Layers of organization of cAMP microdomains in a simple cell

2006 ◽  
Vol 34 (4) ◽  
pp. 480-483 ◽  
Author(s):  
A.C.L. Martin ◽  
D.M.F. Cooper
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Simple Cell ◽  
Protein Protein Interactions ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Phosphodiesterase 4 ◽  
293 Cells ◽  
Phosphatase 2A ◽  
Dependent Protein

Based on a variety of single-cell measurements, the notion that cAMP microdomains exist in cells is being increasingly embraced. The cellular and molecular underpinnings of this organization are also steadily being revealed. A dependence of Ca2+-sensitive ACs (adenylate cyclases) in HEK-293 cells (human embryonic kidney cells) on capacitative Ca2+ entry is enforced by their presence in lipid rafts and protein–protein interactions. In these cells, many of the participants in the cAMP cascade, including AC, phosphodiesterase 4, cAMP-dependent protein kinase [PKA (protein kinase A)] and protein phosphatase 2A, are now seen to be involved in higher order assemblies. Moreover, the presence of Na+/H+ exchanger 1 in these domains creates a microclimate, protected against global swings in cellular pH. The Ca2+-stimulatable AC8, which is targeted to these regions, can sequester calmodulin for its own regulatory purposes. These devices are a sampling of the multiple layers of organization that are in place – even in a simple cell – to ensure faithful and economical communication of the cAMP message.

Phosphodiesterase-4 gates the ability of protein kinase A to phosphorylate G-protein receptor kinase-2 and influence its translocation

2006 ◽  
Vol 34 (4) ◽  
pp. 474-475 ◽  
Author(s):  
M.D. Houslay ◽  
G.S. Baillie
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Fluorescent Protein ◽  
Resting Cells ◽  
Receptor Kinase ◽  
Human Embryonic Kidney Cells ◽  
Phosphodiesterase 4 ◽  
Protein Receptor ◽  
Dependent Protein ◽  
Green Fluorescent

Challenge of the β2Ar (β2-adrenergic receptor) with isoprenaline in HEK-293β2 cells (human embryonic kidney cells stably overexpressing a FLAG- and green fluorescent protein-tagged β2Ar) results in the PKA (cAMP-dependent protein kinase) phosphorylation of GRK2 (G-protein receptor kinase-2). This response was enhanced when PDE4 (phosphodiesterase-4) activity was attenuated using either rolipram, a PDE4-selective inhibitor, or with siRNA (small interfering RNA) knockdown of both PDE4B and PDE4D. Rolipram also facilitated GRK2 recruitment to the membrane and phosphorylation of the β2Ar by GRK2 in response to isoprenaline challenge of cells. In resting cells, rolipram treatment alone is sufficient to promote PKA phosphorylation of GRK2, with consequential effects on GRK2 translocation and GRK2 phosphorylation of the β2Ar. Similar effects are observed in cardiac myocytes. We propose that PDE4 activity protects GRK2 from inappropriate phosphorylation by PKA in resting cells that might have occurred through fluctuations in basal cAMP levels. Thus PDE4 gates the action of PKA to phosphorylate GRK2.

Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14-3-3 binding

2008 ◽  
Vol 410 (1) ◽  
pp. 195-204 ◽  
Author(s):  
Ingeborg Winge ◽  
Jeffrey A. Mckinney ◽  
Ming Ying ◽  
Clive S. D'Santos ◽  
Rune Kleppe ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tryptophan Hydroxylase ◽  
Enzyme Stability ◽  
Enzyme Activation ◽  
Site Directed Mutagenesis ◽  
Peripheral Tissues ◽  
Hek 293 ◽  
Rate Limiting Step ◽  
293 Cells ◽  
Dependent Protein

