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Biochemical as well as immunochemical studies were carried out to quantitatively and qualitatively evaluate the hormonal regulation of nuclear cAMP-dependent protein kinase subunits in ovaries from estrogen-treated hypophysectomized rats. Photoaffinity labeling of nuclear extracts with 8-azido-[32P]cAMP and electrophoretic analysis showed the existence of three variants of the regulatory subunit RI and of a 52,000-dalton RII variant (RII-52) in ovarian nuclei of estrogen-primed hypophysectomized rats. After follicle-stimulating hormone (FSH) stimulation, an additional variant of RII (RII-51, Mr = 51,000) was detected in nuclei. The cytosolic RII-54 variant (Mr = 54,000) could not be identified in nuclei by photoaffinity labeling. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity. Using quantitation by enzyme-linked immunosorbent assay, we identified a marked FSH-mediated increase of nuclear RII variant(s) and confirmed the increase of nuclear catalytic subunit levels. Furthermore, morphometric analysis of nuclear and cytoplasmic antigen density by immunogold electron microscopy demonstrated a cell-specific modulation by FSH of RII and C subunit density. In granulosa cells, both nuclear as well as cytoplasmic RII density was increased by FSH, whereas catalytic subunit density was increased in the nuclear area only. In thecal cells, FSH increased only the nuclear catalytic subunit density. These results provide biochemical as well as immunochemical evidence for a cell-specific FSH regulation of nuclear RII and catalytic subunit levels which may be involved in the molecular events responsible for the FSH-mediated differentiation of the rat ovary.

Utilizing the gel electrophoresis/DNA binding assay and a new technique of direct binding of radioactive DNA to protein blots, we have investigated putative factors selective for the cAMP-responsive element (CRE) of the lactate dehydrogenase A subunit promoter in rat ovary nuclear extracts. Analysis of linker-scanning mutants of lactate dehydrogenase A subunit promoter fragments by DNA binding assay identified DNA binding activity selective for the 11-nucleotide sequence 5′ TCTGACGTCAG 3′ located between positions −51 and −41 relative to the transcription initiation site. This sequence contains the previously identified CRE 5′ TGACGTCA 3′. Probing of protein blots with radioactive promoter fragments containing the CRE demonstrated that ovarian nuclear extracts contain a protein of relative molecular mass 47,000 (Mr47,000) which exhibits selective binding affinity for the CRE. The 47-kDa CRE binding protein was found to be present in comparable levels in the ovaries of normal and hypophysectomized rats. Furthermore, our data suggest the presence of a 37,000-dalton (Mr37,000) protein which possesses selective binding affinity for part of the CRE sequence. The binding activity/level of the 37-kDa CRE binding protein appeared to be modulated by short-term hypophysectomy/follicle-stimulating hormone administration. These results provide evidence for the presence of CRE binding factors in rat ovarian nuclei, which may be involved in the molecular events responsible for transcriptional regulation of ovarian cAMP-inducible genes.

NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids. Although Ku is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (Kd = 0.84 +/- 0.24 nM) than DNA end binding. Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence. Sequences with similarity to NRE1 competed efficiently for NRE1 binding. Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends. Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation. Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527. This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.

An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP-dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density.