Phosphorylation of Nuclear Proteins from Bovine Anterior Pituitary Gland Induced by Adenosine 3′:5′‐monophosphate

Abstract

In the presence of exogenous protein kinase dependent on adenosine 3' : 5'-monophosphate (cyclic AMP), cyclic AMP leads to an important stimulation of 32P incorporation into chromatin purified from bovine anterior pituitary tissue. Over 80% of 32P incorporated into isolated adenohypophyseal histones is associated with Fzb histone while small uptake is measured in F1 and F3 (and its dimer form) histones. Cyclic AMP leads to a 3 to 4-fold stimulation of incorporation of radioactivity into histone Fzb. The radioactivity associated with F2b histone is almost exclusively recovered into two phosphopeptides. Eigthteen bands can be resolved after separation of adenohypophyseal acidic nuclear proteins on 5 % sodium dodecylsulfate polyacrylamide gels and their apparent molecular weights range from 12000 to 300000. Protein kinase catalyses the cyclic-AMP-dependent phosphorylation of a large number of acidic proteins although preferential labeling occurs in five bands. The present data suggest a possible role of cyclic AMP on the transcriptional activity of the anterior pituitary gland through phosphorylation of histones and non-histone acidic nuclear proteins.We have recently obtained direct evidence for a role of the adenylate cyclase system as mediator of the action of at least three hypothalamic releasing hormones in the anterior pituitary gland. These data pertain to our studies on the plasma membrane receptor for thyrotropin-releasing hormone [1,2] and the observed stimulation of adenohypophyseal adenylate cyclase activity by synthetic thyrotropin (P. Borgeat, P. Garneau, and F. Labrie, unpublished data) and luteinizing hormone-releasing hormones [3] and by purified growth hormone-releasing hormone [4]. It is well known that cyclic AMP, or its derivatives, stimulate the release of adenohypophyseal hormones [5].Although the short-term stimulatory effect of cyclic AMP on total adenohypophyseal protein [6], growth hormone and prolactin [4] synthesis seems to be exerted at a translational level, it is likely that the long-term effects of the cyclic nucleotide, in the anterior pituitary gland, will include an action at a transcriptional level.Much evidence has accumulated to support the hypothesis that in many systems, cyclic-AMP-dependent protein kinase mediates the various intracellular effects of the cyclic nucleotide [7]. Many studies indicate that the phosphorylation of histones [8,9] and non-histone [lo--121 nuclear proteins can be correlated with the rate of RNA synthesis. Moreover, glucagon enhances the phosphorylation, at specific sites, of rat liver F1 histone [8] while insulin and prolactin increase the phosphorylation of nuclear proteins (both histones and non-histones) in mouse mammary epithelial cells in organ culture [13]. Chorionic gonadotropin has also been reported to enhance the phosphorylation of both histone and non-histone proteins in rat ovarian tissue [14].In bovine anterior pituitary gland, we have already found a high level of endogenous phosphorylation in the nuclear fraction [15]. It thus seeme...

Studies in vitro of the effects of adenosine 3′:5′-cyclic monophosphate on the phosphorylation of nuclear proteins in isolated rat heart nuclei

“…In view of this it seems difficult to reconcile the reported increased concentration of cyclic AMP with the inhibition of growth in different cell lines. In the presence of cyclic AMP a selective stimulation of phosphorylation of some acidic proteins was observed in rat liver nuclei (Johnson & Allfrey, 1972) and bovine anterior pituitary gland (Jolicoeur & Labrie, 1974). In the present investigations cyclic AMP caused a general increase in phosphorylation ofnearly all non-histoneprotein fractions, but a preferential increase in 32p incorporation was observed in fractions III and IV, which were eluted with 0.35Mand 0.6M-NaCl from the Sephadex column.…”

Regulation of Growth Hormone mRNA and Pro-Opiomelanocortin mRNA Levels by Cyclic AMP in Rat Anterior Pituitary Cells in Culture

“…Such process could be exerted by an increase in nuclear cAMP-dependent protein A-kinase activity and phosphorylation of specific chromatin-associated proteins (Labrie et ai, 1971;Jolicoeur and Labrie, 1974). There is a good evidence for such a mechanism in many tissues (for review, see Cooper and Spaulding, 1985) and especially in GH4 cells (Murdoch et ai, 1983;Waterman et ai, 1985).…”

Effect of thyroliberin on the concentration of adenosine 3′:5′-phosphate and on the activity of adenosine 3′:5′-phosphate-dependent protein kinase in prolactin-producing cells in culture

“…(5) Homogenate of GH4C1 cells contains a cyclic AMP-dependent protein kinase with properties similar to protein kinases from other tissues (Labrie et al, 1971). The natural substrate for this enzyme in the GH4C, cells is unknown, but others (Jolicoeur & Labrie, 1974) have shown that chromatin, histones and acidic proteins purified from bovine anterior pituitary gland can be phosphorylated in vitro with homologous cyclic AMP-dependent protein kinase. (6) In experiments on intact cells, thyroliberin increased the activity ratio (-cyclic AMP/+cyclic AMP) of protein kinase, and the time-course of effect 1977 corresponded to the increase in cyclic AMP concentrations and with initiation of prolactin release.…”