The analysis of thyroid nuclear proteins by polyacrylamide gel electrophoresis has demonstrated that thyrotropin and dibutyryl adenosine 3' : 5'-monophosphate stimulate specifically the phosphorylation of H1 histones in an intact cell system. This effect does not require new protein synthesis and implicates the phosphorylation of serine residue(s) situated in the N-terminal part of H1 histones.It is now well established that cyclic AMP is the intracellular mediator of the action of many hormones on their target cells and, in particular, of the action of thyrotropin on the thyroid [l]. Greengard has postulated that all the effects of cyclic AMP are secondary to the phosphorylation of cellular proteins by one or several cyclic-AMP-activated protein kinases [2]. 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].The purpose of this study has been to use a more efficient method for the separation of nuclear proteins, i.r. polyacrylamide gel electrophoresis, and to characterize the thyrotropin stimulation of H1 histone phosphorylation.
MATERIALS AND METHODS
MethodsDogs were pretreated with thyroid extract (150 mg/ day) for 1 day before the removal of the thyroids.Ahhreviutions. Cyclic AMP, adenosine 3': 5'-monophosphate; HI, H2A, H2B, H3, and H4, five main fractions of histones, international nomenclature.Thyroid slices (about 0.3 mm thick) were immediately prepared at room temperature and incubated at 37 "C, under 95 % 0 2 and 5 % C02, in Krebs-Ringer bicarbonate buffer containing inorganic [32P]phosphate (0.2 pM, specific activity 0