scholarly journals Apoptosis in metanephric development.

1992 ◽  
Vol 119 (5) ◽  
pp. 1327-1333 ◽  
Author(s):  
C Koseki ◽  
D Herzlinger ◽  
Q al-Awqati
Keyword(s):  
Spinal Cord ◽  
Protein Kinase ◽  
Transcriptional Activation ◽  
Phorbol Esters ◽  
Dna Degradation ◽  
Morphological Evidence ◽  
Metanephric Mesenchyme ◽  
Embryonic Spinal Cord

During metanephric development, non-polarized mesenchymal cells are induced to form the epithelial structures of the nephron following interaction with extracellular matrix proteins and factors produced by the inducing tissue, ureteric bud. This induction can occur in a transfilter organ culture system where it can also be produced by heterologous cells such as the embryonic spinal cord. We found that when embryonic mesenchyme was induced in vitro and in vivo, many of the cells surrounding the new epithelium showed morphological evidence of programmed cell death (apoptosis) such as condensed nuclei, fragmented cytoplasm, and cell shrinking. A biochemical correlate of apoptosis is the transcriptional activation of a calcium-sensitive endonuclease. Indeed, DNA isolated from uninduced mesenchyme showed progressive degradation, a process that was prevented by treatment with actinomycin-D or cycloheximide and by buffering intracellular calcium. These results demonstrate that the metanephric mesenchyme is programmed for apoptosis. Incubation of mesenchyme with a heterologous inducer, embryonic spinal cord prevented this DNA degradation. To investigate the mechanism by which inducers prevented apoptosis we tested the effects of protein kinase C modulators on this process. Phorbol esters mimicked the effects of the inducer and staurosporine, an inhibitor of this protein kinase, prevented the effect of the inducer. EGF also prevented DNA degradation but did not lead to differentiation. These results demonstrate that conversion of mesenchyme to epithelial requires at least two steps, rescue of the mesenchyme from apoptosis and induction of differentiation.

ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230

1992 ◽  
Vol 12 (4) ◽  
pp. 1507-1514
Author(s):  
C L Denis ◽  
S C Fontaine ◽  
D Chase ◽  
B E Kemp ◽  
L T Bemis
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Growth Conditions ◽  
Dependent Protein Kinase ◽  
Inactive Form ◽  
Dependent Protein ◽  
Kinase Phosphorylation

Four ADR1c mutations that occur close to Ser-230 of the Saccharomyces cerevisiae transcriptional activator ADR1 and which greatly enhance the ability of ADR1 to activate ADH2 expression under glucose-repressed conditions have been shown to reduce or eliminate cyclic AMP-dependent protein kinase (cAPK) phosphorylation of Ser-230 in vitro. In addition, unregulated cAPK expression in vivo blocks ADH2 depression in an ADR1-dependent fashion in which ADR1c mutations display decreased sensitivity to unregulated cAPK activity. Taken together, these data have suggested that ADR1c mutations enhance ADR1 activity by blocking cAPK phosphorylation and inactivation of Ser-230. We have isolated and characterized an additional 17 ADR1c mutations, defining 10 different amino acid changes, that were located in the region defined by amino acids 227 through 239 of ADR1. Three observations, however, indicate that the ADR1c phenotype is not simply equivalent to a lack of cAPK phosphorylation. First, only some of these newly isolated ADR1c mutations affected the ability of yeast cAPK to phosphorylate corresponding synthetic peptides modeled on the 222 to 234 region of ADR1 in vitro. Second, we observed that strains lacking cAPK activity did not display enhanced ADH2 expression under glucose growth conditions. Third, when Ser-230 was mutated to a nonphosphorylatable residue, lack of cAPK activity led to a substantial increase in ADH2 expression under glucose-repressed conditions. Thus, while cAPK controls ADH2 expression and ADR1 is required for this control, cAPK acts by a mechanism that is independent of effects on ADR1 Ser-230. It was also observed that deletion of the ADR1c region resulted in an ADR1c phenotype. The ADR1c region is, therefore, involved in maintaining ADR1 in an inactive form. ADR1c mutations may block the binding of a repressor to ADR1 or alter the structure of ADR1 so that transcriptional activation regions become unmasked.

scholarly journals Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins.

