Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors

Abstract

Human progesterone receptors (PR) in T47D breast cancer cells are synthesized as two different sized proteins, PR-A [94 kilodaltons (kDa)] and PR-B (120 kDa). Progestin addition to cells (in vivo) causes a 2-fold increase in total phosphorylation of PR and an increase in the apparent mol wt of both PR-A and PR-B on sodium dodecyl sulfate (SDS)-gels. Time-course experiments showed that increased PR phosphorylation that results from hormone addition is a multistep process and involves a rapid increase into total 32P labeling that takes place before the more slowly occurring phosphorylation(s) responsible for the change in electrophoretic mobility of PR on SDS-gels. As an approach to test whether phosphorylation is involved in regulating PR activity, we have examined the effects of cellular modulators of protein phosphorylation on PR-mediated target gene transcription in vivo using a T47D cloned cell line containing a stably transfected mouse mammary tumor virus-chloramphenicol acetyltransferase construct. Treatment with 8-bromo-cAMP (activator of cAMP-dependent protein kinases) or okadaic acid (protein phosphatase-1 and -2A inhibitor) did not stimulate target gene expression in the absence of progestin. When added together with progestin, either compound augmented PR-mediated target gene transcription by 3- to 4-fold. The cyclic nucleotide-dependent protein kinase inhibitor H8 completely blocked target gene responsiveness to hormone. Neither 8-bromo-cAMP, okadaic acid, nor H8 altered the hormone- or DNA-binding activities of PR, as measured in vitro or affected cellular concentrations of PR. These agents, therefore, appeared to selectively modulate PR transcriptional activity. Moreover, none of these compounds altered expression from a control reporter gene, pSV2CAT, indicating that these agents affect PR-mediated processes directly and are not acting through a general effect on transcription. Effects on PR phosphorylation were assessed by measuring 32P labeling of PR in vivo. None of these treatments had a substantial effect on the extent of total 32P labeling of immune isolated PR or on the phosphorylation(s) responsible for PR up-shifts on SDS-gels. This suggests that these agents modulate PR transcriptional activity either through phosphorylation of another protein intimately involved in PR-mediated transcription or through modification of a key site(s) not measurable as a change in total PR phosphorylation or electrophoretic mobility on SDS gels.

Nucleoprotein Structure Influences the Response of the Mouse Mammary Tumor Virus Promoter to Activation of the Cyclic AMP Signalling Pathway

“…Activation of the cAMP signalling pathway or inhibition of cellular phosphatase activity can synergistically stimulate transcription from the MMTV promoter in the presence of glucocorticoids or progestins (10,22,47,53,57). We confirm that the response of transiently introduced MMTV reporter cassettes to GR and PR is cooperatively enhanced by cAMP.…”

“…Briefly, nuclei were thawed on ice and washed once in 10 mM Tris (pH 8.0)-5 mM MgCl 2 -40% glycerol-2.5 mM dithiothreitol (DTT). Nuclear pellets were resuspended in 200 l of the wash buffer, to which an equal volume of reaction buffer (10 Slot blots were prepared as follows. DNA fragments containing sequences from actin, MMTV ras (pM18), MMTV CAT (pM25), or pUC18 were isolated and denatured in 10 mM HEPES (N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid) (pH 8.0)-1 mM EDTA-6% formaldehyde by boiling for 1 min followed by incubation at 60ЊC for 1 h. Fragments were then diluted with 20ϫ SSC and applied to a nylon membrane (Hybond; Amersham) in 1-g aliquots by using a slot blot vacuum apparatus (Hoefer).…”

“…Activation of the cAMP signalling pathway results in a synergistic stimulation of the MMTV promoter in the presence of either activated GR or progesterone receptor (PR) (10,22,47,53,57). This has been shown in various cell types and is probably mediated through the proximal promoter (47).…”

“…This has been shown in various cell types and is probably mediated through the proximal promoter (47). However, activation of the cAMP signalling pathway does not appear to result in changes in phosphorylation of the receptor itself (10,47,57), an observation which has led to the hypothesis that the ultimate target of PKA activation is a factor (or factors) which interacts with the receptor to induce transcription (9,47,49). In any case, these results imply that activation of the GR and the cAMP signalling pathway leads to more efficient interactions between the soluble transcription factors at the MMTV promoter.…”

Multiple mechanisms for regulation of steroid hormone action

“…Indeed, additional evidence suggests that modulators of phosphorylation may not even have the same effects on PR from different species. Beck et al [6] reported that the transcriptional activity of human PR is not stimulated by 8-bromo-CAMP or okadaic acid in the absence of progesterone treatment. However, these compounds were found to enhance progesterone-dependent transactivation of gene expression by three-to fourfold over the levels induced by progesterone alone.…”