scholarly journals Synapsin is required for dense core vesicle capture and cAMP-dependent neuropeptide release

2019 ◽  
Author(s):  
Szi-chieh Yu ◽  
Wagner Steuer Costa ◽  
Jana F. Liewald ◽  
Jiajie Shao ◽  
Alexander Gottschalk
Keyword(s):  
Electron Microscopy ◽  
Protein Kinase ◽  
Motor Neurons ◽  
Phosphorylation Site ◽  
Dense Core ◽  
Behavioral Analysis ◽  
Dense Core Vesicle ◽  
Neuropeptide Release ◽  
Photoactivated Adenylyl Cyclase ◽  
Kinase A

ABSTRACTRelease of neuropeptides from dense core vesicles (DCVs) is important for neuromodulation. By optogenetics, behavioral analysis, electrophysiology, and electron microscopy, we show that synapsin SNN-1 is required for cAMP-dependent neuropeptide release in Caenorhabditis elegans cholinergic motor neurons. In synapsin mutants, behaviors induced by the photoactivated adenylyl cyclase bPAC, which we previously showed to depend on acetylcholine and neuropeptides, are altered like in animals with reduced cAMP. While synapsin mutants have slight alterations in synaptic vesicle distribution, DCVs were affected much more: DCVs were ~30% reduced in synaptic terminals, and not released following bPAC stimulation. Imaging axonal DCV trafficking, also in genome-engineered mutants in the serine-9 protein kinase A phosphorylation site, showed that synapsin captures DCVs at synapses, making them available for release. In non-phosphorylatable SNN-1B(S9A) mutants, DCVs traffic less and accumulate, likely by enhanced tethering to the actin cytoskeleton. Our work establishes synapsin as a key mediator of neuropeptide release.

Four distinct and unusual linker proteins in a mitotically dividing nucleus are derived from a 71-kilodalton polyprotein, lack p34cdc2 sites, and contain protein kinase A sites

1994 ◽  
Vol 14 (1) ◽  
pp. 10-20
Author(s):  
M Wu ◽  
C D Allis ◽  
M T Sweet ◽  
R G Cook ◽  
T H Thatcher ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Phosphorylation Site ◽  
High Mobility ◽  
Single Copy ◽  
Amino Acid Sequences ◽  
Evolutionary Analysis ◽  
Associated Proteins ◽  
Kinase Phosphorylation ◽  
Kinase A

Tetrahymena thermophila micronuclei contain four linker-associated proteins, alpha, beta, gamma, and delta. Synthetic oligonucleotides based on N-terminal protein sequences of beta and gamma were used to clone the micronuclear linker histone (MLH) gene. The MLH gene is single copy and is transcribed into a 2.4-kb message encoding all four linker-associated proteins. The message is translated into a polypeptide (Mic LH) that is processed at the sequence decreases RTK to give proteins whose amino acid sequences differ markedly from each other, from the sequence of macronuclear H1, and from sequences of typical H1s of other organisms. This represents the first example of multiple chromatin proteins derived from a single polyprotein. The delta protein consists largely of two high-mobility-group (HMG) boxes. An evolutionary analysis of HMG boxes indicates that the delta HMG boxes are similar to the HMG boxes of tsHMG, a protein that appears in elongating mouse spermatids when they condense and cease transcription, suggesting that delta could play a similar role in the micronucleus. The micronucleus divides mitotically, while the macronucleus divides amitotically. Surprisingly, macronuclear H1 but not Mic LH contains sequences resembling p34cdc2 kinase phosphorylation sites, while each of the Mic LH-derived proteins contains a typical protein kinase A phosphorylation site in its carboxy terminus.

scholarly journals Endogenous cleavage of the Arg-379-Ala-380 bond in vitronectin results in a distinct conformational change which ‘buries’ Ser-378, its site of phosphorylation by protein kinase A

1991 ◽  
Vol 274 (2) ◽  
pp. 387-394 ◽  
Author(s):  
D Chain ◽  
B Korc-Grodzicki ◽  
T Kreizman ◽  
S Shaltiel
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Blood Platelets ◽  
Phosphorylation Site ◽  
Factor Xa ◽  
Heparin Binding ◽  
Recognition Sequence ◽  
The One ◽  
Chain Form ◽  
Kinase A