1992 ◽  
Vol 12 (10) ◽  
pp. 4478-4485 ◽  
Author(s):  
L Li ◽  
R Heller-Harrison ◽  
M Czech ◽  
E N Olson
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Specific Gene ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins

1992 ◽  
Vol 12 (10) ◽  
pp. 4478-4485
Author(s):  
L Li ◽  
R Heller-Harrison ◽  
M Czech ◽  
E N Olson
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Specific Gene ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

scholarly journals ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230.

1992 ◽  
Vol 12 (4) ◽  
pp. 1507-1514 ◽  
Author(s):  
C L Denis ◽  
S C Fontaine ◽  
D Chase ◽  
B E Kemp ◽  
L T Bemis
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Growth Conditions ◽  
Dependent Protein Kinase ◽  
Inactive Form ◽  
Dependent Protein ◽  
Kinase Phosphorylation

Four ADR1c mutations that occur close to Ser-230 of the Saccharomyces cerevisiae transcriptional activator ADR1 and which greatly enhance the ability of ADR1 to activate ADH2 expression under glucose-repressed conditions have been shown to reduce or eliminate cyclic AMP-dependent protein kinase (cAPK) phosphorylation of Ser-230 in vitro. In addition, unregulated cAPK expression in vivo blocks ADH2 depression in an ADR1-dependent fashion in which ADR1c mutations display decreased sensitivity to unregulated cAPK activity. Taken together, these data have suggested that ADR1c mutations enhance ADR1 activity by blocking cAPK phosphorylation and inactivation of Ser-230. We have isolated and characterized an additional 17 ADR1c mutations, defining 10 different amino acid changes, that were located in the region defined by amino acids 227 through 239 of ADR1. Three observations, however, indicate that the ADR1c phenotype is not simply equivalent to a lack of cAPK phosphorylation. First, only some of these newly isolated ADR1c mutations affected the ability of yeast cAPK to phosphorylate corresponding synthetic peptides modeled on the 222 to 234 region of ADR1 in vitro. Second, we observed that strains lacking cAPK activity did not display enhanced ADH2 expression under glucose growth conditions. Third, when Ser-230 was mutated to a nonphosphorylatable residue, lack of cAPK activity led to a substantial increase in ADH2 expression under glucose-repressed conditions. Thus, while cAPK controls ADH2 expression and ADR1 is required for this control, cAPK acts by a mechanism that is independent of effects on ADR1 Ser-230. It was also observed that deletion of the ADR1c region resulted in an ADR1c phenotype. The ADR1c region is, therefore, involved in maintaining ADR1 in an inactive form. ADR1c mutations may block the binding of a repressor to ADR1 or alter the structure of ADR1 so that transcriptional activation regions become unmasked.

Adenosine and its nucleotides activate wild-type and R117H CFTR through an A2Breceptor-coupled pathway

1999 ◽  
Vol 276 (2) ◽  
pp. C361-C369 ◽  
Author(s):  
John P. Clancy ◽  
Fadel E. Ruiz ◽  
Eric J. Sorscher
Keyword(s):  
Protein Kinase ◽  
Phorbol Esters ◽  
Purinergic Receptor ◽  
Bronchial Epithelium ◽  
Receptor Subtypes ◽  
Wild Type ◽  
Intestinal Epithelia ◽  
Protein Kinase C Inhibitor

ATP and its metabolites stimulate Cl−secretion in human epithelium in vitro and in vivo. The specific purinergic receptor subtypes that govern these effects have been difficult to separate, in part due to multiple parallel pathways for Cl− secretion in respiratory and intestinal epithelia. In a simplified model using COS-7 cells, we demonstrate acquisition of an ATP-, ADP-, AMP-, and adenosine (ADO)-regulated halide permeability specifically following expression of wild-type (wt) cystic fibrosis transmembrane conductance regulator (CFTR). This halide permeability is blocked by the P1 purinergic receptor antagonist 8-phenyl theophylline, sensitive to the protein kinase A inhibitor H-89, and associated with a modest, dose-dependent increase in cellular cAMP concentration. Phorbol esters poorly activate halide permeability compared with ADO, and ADO-stimulated efflux was not affected by treatment with the protein kinase C inhibitor bisindolylmaleimide I. The A2 ADO receptor (AR) agonists 5′- N-ethylcarboxamide adenosine and ADO were strong activators, whereas the A1 AR agonist R-phenylisopropyladenosine failed to activate halide permeability. Metabolic conversion of ADO nucleotides by surface ecto-5′-nucleotidase to more active (less phosphorylated) forms contributes to anion transport activation in these cells. Immunoprecipitation with anti-A2B AR antibody identified a 31-kDa protein in both COS-7 and human bronchial epithelial cells. Together, these findings indicate that ADO and its nucleotides are capable of activating wtCFTR-dependent halide permeability through A2B AR and that this AR subtype is present in human bronchial epithelium. We also present data showing that this pathway can activate clinically significant mutant CFTR molecules such as R117H.

