scholarly journals Protein kinase A catalytic-α and catalytic-β proteins have nonredundant regulatory functions

2020 ◽  
Vol 319 (5) ◽  
pp. F848-F862
Author(s):  
Viswanathan Raghuram ◽  
Karim Salhadar ◽  
Kavee Limbutara ◽  
Euijung Park ◽  
Chin-Rang Yang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Genome Editing ◽  
Collecting Duct ◽  
Water Channel ◽  
Cell Junctions ◽  
Target Proteins ◽  
Renal Collecting Duct ◽  
Regulatory Functions ◽  
Kinase A

Vasopressin regulates osmotic water transport in the renal collecting duct by protein kinase A (PKA)-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA-Cα) and PKA catalytic β (PKA-Cβ). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-Cα or PKA-Cβ was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4,635 phosphopeptides that were quantified, 67 phosphopeptides were significantly altered in abundance with PKA-Cα deletion, whereas 21 phosphopeptides were significantly altered in abundance with PKA-Cβ deletion. However, only four sites were changed in both. The target proteins identified in PKA-Cα-null cells were largely associated with cell membranes and membrane vesicles, whereas target proteins in PKA-Cβ-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-Cα and PKA-Cβ resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in in vivo samples showed that PKA-Cα deletion resulted in a near disappearance of AQP2 protein, whereas PKA-Cβ deletion did not decrease AQP2 abundance. We conclude that PKA-Cα and PKA-Cβ serve substantially different regulatory functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g., mediated by A-kinase anchor proteins, C-kinase anchoring proteins, or PDZ binding.

scholarly journals Protein kinase A catalytic-α and catalytic-β proteins have non-redundant functions

2020 ◽  
Author(s):  
Viswanathan Raghuram ◽  
Karim Salhadar ◽  
Kavee Limbutara ◽  
Euijung Park ◽  
Chin-Rang Yang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Protein Interactions ◽  
Collecting Duct ◽  
Water Channel ◽  
Membrane Vesicles ◽  
Cell Junctions ◽  
Target Proteins ◽  
Renal Collecting Duct

ABSTRACTVasopressin regulates osmotic water transport in the renal collecting duct by PKA-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA-Cα) and PKA catalytic β (PKA-Cβ). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-Cα or PKA-Cβ was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure, but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4635 phosphopeptides that were quantified 67 were significantly altered in abundance with PKA-Cα deletion, while 21 were significantly altered in abundance with PKA-Cβ deletion. However, only four sites were changed in both. The target proteins identified in PKA-Cα-null cells were largely associated with cell membranes and membrane vesicles, while target proteins in the PKA-Cβ-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-Cα and PKA-Cβ resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in the in vivo samples showed that PKA-Cα deletion resulted in a near disappearance of AQP2 protein, while PKA-Cβ deletion did not decrease AQP2 abundance. We conclude that PKA-Cα and PKA-Cβ serve substantially different functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g. mediated by AKAP, C-KAP or PDZ binding.

Conductive properties of a rabbit cortical collecting duct cell line: regulation by isoproterenol

1992 ◽  
Vol 262 (4) ◽  
pp. F578-F582 ◽  
Author(s):  
P. Dietl ◽  
N. Kizer ◽  
B. A. Stanton
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cell Line ◽  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Conductive Properties ◽  
Cl Conductance ◽  
Kinase A

The present study was carried out to characterize the membrane conductive properties of RCCT-28A cells, a continuous cell line derived from rabbit cortical collecting duct (CCD). RCCT-28A cells have many phenotypic properties of acid-secreting intercalated cells (A-IC). Using the whole cell patch-clamp technique, we found that the cells are conductive to Cl-, but not to Na+ or K+. The beta-adrenergic agonists isoproterenol (2 x 10(-6) M) and adenosine 3',5'-cyclic monophosphate (cAMP, 10(-4) M) increased the whole cell Cl- conductance. Protein kinase A (150 nM) in the patch pipette (i.e., intracellular solution) also increased whole cell Cl- conductance. Because isoproterenol increases cAMP levels in these cells, we conclude that isoproterenol stimulates the Cl- conductance by increasing cell cAMP, which in turn activates protein kinase A. In contrast, vasopressin does not increase cAMP in these cells and did not increase the Cl- conductance. In conclusion, these experiments show that RCCT-28A cells, like A-IC, are conductive only to Cl-. Thus RCCT-28A cells are a good model with which to study Cl- channels in the collecting duct.

