Altered cyclic AMP-dependent protein kinase activity in a mutant adrenocortical tumor cell line

A somatic cell genetic approach has been used to evaluate the role of cyclic AMP-dependent protein kinase in ACTH action on adrenal steroidogenesis. A mutant clone, 8BrcAMP^r-1, previously was isolated from an ACTH-sensitive adrenocortical tumor cell line (clone Y1) following mutagenesis and selective growth in 8-bromoadenosine 3′, 5′-monophosphate. This study demonstrates that the 8BrcAMP⁴-1 cells have an altered cyclic AMP-dependent protein kinase. The protein kinase in the cytosol of the mutant characteristically requires, for half-maximal activity, concentrations of cyclic AMP 7-fold higher than those required by the enzyme in preparations from the parent. The cytosolic cyclic AMP-dependent protein kinases of Y1 and 8BrcAMP^r-1 cells chromatograph similarly on columns of DEAE-cellulose. From each cell line, a major peak of activity (≥ 70% of recovered activity), designated as Peak I, elutes with 0.04–0.06 M NaCl; a second peak of activity, designated as Peak II, elutes with 0.12–0.14 M NaCl. Protein kinase activity in the Peak I fraction of mutant cells has a decreased apparent affinity (4-fold) for cyclic AMP relative to the corresponding fraction of parental Y1 cells. The protein kinase activities present in Peak II fractions from Y1 and mutant cells are indistinguishable. The protein kinase mutant exhibits poor steroidogenic responses to added ACTH and cyclic AMP; and as shown previously does not display the growth arrest and morphological changes produced in Y1 by these agents. These results suggest that cyclic AMP-dependent protein kinase is important in the regulation of adrenal steroidogenesis, morphology and growth by ACTH.     

Ezrin is a cyclic AMP-dependent protein kinase anchoring protein.

cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin-related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14-amino-acid amphipathic alpha-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus.     

Modulation of cyclic AMP-dependent protein kinase isozyme expression associated with activation of a macrophage cell line

We have used the RAW 264.7 macrophage (MO) cell line to study cAMPdPK isozymes during activation by lymphokine (LK) and lipopolysaccharide (LPS). Untreated cells were found to have two isozymes of cAMPdPK in their cytosol. PKI and PKII were differentiated based on the Mr of their regulatory subunits (RI, 45,500; and RII, 52,000, respectively) as determined by photoactivated incorporation of the cAMP analog 8-N3-[32P]cAMP. Loss of the RI subunit of PKI occurred in association with activation of the cell line by suboptimal concentrations of LK and LPS (1/40 dilution, 1 ng/ml) or high concentrations of LPS alone (10 ng/ml to 100 micrograms/ml). No modulation of the RII subunit of PKII was observed under these conditions. The loss of RI was dependent on the addition of a triggering signal to the MO. Treatment of RAW 264.7 cells with LK alone at dilutions from 1/10 to 1/1280 was not sufficient to cause a disappearance of the RI subunit from the cytosol or to induce antitumor activity. The addition of a suboptimal concentration of LPS after LK or a high dose of LPS alone was required for acquisition of cytolytic activity and loss of RI. The kinetics for the disappearance of RI from treated cells were found to be identical after activation with either LK and LPS or high concentrations of LPS alone. RI could no longer be detected in the cytosol 8 hr after the addition of activating agents. The antitumor activity of the RAW 264.7 cell line was transiently expressed after activation. Cells no longer exhibited tumoricidal activity 48 hr after the removal of activating agents. It was observed that the loss of cytolytic function was accompanied by the reexpression of RI in the cytosol. This study provides evidence that modulation of cAMPdPK isozymes occurs during activation, suggesting a potential mechanism for controlling the effects of cAMP on the MO.     

A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum. II. Developmental regulation

The cAMP-dependent protein kinase of the cellular slime mold, Dictyostelium discoideum, is developmentally regulated; there is an approximately 4-fold increase in activity during development. The incorporation of [3H]leucine into the enzyme demonstrates that there is de novo synthesis of the cAMP-dependent protein kinase. The activities of the catalytic and regulatory subunits increase in parallel. The maximal rate of increase of cAMP-dependent protein kinase activity precedes "tip" formation, a stage of development characterized by a sharp increase in mRNA complexity. The high level of cAMP-dependent protein kinase activity, attained at this stage of development, persists when aggregates are dispersed and the amoebae are kept in suspension without added cAMP. The synthesis of the developmentally regulated mRNAs under these conditions is dependent on exogenous cAMP. The increase in cAMP-dependent protein kinase activity during development does not require sustained cell-cell contact insofar as it occurs in single cell suspensions of amoebae. Furthermore, the increase does not require exogenous cAMP, although added cAMP stimulates the synthesis of the enzyme to a level higher than that found, when cAMP is not added. These observations support the hypothesis that in D. discoideum cAMP-dependent protein kinase mediates the effects of cAMP on development.     