Activation of blood platelets by thrombin was previously shown to specifically release protein kinase A, which in human plasma singles out and phosphorylates one protein, identified as vitronectin. This protein is known to be involved in processes that follow platelet stimulation, specifically, in the binding of heparin (interfering with the heparin-mediated inhibition of thrombin and Factor Xa by antithrombin III), in the growth of endothelial cells and in fibrinolysis. This paper shows that phosphorylation of vitronectin by protein kinase A is stoichiometric (approx. 1 mol/mol), that it is targeted to one site (Ser-378) at the C-terminal edge of the heparin-binding domain, and that it distinguishes between the two physiologically occurring forms of vitronectin: the one-chain (75 kDa) form, and the nicked two-chain (65 + 10 kDa) form, held together by an interchain disulphide bridge. Protein kinase A phosphorylates the one-chain form but not the two-chain form, although Ser-378 and the complete recognition sequence of the kinase are still present in the clipped 65 kDa chain. Cleavage of the Arg-379-Ala-380 bond results therefore in a conformationally distinct form of vitronectin in which Ser-378 is ‘buried’. This is demonstrated by our finding that Ser-378 is present in the 65 kDa chain of clipped vitronectin but inaccessible to phosphorylation at physiological pH. Upon binding heparin, the phosphorylation site becomes exposed and able to undergo a stoichiometric phosphorylation at physiological pH.

Mutation of the protein kinase A phosphorylation site influences the anti-proliferative activity of mitofusin 2

2010 ◽  
Vol 211 (1) ◽  
pp. 216-223 ◽  
Author(s):  
Wei Zhou ◽  
Kuang-Hueih Chen ◽  
Wenjing Cao ◽  
Jingwei Zeng ◽  
Hua Liao ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Proliferative Activity ◽  
Phosphorylation Site ◽  
Mitofusin 2 ◽  
Kinase A

scholarly journals Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

2011 ◽  
Vol 300 (5) ◽  
pp. C989-C997 ◽  
Author(s):  
Pimthanya Wanichawan ◽  
William E. Louch ◽  
Kristin H. Hortemo ◽  
Bjørg Austbø ◽  
Per Kristian Lunde ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Phosphorylation Site ◽  
Full Length ◽  
Kinase A

The cardiac Na+/Ca2+ exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-32P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.

scholarly journals Synapsin is required for dense core vesicle capture and cAMP-dependent neuropeptide release

2021 ◽  
pp. JN-RM-2631-20
Author(s):  
Szi-chieh Yu ◽  
Jana F. Liewald ◽  
Jiajie Shao ◽  
Wagner Steuer Costa ◽  
Alexander Gottschalk
Keyword(s):  
Dense Core ◽  
Dense Core Vesicle ◽  
Neuropeptide Release

scholarly journals TSH Regulates Pendrin Membrane Abundance and Enhances Iodide Efflux in Thyroid Cells

Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 512-521 ◽  
Author(s):  
Liuska Pesce ◽  
Aigerim Bizhanova ◽  
Juan Carlos Caraballo ◽  
Whitney Westphal ◽  
Maria L. Butti ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Phosphorylation Site ◽  
Physiological Role ◽  
Thyroid Cells ◽  
Hormone Synthesis ◽  
Iodide Transport ◽  
Rat Thyroid ◽  
Kinase A

Thyroid hormones are essential for normal development and metabolism. Their synthesis requires transport of iodide into thyroid follicles. The mechanisms involving the apical efflux of iodide into the follicular lumen are poorly elucidated. The discovery of mutations in the SLC26A4 gene in patients with Pendred syndrome (congenital deafness, goiter, and defective iodide organification) suggested a possible role for the encoded protein, pendrin, as an apical iodide transporter. We determined whether TSH regulates pendrin abundance at the plasma membrane and whether this influences iodide efflux. Results of immunoblot and immunofluorescence experiments reveal that TSH and forskolin rapidly increase pendrin abundance at the plasma membrane through the protein kinase A pathway in PCCL-3 rat thyroid cells. The increase in pendrin membrane abundance correlates with a decrease in intracellular iodide as determined by measuring intracellular 125iodide and can be inhibited by specific blocking of pendrin. Elimination of the putative protein kinase A phosphorylation site T717A results in a diminished translocation to the membrane in response to forskolin. These results demonstrate that pendrin translocates to the membrane in response to TSH and suggest that it may have a physiological role in apical iodide transport and thyroid hormone synthesis.