In mpkCCD cells, long-term regulation of aquaporin-2 by vasopressin occurs independent of protein kinase A and CREB but may involve Epac

2012 ◽  
Vol 302 (11) ◽  
pp. F1395-F1401 ◽  
Author(s):  
Marleen L. A. Kortenoeven ◽  
Christiane Trimpert ◽  
Michiel van den Brand ◽  
Yuedan Li ◽  
Jack F. M. Wetzels ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Collecting Duct ◽  
Urine Concentration ◽  
Creb Phosphorylation ◽  
Aquaporin 2 ◽  
Element Binding Protein ◽  
Responsive Element ◽  
Kinase A

Urine concentration involves the hormone vasopressin (AVP), which stimulates cAMP production in renal principal cells, resulting in translocation and transcription of aquaporin-2 (AQP2) water channels, greatly increasing the water permeability, leading to a concentrated urine. As cAMP levels decrease shortly after AVP addition, whereas AQP2 levels still increase and are maintained for days, we investigated in the present study the mechanism responsible for the AQP2 increase after long-term 1-desamino-8-d-arginine vasopressin (dDAVP) application using mouse collecting duct (mpkCCD) cells. While 30 min of dDAVP incubation strongly increased cAMP, cAMP was lower with 1 day and was even further reduced with 4 days of dDAVP, although still significantly higher than in control cells. One day of dDAVP incubation increased AQP2 promoter-dependent transcription, which was blocked by the protein kinase A (PKA) inhibitor H89. Moreover, phosphorylation of the cAMP-responsive element binding protein (CREB) and CRE-dependent transcription was observed after short-term dDAVP stimulation. With 4 days of dDAVP, AQP2 transcription remained elevated, but this was not blocked by H89, and CRE-dependent transcription and CREB phosphorylation were not increased. Exchange factor directly activated by cAMP (Epac) 1 and 2 were found to be endogenously expressed in mpkCCD cells. Application of dDAVP increased the expression of Epac1, while Epac2 was reduced. Incubation with a specific Epac activator after dDAVP pretreatment increased both AQP2 abundance and transcription compared with cells left unstimulated the last day. In conclusion, the PKA-CRE pathway is involved in the initial rise in AQP2 levels after dDAVP stimulation but not in the long-term effect of dDAVP. Instead, long-term regulation of AQP2 may involve the activation of Epac.

scholarly journals Protein Kinase A (PKA) Catalytic Subunits a and b Have Non‐Redundant Functions in Collecting Duct Cells

2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Karim Salhadar ◽  
V. Raghuram ◽  
Chin-Rang Yang ◽  
Kavee Limbutara ◽  
Mark Knepper
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Collecting Duct ◽  
Catalytic Subunits ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Redundant Functions ◽  
Kinase A

Psychotropic drugs upregulate aquaporin-2 via vasopressin-2 receptor/cAMP/protein kinase A signaling in inner medullary collecting duct cells

2021 ◽  
Author(s):  
Sua Kim ◽  
Chor Ho Jo ◽  
Gheun-Ho Kim
Keyword(s):  
Protein Kinase ◽  
Protein Expression ◽  
Protein Kinase A ◽  
Psychotropic Drugs ◽  
Collecting Duct ◽  
Camp Production ◽  
Inner Medullary Collecting Duct ◽  
Aquaporin 2 ◽  
Medullary Collecting Duct ◽  
Kinase A

