[Show abstract][Hide abstract] ABSTRACT: Novel (Rp)-cAMPS analogs differed widely in ability to antagonize cAMP activation of pure cAMP-dependent protein kinase I and II and to antagonize actions of cAMP on gene expression, shape change, apoptosis, DNA replication, and protein phosphorylation in intact cells. These differences were related to different abilities of the analogs to stabilize the holoenzyme form relative to the dissociated form of cAMP kinase type I and II. (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were the most potent cAMP antagonists for isolated type I kinase and for cells expressing mostly type I kinase, like IPC-81 leukemia cells, fibroblasts transfected with type I regulatory subunit (RI), and primary hepatocytes. It is proposed that (Rp)-8-Br-cAMPS or (Rp)-8-Cl-cAMPS should replace (Rp)-cAMPS as the first line cAMP antagonist, particularly for studies in cells expressing predominantly type I kinase. The phosphorylation of endogenous hepatocyte proteins was affected oppositely by (Rp)-8-Br-cAMPS and increased cAMP, indicating that (Rp)-8-Br-cAMPS inhibited basal cAMP-kinase activity. The inhibition of basal kinase activity was accompanied by enhanced DNA replication, an effect which could be reproduced by microinjected mutant cAMP-subresponsive RI. It is concluded that the basal cAMP-kinase activity exerts a tonic inhibition of hepatocyte replication. (Rp)-8-Br-cAMPS and microinjected RI also desensitized hepatocytes toward inhibition of DNA synthesis by interleukin-1 beta. This indicates that basal cAMP-kinase activity can have a permissive role for the action of another (interleukin-1 beta) signaling pathway.
[Show abstract][Hide abstract] ABSTRACT: ras-Transformed NIH3T3 (R3T3) cells were transfected with expression vectors for the RII alpha and RII beta regulatory subunits of the type II isozyme of cAMP-dependent protein kinase, and the effects on gene activation by corticotropin-releasing factor (CRF) and prostaglandin E1 (PGE1) were analyzed. In RII alpha and RII beta-overexpressing cells, type II isozyme levels were increased, and type I isozyme levels were eliminated, demonstrating that both RII regulatory subunits compete efficiently with RI for catalytic subunit. The type II isozyme separated into three peaks on high performance liquid chromatography, referred to as A, B, and C. Western blot analysis strongly suggests that peak A and peak C correspond to holoenzymes containing RII beta and RII alpha, respectively. Overexpression of RII alpha resulted in the loss of peak A and a dramatic reduction in RII beta protein with no change in RII beta mRNA, indicating that the level of RII beta protein is controlled posttranscriptionally and that RII beta protein may become unstable when displaced from C. The role of type I and II kinases in transcriptional activation was investigated by comparing the response of control and RII expressing clones to site-selective cAMP analogs and the hormones, CRF and PGE1. The site-selective analogs demonstrated that either type I or type II kinase could activate the cAMP-responsive alpha-subunit promoter. The response to various concentrations of CRF or PGE1 was identical in control cells and transfected clones containing very little type I kinase. These experiments suggest that in the CRF and PGE1 response pathways leading to gene induction, the magnitude and sensitivity of the response are not influenced by the presence or absence of type I cAMP-dependent protein kinase.
Journal of Biological Chemistry 01/1992; 266(34):23074-82. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian tissues and cell lines express two major types of cAMP-dependent protein kinase, PKA-I and PKA-II, which can be distinguished at the molecular level by the presence of either type I or type II regulatory subunits in the holoenzyme. An expression vector for the mouse type II regulatory subunit (RII alpha) was transfected into ras-transformed NIH3T3 (R3T3) cells, which contain approximately equal amounts of both holoenzymes, PKA-I and PKA-II. In RII alpha-overexpressing R3T3 cells, PKA-II levels were increased, and the level of PKA-I declined. The decrease in PKA-I was dependent on the amount of RII alpha expressed, and at high levels of RII alpha expression, PKA-I was completely eliminated. In contrast, overexpression of the type I regulatory subunit (RI alpha) did not alter PKA isozyme levels. We propose that competition between RII alpha and RI alpha for a limited pool of catalytic subunit results in preferential assembly of PKA-II and that significant amounts of PKA-I are formed only if catalytic subunit is present in excess of the RII alpha subunit. The PKA-I isozyme, which is absent in untransformed 3T3 cells, is not essential for the transformed phenotype of R3T3 cells. RII alpha-overexpressing R3T3 cells that are devoid of PKA-I continued to exhibit a transformed phenotype including anchorage-independent growth. Overexpression of RII alpha provides a genetic approach that may prove useful in demonstrating specific functions for the two PKA isozymes in cAMP-dependent signal transduction pathways.
Journal of Biological Chemistry 01/1990; 264(34):20255-60. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Induction of the mouse mammary tumor virus (MMTV) promoter by steroid hormones was examined in chick oviduct primary cell cultures transfected with MMTV-chloramphenicol acetyl transferase fusion constructs. Our results demonstrate that in this system glucocorticoids, progesterone and dihydrotestosterone are all able to stimulate MMTV transcription; induction by progesterone and dihydrotestosterone is not mediated by glucocorticoid receptors, since the specific glucocorticoid antagonist RU486 did not inhibit the response. In contrast, estrogen does not stimulate MMTV transcription, although estrogen does induce the endogenous ovalbumin gene in the same cells. While progesterone effects are mediated by the same response elements within the MMTV long terminal repeat that were originally characterized for the glucocorticoid receptor, an androgen response element has not yet been defined. Our data indicate that the MMTV long terminal repeat does not contain an estrogen response element.