scholarly journals Acetylation-Mediated Transcriptional Activation of the ETS Protein ER81 by p300, P/CAF, and HER2/Neu

2003 ◽  
Vol 23 (17) ◽  
pp. 6243-6254 ◽  
Author(s):  
Apollina Goel ◽  
Ralf Janknecht
Keyword(s):  
Protein Kinase ◽  
Transcriptional Activation ◽  
Binding Activity ◽  
Tumor Formation ◽  
Mitogen Activated Protein Kinase ◽  
Transactivation Domain ◽  
Dna Binding Activity ◽  
Mitogen Activated Protein

ABSTRACT The regulated expression of the ETS transcription factor ER81 is a prerequisite for normal development, and its dysregulation contributes to neoplasia. Here, we demonstrate that ER81 is acetylated by two coactivators/acetyltransferases, p300 and p300- and CBP-associated factor (P/CAF) in vitro and in vivo. Whereas p300 acetylates two lysine residues (K33 and K116) within the ER81 N-terminal transactivation domain, P/CAF targets only K116. Acetylation of ER81 not only enhances its ability to transactivate but also increases its DNA binding activity and in vivo half-life. Furthermore, oncogenic HER2/Neu, which induces phosphorylation and thereby activation of ER81, was less able to activate acetylation-deficient ER81 mutants, indicating that both acetyltransferase and protein kinase-specific regulatory mechanisms control ER81 activity. Importantly, HER2/Neu overexpression stimulates the ability of p300 to acetylate ER81, likely by inducing phosphorylation of p300 through the Ras→Raf→mitogen-activated protein kinase pathway. This represents a novel mechanism by which oncogenic HER2/Neu, Ras, or Raf may promote tumor formation by enhancing acetylation not only of ER81 but also of other downstream effector transcription factors as well as histones.

scholarly journals Differential spatial and temporal phosphorylation of the visual receptor, rhodopsin, at two primary phosphorylation sites in mice exposed to light

2003 ◽  
Vol 374 (2) ◽  
pp. 537-543 ◽  
Author(s):  
Ryan A. ADAMS ◽  
Xinran LIU ◽  
David S. WILLIAMS ◽  
Alexandra C. NEWTON
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Outer Segment ◽  
Phorbol Esters ◽  
Phosphorylation Sites ◽  
Rod Outer Segment ◽  
Kinase C ◽  
Rhodopsin Kinase

Phosphorylation of rhodopsin critically controls the visual transduction cascade by uncoupling it from the G-protein transducin. The kinase primarily responsible for this phosphorylation is rhodopsin kinase, a substrate-regulated kinase that phosphorylates light-activated rhodopsin. Protein kinase C has been implicated in controlling the phosphorylation of both light-activated and dark-adapted rhodopsin. Two of the major rhodopsin phosphorylation sites in vivo, Ser334 and Ser338, are effective protein kinase C phosphorylation sites in vitro, while the latter is preferentially phosphorylated by rhodopsin kinase in vitro. Using phosphospecific antibodies against each of these two sites, we show that both sites are under differential spatial and temporal regulation. Exposure of mice to light results in rapid phosphorylation of Ser338 that is evenly distributed along the rod outer segment. Phosphorylation of Ser334 is considerably slower, begins at the base of the rod outer segment, and spreads to the top of the photoreceptor over time. In addition, we show that phosphorylation of both sites is abolished in rhodopsin kinase−/− mice, revealing an absolute requirement for rhodopsin kinase to phosphorylate rhodopsin. This requirement may reflect the need for priming phosphorylations at rhodopsin kinase sites allowing for subsequent phosphorylation by protein kinase C at Ser334. In this regard, treatment of mouse retinas with phorbol esters results in a 4-fold increase in phosphorylation on Ser334, with no significant effect on the phosphorylation of Ser338. Our results are consistent with light triggering rapid priming phosphorylations of rhodopsin by rhodopsin kinase, followed by a slower phosphorylation on Ser334, which is regulated by protein kinase C.

