Spatial regulation of a zebrafish patched homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2835-2846 ◽  
Author(s):  
J.P. Concordet ◽  
K.E. Lewis ◽  
J.W. Moore ◽  
L.V. Goodrich ◽  
R.L. Johnson ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Sonic Hedgehog ◽  
Dominant Negative ◽  
Spatial Regulation ◽  
Spatiotemporal Regulation ◽  
Segment Polarity ◽  
Ectopic Activation ◽  
Hedgehog Signal ◽  
Kinase A

Signalling by members of the Hedgehog family of secreted proteins plays a central role in the development of vertebrate and invertebrate embryos. In Drosophila, transduction of the Hedgehog signal is intimately associated with the activity of protein kinase A and the product of the segment polarity gene patched. We have cloned a homologue of patched from the zebrafish Danio rerio and analysed the spatiotemporal regulation of its transcription during embryonic development in both wild-type and mutant animals. We find a striking correlation between the accumulation of patched1 transcripts and cells responding to sonic hedgehog activity both in the neurectoderm and mesoderm, suggesting that like its Drosophila counterpart, patched1 is regulated by sonic hedgehog activity. Consistent with this interpretation, mis-expression of sonic hedgehog results in ectopic activation of patched1 transcription. Using dominant negative and constitutively active forms of the protein kinase A subunits, we also show that expression of patched1 as well as of other sonic hedgehog targets, is regulated by protein kinase A activity. Taken together, our findings suggest that the mechanism of signalling by Hedgehog family proteins has been highly conserved during evolution.

Control of CCK gene transcription by PACAP in STC-1 cells

2000 ◽  
Vol 279 (3) ◽  
pp. G605-G612 ◽  
Author(s):  
Damian G. Deavall ◽  
Raktima Raychowdhury ◽  
Graham J. Dockray ◽  
Rod Dimaline
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Gene Transcription ◽  
Transcriptional Start Site ◽  
Dominant Negative ◽  
Activator Protein 1 ◽  
Mobility Shift ◽  
Creb Phosphorylation ◽  
Mobility Shift Assay ◽  
Kinase A

The mechanisms by which neuroendocrine stimulants regulate CCK gene transcription are unclear. We examined promoter activation by pituitary adenylate cyclase-activating polypeptide (PACAP), a known CCK secretagogue, in the enteroendocrine cell line STC-1. The promoter region from −70 to −87 bp, relative to the transcriptional start site, contains a composite calcium/cyclic AMP response element (CRE)/activator protein 1 (AP1) site that may bind CRE binding protein (CREB) and AP1. PACAP (with IBMX) stimulated expression of an 87-bp construct 3.35 ± 0.36-fold but had no effect on a −70 construct. The effect was blocked by the protein kinase A inhibitor H-89 and by a dominant-negative CREB plasmid. Mutation of the CRE/AP1 site to a canonical CRE site did not affect the response to PACAP, but mutation to a canonical AP1 site prevented it. CREB phosphorylation was increased after PACAP treatment. Electrophoretic mobility shift assay and supershift analysis revealed that CREB and not AP1 bound to the CRE/AP1 site and that PACAP increased the proportion of phosphorylated CREB that was bound. We conclude that PACAP increases CCK gene expression via a cAMP-mediated pathway involving CREB phosphorylation by protein kinase A and activation of a composite CRE/AP1 site.

Interactions Between the Inositol 1,4,5-Trisphosphate and Cyclic AMP Signaling Pathways Regulate Larval Molting in Drosophila

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 309-318 ◽  
Author(s):  
K Venkatesh ◽  
G Siddhartha ◽  
Rohit Joshi ◽  
Sonal Patel ◽  
Gaiti Hasan
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Signaling Pathways ◽  
Feedback Inhibition ◽  
Receptor Gene ◽  
Dominant Negative ◽  
Neural Signals ◽  
Inositol 1,4,5 Trisphosphate ◽  
Kinase A ◽  
Insp3 Receptor

Abstract Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. In this study we have analyzed, using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting in Drosophila. Previous studies have shown that mutants for the inositol 1,4,5-trisphosphate receptor gene (itpr) are larval lethals. In addition they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Here we show that mutants for adenylate cyclase (rut) synergize, during larval molting, with itpr mutant alleles, indicating that both cAMP and InsP3 signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP3 receptor. Furthermore, our studies predict the existence of feedback inhibition through protein kinase A on the InsP3 receptor by increased levels of 20-hydroxyecdysone.