Psychotropic drugs may be associated with hyponatremia, but an understanding of how they induce water retention in the kidney remains elusive. Previous studies have postulated that they may increase vasopressin production in the hypothalamus without supporting evidences. In this study, we investigated the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis (NSIAD) using haloperidol, sertraline, and carbamazepine. Haloperidol, sertraline, or carbamazepine were treated in inner medullary collecting duct (IMCD) suspensions and primary cultured IMCD cells prepared from male Sprague-Dawley rats. The responses of intracellular cAMP production, aquaporin-2 (AQP2) protein expression and localization, vasopressin-2 receptor (V2R) and AQP2 mRNA, and cAMP-responsive element binding protein (CREB) were tested with and without tolvaptan, and the protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. In IMCD suspensions, cAMP production was increased by haloperidol, sertraline, or carbamazepine and was relieved by tolvaptan cotreatment. In primary cultured IMCD cells, haloperidol, sertraline, or carbamazepine treatment increased total-AQP2 and decreased pSer261-AQP2 protein expression. Notably, these responses were reversed by cotreatment with tolvaptan or a PKA inhibitor. AQP2 membrane trafficking was induced by haloperidol, sertraline, or carbamazepine and was also blocked by cotreatment with tolvaptan or a PKA inhibitor. Furthermore, upregulation of V2R and AQP2 mRNA and phosphorylated CREB was induced by haloperidol, sertraline, or carbamazepine and was blocked by tolvaptan cotreatment. We conclude that, in the rat IMCD, psychotropic drugs upregulate AQP2 via V2R-cAMP-PKA signaling in the absence of vasopressin stimulation. The vasopressin-like action on the kidney appears to accelerate AQP2 transcription and dephosphorylate AQP2 at serine 261.

scholarly journals Activation of AQP2 water channels by protein kinase A: therapeutic strategies for congenital nephrogenic diabetes insipidus

2021 ◽  
Author(s):  
Fumiaki Ando
Keyword(s):  
Protein Kinase ◽  
Diabetes Insipidus ◽  
Protein Kinase A ◽  
Nephrogenic Diabetes Insipidus ◽  
Water Channel ◽  
Adenosine Monophosphate ◽  
Loss Of Function ◽  
Collecting Ducts ◽  
Congenital Nephrogenic Diabetes Insipidus ◽  
Kinase A

Abstract Background Congenital nephrogenic diabetes insipidus (NDI) is primarily caused by loss-of-function mutations in the vasopressin type 2 receptor (V2R). Renal unresponsiveness to the antidiuretic hormone vasopressin impairs aquaporin-2 (AQP2) water channel activity and water reabsorption from urine, resulting in polyuria. Currently available symptomatic treatments inadequately reduce patients’ excessive amounts of urine excretion, threatening their quality of life. In the past 25 years, vasopressin/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) has been believed to be the most important signaling pathway for AQP2 activation. Although cAMP production without vasopressin is the reasonable therapeutic strategy for congenital NDI caused by V2R mutations, the efficacy of candidate drugs on AQP2 activation is far less than that of vasopressin. Results Intracellular distribution and activity of PKA are largely controlled by its scaffold proteins, A-kinase anchoring proteins (AKAPs). Dissociating the binding of AKAPs and PKA significantly increased PKA activity in the renal collecting ducts and activated AQP2 phosphorylation and trafficking. Remarkably, the AKAPs–PKA disruptor FMP-API-1 increased transcellular water permeability in isolated renal collecting ducts to the same extent as vasopressin. Moreover, derivatives of FMP-API-1 possessed much more high potency. FMP-API-1/27 is the first low-molecular-weight compound to be discovered that can phosphorylate AQP2 more effectively than preexisting drug candidates. Conclusion AKAP-PKA disruptors are a promising therapeutic target for congenital NDI. In this article, we shall discuss the pathophysiological roles of PKA and novel strategies to activate PKA in renal collecting ducts.

scholarly journals Regulation of the formation and water permeability of endosomes from toad bladder granular cells.