TPH (tryptophan hydroxylase) catalyses the rate-limiting step in the synthesis of serotonin, and exists in two isoforms: TPH1, mainly found in peripheral tissues and the pineal body, and TPH2, a neuronal form. In the present study human TPH2 was expressed in Escherichia coli and in HEK (human embryonic kidney)-293 cells and phosphorylated using several different mammalian protein kinases. TPH2 was rapidly phosphorylated to a stoichiometry of 2 mol of phosphate/mol of subunit by PKA (protein kinase A), but only to a stoichiometry of 0.2 by Ca2+/calmodulin dependent protein kinase II. Both kinases phosphorylated Ser19, but PKA also phosphorylated Ser104, as determined by MS, phosphospecific antibodies and site-directed mutagenesis of several possible phosphorylation sites, i.e. Ser19, Ser99, Ser104 and Ser306. On average, purified TPH2 WT (wild-type) was activated by 30% after PKA phosphorylation and studies of the mutant enzymes showed that enzyme activation was mainly due to phosphorylation at Ser19. This site was phosphorylated to a stoichiometry of up to 50% in HEK-293 cells expressing TPH2, and the enzyme activity and phosphorylation stoichiometry was further increased upon treatment with forskolin. Purified PKA-phosphorylated TPH2 bound to the 14-3-3 proteins γ, ϵ and BMH1 with high affinity, causing a further increase in enzyme stability and activity. This indicates that 14-3-3 proteins could play a role in consolidating and strengthening the effects of phosphorylation on TPH2 and that they may be important for the regulation of serotonin function in the nervous system.

scholarly journals Molecular mechanism of TMEM16A regulation: role of CaMKII and PP1/PP2A

2019 ◽  
Vol 317 (6) ◽  
pp. C1093-C1106 ◽  
Author(s):  
Ramon J. Ayon ◽  
Matthew B. Hawn ◽  
Joydeep Aoun ◽  
Michael Wiwchar ◽  
Abigail S. Forrest ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Inhibitory Peptide ◽  
Dependent Protein Kinase ◽  
Initial Current ◽  
Pipette Solution ◽  
Hek 293 ◽  
293 Cells ◽  
Dependent Protein

This study explored the mechanism by which Ca2+-activated Cl− channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl− currents ( IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCa were evoked using a pipette solution in which free Ca2+ concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. IClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCa recorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of IClCa by CaMKII.

Subcellular Localization of the Type II cAMP-Dependent Protein Kinase

Physiology ◽  
1992 ◽  
Vol 7 (4) ◽  
pp. 143-148 ◽  
Author(s):  
JD Scott ◽  
DW Carr
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Regulatory Subunit ◽  
Type Ii ◽  
Protein Protein Interactions ◽  
Dependent Protein Kinase ◽  
Biochemical Effects ◽  
Camp Dependent Protein Kinase ◽  
Anchoring Proteins ◽  
Dependent Protein

Diverse biochemical effects of different neurotransmitters or hormones that stimulate cAMP production may occur through activation of compartmentalized pools of cAMP-dependent protein kinase (PKA). Evidence suggests that compartmentalization of type II PKA is maintained through protein-protein interactions between the regulatory subunit and specific anchoring proteins.

Regulation of the exocytotic machinery by cAMP-dependent protein kinase: implications for presynaptic plasticity

2003 ◽  
Vol 31 (4) ◽  
pp. 824-827 ◽  
Author(s):  
G.J.O. Evans ◽  
A. Morgan
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Cysteine String Protein ◽  
Presynaptic Plasticity ◽  
Dependent Protein

For over a decade, the enhancement of regulated exocytosis by cAMP-dependent protein kinase (PKA) has remained unexplained at the molecular level. The fact that this phenomenon has been observed in such a wide variety of secretory cell types, from pancreatic β-cells to neurons, suggests that it is an important and fundamental mechanism. Extensive analysis of the phosphorylation of exocytotic proteins has yielded few substrates of PKA in vitro, and fewer still have had physiological effects attributed to their phosphorylation. Here we review two proteins that do fulfil these criteria: the synaptic vesicle proteins cysteine string protein (CSP) and Snapin. Phosphorylation of these proteins by PKA produces changes in their respective protein–protein interactions, and has been attributed to modulation of the vesicle priming (Snapin) and vesicle fusion (CSP) stages of exocytosis. We also discuss how the function of CSP and Snapin phosphorylation might fit into an interesting aspect of the PKA-dependent enhancement of exocytosis: presynaptic plasticity in the brain.