scholarly journals Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase promoter contains a CREB binding site that regulates cAMP action in Caco-2 cells

2000 ◽  
Vol 345 (2) ◽  
pp. 201-206 ◽  
Author(s):  
Anke EGGERS ◽  
Concha CAUDEVILLA ◽  
Guillermina ASINS ◽  
Fausto G. HEGARDT ◽  
Dolors SERRA
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Camp Response Element Binding ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mrna Levels ◽  
Camp Response Element ◽  
Response Element ◽  
Kinase A

cAMP increases transcription of the mitochondrial (mit.) gene for 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase, which encodes an enzyme that has been proposed as a control site of ketogenesis. The incubation of Caco-2 cells with cAMP increased mit.HMG-CoA synthase mRNA levels 4-fold within 24 h. We have identified an active cAMP-response element (CRE) located 546 bp upstream of the mit.HMG-CoA synthase promoter that is necessary for the induction of expression by dibutyryl cAMP. Co-transfections of constructs, containing the CRE element of the mit.HMG-CoA synthase promoter fused to the gene for chloramphenicol acetyltransferase, with protein kinase A and a dominant-negative mutant of cAMP-response-element-binding protein (CREB) show that the response to cAMP is mediated by the transcription factor CREB. The CRE element confers responsiveness of protein kinase A to a heterologous promoter in transfection assays in Caco-2 cells. Gel-retardation assays revealed that the mit.HMG-CoA synthase CRE binds to recombinant CREB. The shifted band obtained with the putative mit.HMG-CoA synthase CRE sequence and nuclear proteins from Caco-2 cells competed with CRE sequences of other genes such as somatostatin and phosphoenolpyruvate carboxykinase. We conclude that the regulation of the expression of the gene for mit.HMG-CoA synthase in Caco-2 cells by cAMP is mediated by a CRE sequence in the promoter.

cAMP, an Activator of Protein Kinase A, Suppresses the Expression of Sonic Hedgehog

1996 ◽  
Vol 219 (1) ◽  
pp. 180-185 ◽  
Author(s):  
Alexander Noveen ◽  
Ting-Xin Jiang ◽  
Cheng-Ming Chuong
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Sonic Hedgehog ◽  
Kinase A

scholarly journals Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development

Cell ◽  
1995 ◽  
Vol 80 (4) ◽  
pp. 553-562 ◽  
Author(s):  
Willis Li ◽  
Johanna Talavera Ohlmeyer ◽  
Mary Ellen Lane ◽  
Daniel Kalderon
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Imaginal Disc ◽  
Hedgehog Signal Transduction ◽  
Hedgehog Signal ◽  
Kinase A

Two distinct caveolin-1 domains mediate the functional interaction of caveolin-1 with protein kinase A

2001 ◽  
Vol 281 (4) ◽  
pp. C1241-C1250 ◽  
Author(s):  
Babak Razani ◽  
Michael P. Lisanti
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Wild Type ◽  
Functional Interaction ◽  
Caveolin 1 ◽  
Kinase A

Numerous components of the cAMP-based signaling cascade, namely G-proteins and G- protein coupled receptors, adenylyl cyclase, and protein kinase A (PKA) have been localized to caveolae and shown to be regulated by the caveolar marker proteins, the caveolins. In order to gain mechanistic insights into these processes in vivo, we have assessed the functional interaction of caveolin-1 (Cav-1) with PKA using mutational analysis. As two regions of Cav-1 had previously been implicated in PKA signaling in vitro, we constructed Cav-1 molecules with mutations/deletions in one or both of these domains. Examination of these mutants shows that Cav-1 requires the presence of either the scaffolding domain or the COOH-terminal domain (but not both) to functionally interact with and inhibit PKA. Interestingly, in contrast to the wild-type protein, these Cav-1 mutants are not localized to caveolae microdomains. However, upon coexpression with wild-type Cav-1, a substantial amount of the mutants was recruited to the caveolae membrane fraction. Using the Cav-1 double mutant with both disrupted scaffolding and COOH-terminal domains, we show that wild-type Cav-1's inhibition of PKA signaling can be partially abrogated in a dose-responsive manner; i.e., the mutant acts in a dominant-negative fashion. Thus, this dominant-negative caveolin-1 mutant will be extremely valuable for assessing the functional role of endogenous caveolin-1 in regulating a variety of other signaling cascades.

Sign in / Sign up

Export Citation Format

Share Document