In vitro regulation of serotonin transporter activity by protein kinase A and nicotinic acetylcholine receptors in the prefrontal cortex of rats

Synapse ◽  
2006 ◽  
Vol 59 (6) ◽  
pp. 342-349 ◽  
Author(s):  
Tammy L. Awtry ◽  
Julie G. Frank ◽  
Linda L. Werling
Keyword(s):  
Prefrontal Cortex ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Serotonin Transporter ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Nicotinic Acetylcholine ◽  
Kinase A

scholarly journals Evaluation of the Phosphoproteome of Mouse Alpha 4/Beta 2-Containing Nicotinic Acetylcholine Receptors In Vitro and In Vivo

Proteomes ◽  
2018 ◽  
Vol 6 (4) ◽  
pp. 42 ◽  
Author(s):  
Megan Miller ◽  
Rashaun Wilson ◽  
TuKiet Lam ◽  
Angus Nairn ◽  
Marina Picciotto
Keyword(s):  
Protein Kinase ◽  
Mouse Brain ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Mammalian Brain ◽  
Phosphorylation Sites ◽  
Nicotinic Acetylcholine ◽  
Hek Cells

Activation of nicotinic acetylcholine receptors containing α4 and β2 subunits (α4/β2* nAChRs) in the mammalian brain is necessary for nicotine reinforcement and addiction. We previously identified interactions between α4/β2* nAChRs and calcium/calmodulin-dependent protein kinase II (CaMKII) in mouse and human brain tissue. Following co-expression of α4/β2 nAChR subunits with CaMKII in HEK cells, mass spectrometry identified 8 phosphorylation sites in the α4 subunit. One of these sites and an additional site were identified when isolated α4/β2* nAChRs were dephosphorylated and subsequently incubated with CaMKII in vitro, while 3 phosphorylation sites were identified following incubation with protein kinase A (PKA) in vitro. We then isolated native α4/β2* nAChRs from mouse brain following acute or chronic exposure to nicotine. Two CaMKII sites identified in HEK cells were phosphorylated, and 1 PKA site was dephosphorylated following acute nicotine administration in vivo, whereas phosphorylation of the PKA site was increased back to baseline levels following repeated nicotine exposure. Significant changes in β2 nAChR subunit phosphorylation were not observed under these conditions, but 2 novel sites were identified on this subunit, 1 in HEK cells and 1 in vitro. These experiments identified putative CaMKII and PKA sites on α4/β2* nAChRs and novel nicotine-induced phosphorylation sites in mouse brain that can be explored for their consequences on receptor function.

scholarly journals Activity, expression and function of a second Drosophila protein kinase A catalytic subunit gene.

Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1507-1520 ◽  
Author(s):  
A Meléndez ◽  
W Li ◽  
D Kalderon
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Essential Gene ◽  
Sequence Similarity ◽  
Catalytic Subunit ◽  
Subunit Gene ◽  
Drosophila Protein ◽  
Pka Catalytic Subunit ◽  
Kinase A

Abstract The DC2 gene was isolated previously on the basis of sequence similarity to DC0, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development.

scholarly journals NMR Structure and Action on Nicotinic Acetylcholine Receptors of Water-soluble Domain of Human LYNX1

2011 ◽  
Vol 286 (12) ◽  
pp. 10618-10627 ◽  
Author(s):  
Ekaterina N. Lyukmanova ◽  
Zakhar O. Shenkarev ◽  
Mikhail A. Shulepko ◽  
Konstantin S. Mineev ◽  
Dieter D'Hoedt ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Nmr Structure ◽  
Water Soluble ◽  
Gpi Anchor ◽  
Nicotinic Acetylcholine ◽  
Brain Functions ◽  
The Central Nervous System ◽  
High Concentrations

Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake α-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5–30 μm, ws-LYNX1 competed with 125I-α-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing α7 nAChRs to 1 μm ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on α4β2 and α3β2 nAChRs. Increasing ws-LYNX1 concentration to 10 μm caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.

scholarly journals Membrane-Bound Protein Kinase A Inhibits Smooth Muscle Cell Proliferation In Vitro and In Vivo by Amplifying cAMP–Protein Kinase A Signals

2001 ◽  
Vol 88 (3) ◽  
pp. 319-324 ◽  
Author(s):  
Ciro Indolfi ◽  
Eugenio Stabile ◽  
Carmela Coppola ◽  
Adriana Gallo ◽  
Cinzia Perrino ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Smooth Muscle Cell ◽  
Protein Kinase A ◽  
Membrane Bound ◽  
Proliferation In Vitro ◽  
Kinase A

scholarly journals The cross-regulation of Gi-protein by cholera toxin involves a phosphorylation by protein kinase A

1995 ◽  
Vol 306 (3) ◽  
pp. 765-769 ◽  
Author(s):  
R Levistre ◽  
M Berguerand ◽  
G Bereziat ◽  
J Masliah
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Cholera Toxin ◽  
Inhibitory Effect ◽  
Negative Control ◽  
Adp Ribosylation ◽  
Gi Proteins ◽  
Kinase A

Pretreatment of alveolar macrophages with cholera toxin inhibits the release of arachidonic acid induced by the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine. The results presented here show that cholera toxin might exert its inhibitory effect through the phosphorylation of Gi alpha by protein kinase A (PKA). (1) Gi-proteins from cells pretreated with cholera toxin showed parallel increases in their sensitivity to ADP-ribosylation by toxins in vitro and in Gi alpha phosphorylation. By contrast, the Gi alpha concentration was unchanged. (2) Cholera toxin pretreatment also decreased the functional activity of Gi, as assessed by the inhibition (80%) of agonist-induced binding of guanosine-5′-[gamma-thio]triphosphate (GTP[gamma S]). (3) These effects of cholera toxin were blocked by a specific PKA inhibitor, N-(2-[methyl-amino]ethyl)-3-isoquinolinesulphonamide dihydrochloride (H8) and mimicked by a cyclic AMP (cAMP) analogue and a phosphatase inhibitor. (4) Gi alpha was also phosphorylated in vitro by the catalytic subunit of PKA. In contrast with other cell systems, the stimulation of protein kinase C seems to have no effect on the sensitivity of Gi to ADP-ribosylation or on its phosphorylation. Therefore, the phosphorylation of Gi-proteins by PKA seems to be the actual target of the negative control of arachidonic acid release via the cAMP-mediated pathway.

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