scholarly journals Effects of Single-Nucleotide Polymorphisms in Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2): A Comprehensive Study

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Zoya Khalid ◽  
Omar Almaghrabi
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Bipolar Disorders ◽  
Docking Studies ◽  
Nucleotide Polymorphisms ◽  
Protein Protein Interactions ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Healthy Activity ◽  
Protein Kinase Kinase

Calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a protein kinase that belongs to the serine/threonine kinase family. It phosphorylates kinases like CAMK1, CAMK2, and AMP, and this signaling cascade is involved in various biological processes including cell proliferation, apoptosis, and proliferation. Also, the CAMKK2 signaling activity is required for the healthy activity of the brain which otherwise can cause diseases like bipolar disorders and anxiety. The current study is based on in silico bioinformatics analysis that combines sequence- and structure-based predictions to mark a SNP as damaging or neutral. The combined results from sequence-based, evolutionary conservation-based, and consensus-based tools have predicted a total of 18 nsSNPs as deleterious, and these nsSNPs were further subjected to structure-based analysis. The six mutant models of V195A, V249M, R311C, F366Y, P389T, and W445C showed a higher deviation from the wildtype protein model and hence were further taken for docking studies. The molecular docking analysis has predicted that these mutations will also be disruptive to the protein-protein interactions between CAMKK2 and PRKAG1 which will create an evident reduction in the kinase activity. The current study has enlightened us that a few of the significant mutations are prime candidates in CAMKK2 which could be the fundamental cause of various bipolar and psychiatric disorders. This is the first detailed study that predicts the deleterious nsSNPs in CAMKK2 and contributes positively in providing a better understanding of disease mechanisms.

scholarly journals Protein interaction landscapes revealed by advanced in vivo cross-linking–mass spectrometry

2021 ◽  
Vol 118 (32) ◽  
pp. e2023360118
Author(s):  
Andrew Wheat ◽  
Clinton Yu ◽  
Xiaorong Wang ◽  
Anthony M. Burke ◽  
Ilan E. Chemmama ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Cross Linking ◽  
Technological Advancement ◽  
Protein Protein Interactions ◽  
Hek 293 ◽  
293 Cells ◽  
And Function

Defining protein–protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking–mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platform to facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry–based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine–lysine linkages from in vivo cross-linked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.

scholarly journals Characterization of A-kinase-anchoring disruptors using a solution-based assay

2006 ◽  
Vol 400 (3) ◽  
pp. 493-499 ◽  
Author(s):  
Anne J. Stokka ◽  
Frank Gesellchen ◽  
Cathrine R. Carlson ◽  
John D. Scott ◽  
Friedrich W. Herberg ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Regulatory Subunits ◽  
Anchoring Proteins ◽  
Ic50 Values ◽  
Order Of Magnitude ◽  
Dependent Protein

Subcellular localization of PKA (cAMP-dependent protein kinase or protein kinase A) is determined by protein–protein interactions between its R (regulatory) subunits and AKAPs (A-kinase-anchoring proteins). In the present paper, we report the development of the Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen™) as a means to characterize AKAP-based peptide competitors of PKA anchoring. In this assay, the prototypic anchoring disruptor Ht31 efficiently competed in RIIα isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4±0.2 nM and 6±1 nM respectively). In contrast, RIα isoform binding to a dual-specific AKAP was less efficiently competed (IC50 of 156±10 nM). Characterization of two RI-selective anchoring disruptors, RIAD (RI-anchoring disruptor) and PV-38 revealed that RIAD (IC50 of 13±1 nM) was 20-fold more potent than PV-38 (IC50 of 304±17 nM) and did not compete in the RIIα–AKAP interaction. We also observed that the kinetics of RII displacement from pre-formed PKA–AKAP complexes and competition of RII–AKAP complex formation by Ht31 differed by an order of magnitude when the component parts were mixed in vitro. No such difference in potency was seen for RIα–AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA–AKAP complexes.

Sign in / Sign up

Export Citation Format

Share Document