Endoplasmic reticulum contribution to the relaxant effect of cGMP- and cAMP-elevating agents in feline aorta

2000 ◽  
Vol 278 (6) ◽  
pp. H1856-H1865 ◽  
Author(s):  
Cecilia Mundiña-Weilenmann ◽  
Leticia Vittone ◽  
Gustavo Rinaldi ◽  
Matilde Said ◽  
Gladys Chiappe de Cingolani ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Protein Kinase G ◽  
Site Specific ◽  
Relaxant Effect ◽  
Specific Antibodies ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Kinase A

The contribution of endoplasmic reticulum (ER) and phosphorylation of phospholamban (PLB) to the relaxant effect of cGMP- and cAMP-elevating agents was studied in feline aorta. Sodium nitroprusside (NP, 100 μM) completely relaxed contracture induced by 10 μM norepinephrine. This NP-induced relaxation was partially prevented by tetraethylammonium, suggesting that a fraction of NP-induced relaxation was mediated by activation of K+ channels. In the absence and presence of tetraethylammonium, the relaxant effect of NP was associated with a significant increase in Ser16 phosphorylation of PLB immunodetected by phosphorylation site-specific antibodies. The relaxant effect of NP on aortic strips precontracted with 80 mM KCl was significantly reduced by 1 μM thapsigargin. This decrease, which represents the ER contribution to the relaxant effect of NP, reached 23 ± 9% at 100 μM NP and was closely associated with a dose-dependent increase in Ser16 phosphorylation (128 ± 49% over control at 100 μM NP). Effects of NP were associated with a significant increase in activity of protein kinase G and were mimicked by 8-bromo-cGMP. Forskolin produced a dose-dependent relaxant effect on KCl-induced contracture, which reached 64 ± 8% at 50 μM and was associated with an increase in phosphorylation of Ser16residue of PLB (88 ± 18% over control). Thapsigargin reduced this relaxant effect by 38 ± 9%. 8-Bromo-cAMP mimicked effects of forskolin. The ER-mediated relaxant effect and the increase in Ser16 phosphorylation produced by forskolin were partially blocked by the protein kinase A inhibitor H-89 (5 μM). The results indicate that ER partially contributes to the relaxant effect of NP and forskolin in feline aorta. This effect may be mediated by the associated increase in Ser16 phosphorylation of PLB.

scholarly journals Alternate splicing in human Na+-MI cotransporter gene yields differentially regulated transport isoforms

1999 ◽  
Vol 276 (6) ◽  
pp. C1325-C1337 ◽  
Author(s):  
Francesca Porcellati ◽  
Yoshiyuki Hosaka ◽  
Tommy Hlaing ◽  
Masaki Togawa ◽  
Dennis D. Larkin ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Phosphorylation Site ◽  
Open Reading Frame ◽  
Phosphorylation Sites ◽  
Reading Frame ◽  
Kinase C ◽  
Multiple Protein ◽  
Kinase A

myo-Inositol is a ubiquitous intracellular organic osmolyte and phosphoinositide precursor maintained at millimolar intracellular concentrations through the action of membrane-associated Na+- myo-inositol cotransporters (SMIT). Functional cloning and expression of a canine SMIT cDNA, which conferred SMIT activity in Xenopus oocytes, predicted a 718-amino acid peptide homologous to the Na+-glucose cotransporter with a potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites. A consistent ∼1.0- to 13.5-kb array of transcripts hybridizing with this cDNA are osmotically induced in a variety of mammalian cells and species, yet SMIT activity appears to vary among different tissues and species. An open reading frame on human chromosome 21 (SLC5A3) homologous to that of the canine cDNA (96.5%) is thought to comprise an intronless human SMIT gene. Recently, this laboratory ascribed multiply sized, osmotically induced SMIT transcripts in human retinal pigment epithelial cells to the alternate utilization of several 3′-untranslated SMIT exons. This article describes an alternate splice donor site within the coding region that extends the open reading frame into the otherwise untranslated 3′ exons, potentially generating novel SMIT isoforms. In these isoforms, the last putative transmembrane domain is replaced with intracellular carboxy termini containing a novel potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites, and this could explain the heterogeneity in the regulation and structure of the SMIT.

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