Rabbit red cell cyclic AMP-dependent protein kinase. I. Reversible subunit interaction

Cyclic AMP-dependent protein kinase I consists of two dissimilar functional subunits: a catalytic subunit and a cyclic AMP binding subunit. The interaction of the two subunits appears to be reversible.     

Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase.

Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.     

Phosphorylation of casein by cyclic AMP-dependent protein kinase.

The catalytic subunit of rabbit muscle cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein transferase) has been tested on a variety of caseins. The B variant of β-casein was phosphorylated at a much greater rate than other β-caseins, α_s1-caseins, and κ-caseins. Whole casein homozygous for β-casein B was phosphorylated at 2.5 times the rate of commercial whole casein. Gel electrophoresis experiments indicate that β-casein is the predominant component phosphorylated in commerical casein. It is therefore suggested that phosphorylation of whole casein depends on its content of the specific genetic variant, β-casein B.     

Hormonal regulation of nuclear cyclic AMP-dependent protein kinase subunit levels in rat ovaries

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.     

Negative regulation of histone deacetylase 8 activity by cyclic AMP-dependent protein kinase A

Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from lysine residues of histone and nonhistone proteins. Recent studies suggest that they are key regulators of many cellular events, including cell proliferation and cancer development. Human class I HDACs possess homology to the yeast RPD3 protein and include HDAC1, HDAC2, HDAC3, and HDAC8. While HDAC1, HDAC2, and HDAC3 have been characterized extensively, almost nothing is known about HDAC8. Here we report that HDAC8 is phosphorylated by cyclic AMP-dependent protein kinase A (PKA) in vitro and in vivo. The PKA phosphoacceptor site of HDAC8 is Ser(39), a nonconserved residue among class I HDACs. Mutation of Ser(39) to Ala enhances the deacetylase activity of HDAC8. In contrast, mutation of Ser(39) to Glu or induction of HDAC8 phosphorylation by forskolin, a potent activator of adenyl cyclase, decreases HDAC8's enzymatic activity. Remarkably, inhibition of HDAC8 activity by hyperphosphorylation leads to hyperacetylation of histones H3 and H4, suggesting that PKA-mediated phosphorylation of HDAC8 plays a central role in the overall acetylation status of histones.     

Concentrations of cyclic AMP-dependent protein kinase subunits in various tissues.

The concentrations of the regulatory (R) and catalytic (C) subunits of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase(s) were measured in extracts of skeletal muscle, heart, liver, kidney, and brain. These concentrations were also estimated for the particulate fraction from brain, the only tissue in which a major part of the total activity was not readily extracted in a soluble form. Values for R were determined by measuring the amount of cyclic [3H]amp bound to protein in these tissue fractions under specified conditions; it was assumed that 1 mol of cyclic AMP binds to 1 mol of R. Values for C were determined from measurements of the specific protein kinase activity of the fractions utilizing the turnover number of pure C in the calculations. Turnover numbers for C were found to be identical for this subunit obtained in the pure form from rabbit skeletal muscle, rabbit liver, and beef heart. The methods used for measuring C were evaluated by kinetic studies and through the use of the specific heatstable protein inhibitor of cyclic AMP-dependent protein kinase(s). R and C were found to exist in a 1:1 molar ratio in all of the tissue fractions that were studied. the absolute concentrations of R and C ranged from 0.23 mumol/kg wet weight for liver to 0.78 mumol/kg wet weight for brain. For brain this value was based on the amount of each subunit in the particulate as well as the soluble fraction. For other tissues the values were based solely on the subunit content of the latter fraction. It was noted that the molar concentrations of R are close to those of cyclic AMP under basal conditions in the various tissues.     

Oligonucleotides antisense to catalytic subunit of cyclic AMP-dependent protein kinase inhibit mouse mammary epithelial cell DNA synthesis

Mouse mammary epithelial cells were plated onto 24-well culture plates (50,000 per well), allowed to attach and serum starved for 24 h. Following serum starvation, DNA synthesis was induced by the addition of 10% fetal calf serum and determined by a 1-h pulse with [3H]thymidine from 17 to 18 h after serum addition. Addition of oligonucleotides antisense to the translation start region of cyclic AMP-dependent protein kinase (kinase A) mRNA inhibited thymidine incorporation into DNA (total or percentage of cells incorporating thymidine, as measured by autoradiography). This inhibition was apparent whether compared to controls with no oligonucleotide addition, sense oligonucleotides, or mismatch oligonucleotides. Enzymatic assays indicated that the antisense oligonucleotides lowered kinase A activity in cells. Time course studies indicated that the inhibition in DNA synthesis was not an artifact of the time at which DNA synthesis was estimated. Long-term (4 day) cultures indicated that effects on induction of DNA synthesis were reflected in long-term cell proliferation.