scholarly journals AKAP79 Orchestrates a Cyclic AMP Signalosome Adjacent to Orai1 Ca2+ Channels

Function ◽  
2021 ◽  
Author(s):  
Pulak Kar ◽  
Pradeep Barak ◽  
Anna Zerio ◽  
Yu-Ping Lin ◽  
Amy J Parekh ◽  
...  

Abstract To avoid conflicting and deleterious outcomes, eukaryotic cells often confine signalling molecules to spatially restricted sub-compartments. The smallest signalling unit is the Ca2+ nanodomain, forming near open Ca2+ channels. Ca2+ nanodomains near store-operated Orai1 channels stimulate calcineurin, which activates the transcription factor NFAT1, and both enzyme and target are initially attached to the plasma membrane through the scaffolding protein AKAP79. Here we show that a cAMP signalling nexus also forms adjacent to Orai1. Protein kinase A and phosphodiesterase 4, an enzyme that rapidly breaks down cAMP, both associate with AKAP79 and realign close to Orai1 after stimulation. PCR and mass spectrometry failed to show expression of Ca2+-activated adenylyl cyclase 8 in HEK293 cells, whereas the enzyme was observed in neuronal cell lines. FRET and biochemical measurements of bulk cAMP and protein kinase A activity consistently failed to show an increase in adenylyl cyclase activity following even a large rise in cytosolic Ca2+. Furthermore, expression of AKAP79-CUTie, a cAMP FRET sensor tethered to AKAP79, did not report a rise in cAMP after stimulation, despite AKAP79 association with Orai1. Hence HEK293 cells do not express functionally active Ca2+-activated adenylyl cyclases including adenylyl cyclase 8. Our results show that two ancient second messengers are independently generated in nanodomains close to Orai1 Ca2+ channels.

2021 ◽  
Vol 8 (1) ◽  
pp. 23-31
Author(s):  
Jefrin Ahmed ◽  
Judith Mary Lamo ◽  
Baphilinia Jones Mylliemngap

Protein kinases are key regulators of cell function that constitute one of the largest and most functionally diverse gene families. By adding phosphate groups to substrate proteins, they direct the activity, localization and overall function of many proteins, and serve to orchestrate the activity of almost all cellular processes. The main protein kinases consist of protein kinase A (PKA), protein kinase B (PKB), and protein kinase C (PKC) and are distinguished from each other by the different intracellular second messengers involved in their regulation and by the selective substrates they use. They all have a binding site for Mg2+-ATP (phosphate donor) and for substrate protein as well as various regulatory sites. We formulated to compare the binding capacity of protein kinases at the active site to allosteric sites. By comparing the active site and allosteric site of the protein kinases – A, B and C, using molecular docking it was found that in most of the cases the binding energy is high when an inhibitor binds to an active site as compared to the allosteric site. This comparison gave us an understanding of the interaction and inhibition of compounds to protein kinases in order to inhibit the activity of protein kinase A, B and C. It was concluded that for inhibiting the protein kinase function such as cell division and proliferation, binding of inhibitor to the allosteric site will be more effective.


2019 ◽  
Vol 93 (8) ◽  
pp. 2279-2294
Author(s):  
Romina Belén Andermatten ◽  
Nadia Ciriaci ◽  
Virginia Soledad Schuck ◽  
Nicolás Di Siervi ◽  
María Valeria Razori ◽  
...  

2010 ◽  
Vol 299 (4) ◽  
pp. H1146-H1152 ◽  
Author(s):  
Meera Sridharan ◽  
Shaquria P. Adderley ◽  
Elizabeth A. Bowles ◽  
Terrance M. Egan ◽  
Alan H. Stephenson ◽  
...  

Erythrocytes release ATP in response to exposure to the physiological stimulus of lowered oxygen (O2) tension as well as pharmacological activation of the prostacyclin receptor (IPR). ATP release in response to these stimuli requires activation of adenylyl cyclase, accumulation of cAMP, and activation of protein kinase A. The mechanism by which ATP, a highly charged anion, exits the erythrocyte in response to lowered O2 tension or receptor-mediated IPR activation by iloprost is unknown. It was demonstrated previously that inhibiting pannexin 1 with carbenoxolone inhibits hypotonically induced ATP release from human erythrocytes. Here we demonstrate that three structurally dissimilar compounds known to inhibit pannexin 1 prevent ATP release in response to lowered O2 tension but not to iloprost-induced ATP release. These results suggest that pannexin 1 is the conduit for ATP release from erythrocytes in response to lowered O2 tension. However, the identity of the conduit for iloprost-induced ATP release remains unknown.


2006 ◽  
Vol 20 (9) ◽  
pp. 2231-2246 ◽  
Author(s):  
Arturo E. Gonzalez-Iglesias ◽  
Yonghua Jiang ◽  
Melanija Tomić ◽  
Karla Kretschmannova ◽  
Silvana A. Andric ◽  
...  

Abstract Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+ influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenyltio)-2′-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.


2021 ◽  
Vol 17 (8) ◽  
pp. e1009861
Author(s):  
Kyunghun Min ◽  
Thomas F. Jannace ◽  
Haoyu Si ◽  
Krishna R. Veeramah ◽  
John D. Haley ◽  
...  

Microbial pathogens grow in a wide range of different morphologies that provide distinct advantages for virulence. In the fungal pathogen Candida albicans, adenylyl cyclase (Cyr1) is thought to be a master regulator of the switch to invasive hyphal morphogenesis and biofilm formation. However, faster growing cyr1Δ/Δ pseudorevertant (PR) mutants were identified that form hyphae in the absence of cAMP. Isolation of additional PR mutants revealed that their improved growth was due to loss of one copy of BCY1, the negative regulatory subunit of protein kinase A (PKA) from the left arm of chromosome 2. Furthermore, hyphal morphogenesis was improved in some of PR mutants by multigenic haploinsufficiency resulting from loss of large regions of the left arm of chromosome 2, including global transcriptional regulators. Interestingly, hyphal-associated genes were also induced in a manner that was independent of cAMP. This indicates that basal protein kinase A activity is an important prerequisite to induce hyphae, but activation of adenylyl cyclase is not needed. Instead, phosphoproteomic analysis indicated that the Cdc28 cyclin-dependent kinase and the casein kinase 1 family member Yck2 play key roles in promoting polarized growth. In addition, integrating transcriptomic and proteomic data reveals hyphal stimuli induce increased production of key transcription factors that contribute to polarized morphogenesis.


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