1990 ◽  
Vol 96 (4) ◽  
pp. 789-808 ◽  
Author(s):  
L B Shi ◽  
Y X Wang ◽  
A S Verkman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Water Permeability ◽  
Apical Membrane ◽  
Water Channel ◽  
Water Channels ◽  
Granular Cells ◽  
Kinase C ◽  
Kinase A

Osmotic water permeability (Pf) in toad bladder is regulated by the vasopressin (VP)-dependent movement of vesicles containing water channels between the cytoplasm and apical membrane of granular cells. Apical endosomes formed in the presence of serosal VP have the highest Pf of any biological or artificial membrane (Shi and Verkman. 1989. J. Gen. Physiol. 94:1101-1115). We examine here: (a) the influence of protein kinase A and C effectors on transepithelial Pf (Pfte) in intact bladders and on the number and Pf of labeled endosomes, and (b) whether endosome Pf can be modified physically or biochemically. In paired hemibladder studies, Pfte induced by maximal serosal VP (50 mU/ml, 0.03 cm/s) was not different than that induced by 8-Br-cAMP (1 mM), forskolin (50 microM), VP + 8-Br-cAMP, or VP + forskolin. Pf was measured in endosomes labeled in intact bladders with carboxyfluorescein by a stopped-flow, fluorescence-quenching assay using an isolated microsomal suspension; the number and Pf (0.08-0.11 cm/s, 18 degrees C) of labeled endosomes was not different in bladders treated with VP, forskolin, and 8-Br-cAMP. Protein kinase C activation by 1 microM mucosal phorbol myristate acetate (PMA) induced submaximal bladder Pfte (0.015 cm/s) and endosome Pf (0.022 cm/s) in the absence of VP, but had little effect on maximal Pfte and endosome Pf induced by VP. However, PMA increased by threefold the number of apical endosomes with high Pf formed in response to serosal VP. Pf of endosomes containing the VP-sensitive water channel decreased fourfold by increasing membrane fluidity with hexanol or chloroform (0-75 mM); Pf of phosphatidylcholine liposomes (0.002 cm/s) increased 2.5-fold under the same conditions. Endosome Pf was mildly pH dependent, strongly inhibited by HgCl2, but not significantly altered by GTP gamma S, Ca, ATP + protein kinase A, and phosphatase action. We conclude that: (a) water channels cycled in endocytic vesicles are functional and not subject to physiological regulation, (b) VP and forskolin do not have cAMP-independent cellular actions, (c) activation of protein kinase C stimulates trafficking of water channels, but does not increase the number of apical membrane water channels induced by maximal VP, and (d) water channel function is sensitive to membrane fluidity. By using VP and PMA together, large quantities of endosomes containing the VP-sensitive water channel are labeled with fluid-phase endocytic markers.

N-ethylmaleimide causes aquaporin-2 trafficking in the renal inner medullary collecting duct by direct activation of protein kinase A

2005 ◽  
Vol 288 (4) ◽  
pp. F832-F839 ◽  
Author(s):  
Stephen Shaw ◽  
David Marples
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Arginine Vasopressin ◽  
Water Permeability ◽  
Collecting Duct ◽  
Direct Activation ◽  
Aquaporin 2 ◽  
Collecting Ducts ◽  
Kinase A

The antidiuretic hormone arginine vasopressin increases the osmotic water permeability of the renal collecting ducts by inducing the shuttling of aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical plasma membrane of the principal cells. This process has been demonstrated to be dependent on the cytoskeleton and protein kinase A (PKA). Previous studies in the toad urinary bladder, a functional homologue of the renal collecting duct, have demonstrated that the sulfhydryl reagent N-ethylmaleimide (NEM) is also able to activate the vasopressin-sensitive water permeability pathway in this tissue. The aim of the present study was to investigate the effects of NEM on AQP2 trafficking in a mammalian system. We show that NEM causes translocation of AQP2 from the cytosol to the plasma membrane in rat inner medullary collecting ducts; like the response to arginine vasopressin, this action was also dependent on an intact cytoskeleton and PKA. This effect is not mediated by cAMP but results from direct activation of PKA by NEM.

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