scholarly journals Effects of insulin on the translocation of protein kinase C-θ and other protein kinase C isoforms in rat skeletal muscles

1995 ◽  
Vol 308 (1) ◽  
pp. 177-180 ◽  
Author(s):  
K Yamada ◽  
A Avignon ◽  
M L Standaert ◽  
D R Cooper ◽  
B Spencer ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Skeletal Muscles ◽  
Phorbol Esters ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Isoforms ◽  
Pkc Alpha

Protein kinase C (PKC)-theta is a newly recognized major PKC isoform in skeletal muscle. In this study we found that insulin provoked rapid biphasic increases in membrane-associated immunoreactive PKC-theta, as well as PKC-alpha, PKC-beta and PKC-epsilon, in rat soleus muscles incubated in vitro. Effects of insulin on PKC isoforms in the soleus were comparable in magnitude with those of phorbol esters. Increases in membrane-associated PKC-theta, PKC-alpha, PKC-beta and PKC-epsilon were also observed in rat gastrocnemius muscles after insulin treatment in vivo. Our findings suggest that PKC-theta, like other diacylglycerol-sensitive PKC isoforms (alpha, beta and epsilon), may play a role in insulin action in skeletal muscles.

Calcitonin causes a sustained inhibition of protein kinase C-stimulated bone resorption in contrast to the transient inhibition of parathyroid hormone-induced bone resorption

1990 ◽  
Vol 123 (3) ◽  
pp. 251-256 ◽  
Author(s):  
Maria Ransjö ◽  
Ulf H. Lerner
Keyword(s):  
Protein Kinase C ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Bone Resorption ◽  
Phorbol Esters ◽  
Inhibitory Effect ◽  
Prostaglandin E ◽  
Kinase C

Abstract. Calcitonin is a well known inhibitor of osteoclastic bone resorption, both in vivo and in vitro. However, it is also known that calcitonin has only a transient inhibitory effect on bone resorption. The mechanism for this so-called "escape from inhibition" phenomenon is not clear. In the present study, the inhibitory effect of calcitonin on phorbol ester-induced bone resorption was examined in cultured neonatal mouse calvaria. Bone resorption was assessed as the release of radioactivity from bones prelabelled in vivo with 45Ca. Two protein kinase C-activating phorbol esters, phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate, both stimulated 45Ca release in 120-h cultures at a concentration of 10 nmol/l. Calcitonin (30 nmol/l) inhibited phorbol esterstimulated bone resorption without any "escape from inhibition". This was in contrast to the transient inhibitory effect of calcitonin on bone resorption stimulated by parathyroid hormone (10 nmol/l), prostaglandin E2 (2 μmol/l), and bradykinin (1 μmol/l). Our results suggest that activation of protein kinase C produces a sustained inhibitory effect of calcitonin on bone resorption.

scholarly journals Repression of GCN5 Histone Acetyltransferase Activity via Bromodomain-Mediated Binding and Phosphorylation by the Ku–DNA-Dependent Protein Kinase Complex

1998 ◽  
Vol 18 (3) ◽  
pp. 1349-1358 ◽  
Author(s):  
Nickolai A. Barlev ◽  
Vladimir Poltoratsky ◽  
Tom Owen-Hughes ◽  
Carol Ying ◽  
Lin Liu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Transcriptional Activation ◽  
Histone Acetyltransferase ◽  
Dependent Protein Kinase ◽  
Nuclear Extracts ◽  
Dependent Protein ◽  
Endogenous Proteins ◽  
Hybrid Screening

ABSTRACT GCN5, a putative transcriptional adapter in humans and yeast, possesses histone acetyltransferase (HAT) activity which has been linked to GCN5’s role in transcriptional activation in yeast. In this report, we demonstrate a functional interaction between human GCN5 (hGCN5) and the DNA-dependent protein kinase (DNA-PK) holoenzyme. Yeast two-hybrid screening detected an interaction between the bromodomain of hGCN5 and the p70 subunit of the human Ku heterodimer (p70-p80), which is the DNA-binding component of DNA-PK. Interaction between intact hGCN5 and Ku70 was shown biochemically using recombinant proteins and by coimmunoprecipitation of endogenous proteins following chromatography of HeLa nuclear extracts. We demonstrate that the catalytic subunit of DNA-PK phosphorylates hGCN5 both in vivo and in vitro and, moreover, that the phosphorylation inhibits the HAT activity of hGCN5. These findings suggest a possible regulatory mechanism of HAT activity.

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