Action of Thyrotropin on Phosphate Incorporation into Thyroid Proteins in vitro

Abstract

The kinetics of [32P]phosphate incorporation into dog thyroid slices, in vitro, has been studied.Phosphate penetration was saturable which suggests a mediated transport. This transport appeared to be the limiting step in the slow incorporation of [32P]phosphate in the trichloroacetic-acidsoluble fraction of the slices. The incorporation of [32P]phosphate in thyroid proteins was a fast process (half equilibrium with the trichloroacetic-acid-soluble fraction radioactivity in 30 min). About 50°/, of this incorporation took place in the nuclear proteins, 2501, of it in the histones (mostly in the arginine-rich histones). The turnover of protein P was fast in all classes of proteins.Thyrotropin, in vitro, often enhanced the incorporation of [32P]phosphate in the trichloroacetic-acid-soluble fraction and into total proteins of the slices. However, this effect was delayed and rather small. It is ascribed mainly to an enhancement of phosphate penetration into the cells.This suggests, that, in dog thyroid cells, most of protein phosphorylation is independent of thyrotropin and cyclic AMP. Thyrotropin markedly enhanced the phosphorylation of Fl (lysine-rich) histones. This effect was fast, specific and mimicked by dibutyryl cyclic AMP; it required higher concentrations of thyrotropin than the activation of iodide binding to proteins. The role pf this action of thyrotropin is discussed; the hypothesis is proposed that it is related to thyrotropin-induced growth of thyroid tissue. NaF (5 mM) enhanced total protein phosphorylation in the thyroid slices. This effect, which may be due to unspecific inhibition of protein phosphatases, may explain the fact that NaF mimics several effects of thyrotropin. Cyclic-AMP-activated protein kinases have been demonstrated in thyroid tissue [3,7-121. To investigate the role of cyclic-AMP-induced protein phosphorylation in the activation of thyroid by thyrotropin, two approaches may be considered: the study of the enzymes involved (kinases and phosphatases) and the study of the substrate. Whereas cyclic-AMP-activated protein kinases have Abbreviation. Cyclic AMP, adenosine 3': 5'-monophosphate. been much investigated, little is known about their natural substrates. I n intact rat liver cells, glucagon, dibutyryl cyclic AMP and cyclic AMP activate the phosphorypation of the lysine-rich histones (FI) [13,14] [23,24] are substrates in acellular systems. The purpose of this work is to define the nature of the phosphorylated proteins in the thyroid, the kinetics of these phosphorylations and their dependence on cyclic AMP and thyrotropin.Eur. J. Biochem. 45 (1974)

Thyrotropin Stimulation of the Phosphorylation of Serine in the N‐Terminal Region of Thyroid H1 Histones

“…In a previous paper [4] we have shown that all the ["P]phosphate incorporated into the HI histones is alkali labile, which is a characteristic of ester bonds. In order to verify if both serine and threonine residues were implicated in the phosphorylation of H1 histones, these histones were extracted after polyacrylamide gel electrophoresis of the whole histone fraction and partially hydrolysed in 6 M HCl.…”

“…This phenomenon is not particular to dog thyroid but seems to be a general characteristic of all thyroid tissues. The identification of the H1 histones is based on three criteria : first their solubility properties [4], secondly when the whole histone fraction extracted from thyroid slices was subjected to polyacrylamide gel electrophoresis, the distance of migration of the thyrotropininduced radioactive peak (Fig. 2) was identical to the distance of migration of calf thymus HI histones, thirdly the amino acid analysis of this material established unequivocally that it was mainly constituted of H1 histones.…”

“…Whereas cyclic-AMP-dependent protein kinases have been widely studied, little is known about their natural substrates 131. This question is particularly pertinent in the thyroid where thyrotropin not only activates function but also induces growth the latter by unknown mechanisms but presumably through cyclic AMP [l].In work reported previously protein phosphorylation has been studied in thyroid slices in v i m [4]. The only marked effect of thyrotropin was an enhancement of the phosphorylation of the lysine-rich histone fraction (HI) as identified by their solubility properties [4].…”

“…In work reported previously protein phosphorylation has been studied in thyroid slices in v i m [4]. The only marked effect of thyrotropin was an enhancement of the phosphorylation of the lysine-rich histone fraction (HI) as identified by their solubility properties [4].…”

“…The only marked effect of thyrotropin was an enhancement of the phosphorylation of the lysine-rich histone fraction (HI) as identified by their solubility properties [4].…”