen-regulated gene expression in the absence of cell growth and differentiation. Whereas studies designed to evaluate the presence of cytoplasmic estrogen receptors have proven to be negative, administration of estrogen to immature chickens results in a rapid but transient increase in nuclear receptor content (1). Because of the extreme complexity of the nuclear matrix protein composition, it seemed likely that qualitative changes in various two-dimensional gel spots might be difficult to interpret. It was desirable, to focus on a subset of nuclear proteins. Immunofluorescence studies have been interpreted to indicate an increase in the nuclear content of calmodulin in the rat uterus within 12 h after estrogen administration (11), and calmodulin is ' .4hhreviations used in this paper: CaM, calmodulin ; MLCK, myosin light chain kinase
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