scholarly journals Unexpected localization of AQP3 and AQP4 induced by migration of primary cultured IMCD cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ralph Rose ◽  
Björn Kemper ◽  
Albrecht Schwab ◽  
Eberhard Schlatter ◽  
Bayram Edemir
Keyword(s):  
Protein Kinase ◽  
Marked Reduction ◽  
Collecting Duct ◽  
Leading Edge ◽  
Migration Behavior ◽  
Migration Speed ◽  
Lysosomal Degradation ◽  
Acute Increase ◽  
Migration Of Cells ◽  
Kinase A

AbstractAquaporin-2–4 (AQP) are expressed in the principal cells of the renal collecting duct (CD). Beside their role in water transport across membranes, several studies showed that AQPs can influence the migration of cells. It is unknown whether this also applies for renal CD cells. Another fact is that the expression of these AQPs is highly modulated by the external osmolality. Here we analyzed the localization of AQP2–4 in primary cultured renal inner medullary CD (IMCD) cells and how osmolality influences the migration behavior of these cells. The primary IMCD cells showed a collective migration behavior and there were no differences in the migration speed between cells cultivated either at 300 or 600 mosmol/kg. Acute increase from 300 to 600 mosmol/kg led to a marked reduction and vice versa an acute decrease from 600 to 300 mosmol/kg to a marked increase in migration speed. Interestingly, none of the analyzed AQPs were localized at the leading edge. While AQP3 disappeared within the first 2–3 rows of cells, AQP4 was enriched at the rear end. Further analysis indicated that migration induced lysosomal degradation of AQP3. This could be prevented by activation of the protein kinase A, inducing localization of AQP3 and AQP2 at the leading edge and increasing the migration speed.

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.

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 Integrin-mediated Protein Kinase A Activation at the Leading Edge of Migrating Cells

2008 ◽  
Vol 19 (11) ◽  
pp. 4930-4941 ◽  
Author(s):  
Chinten J. Lim ◽  
Kristin H. Kain ◽  
Eugene Tkachenko ◽  
Lawrence E. Goldfinger ◽  
Edgar Gutierrez ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Leading Edge ◽  
Pi3 Kinase ◽  
Early Event ◽  
Type I ◽  
Cell Protrusion ◽  
Kinase A ◽  
Migrating Cells

cAMP-dependent protein kinase A (PKA) is important in processes requiring localized cell protrusion, such as cell migration and axonal path finding. Here, we used a membrane-targeted PKA biosensor to reveal activation of PKA at the leading edge of migrating cells. Previous studies show that PKA activity promotes protrusion and efficient cell migration. In live migrating cells, membrane-associated PKA activity was highest at the leading edge and required ligation of integrins such as α4β1 or α5β1 and an intact actin cytoskeleton. α4 integrins are type I PKA-specific A-kinase anchoring proteins, and we now find that type I PKA is important for localization of α4β1 integrin-mediated PKA activation at the leading edge. Accumulation of 3′ phosphorylated phosphoinositides [PtdIns(3,4,5)P3] products of phosphatidylinositol 3-kinase (PI3-kinase) is an early event in establishing the directionality of migration; however, polarized PKA activation did not require PI3-kinase activity. Conversely, inhibition of PKA blocked accumulation of a PtdIns(3,4,5)P3-binding protein, the AKT-pleckstrin homology (PH) domain, at the leading edge; hence, PKA is involved in maintaining cell polarity during migration. In sum, we have visualized compartment-specific PKA activation in migrating cells and used it to reveal that adhesion-mediated localized activation of PKA is an early step in directional cell migration.

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 Protein Kinase A Gating of a Pseudopodial-located RhoA/ROCK/p38/NHE1 Signal Module Regulates Invasion in Breast Cancer Cell Lines

2005 ◽  
Vol 16 (7) ◽  
pp. 3117-3127 ◽  
Author(s):  
Rosa A. Cardone ◽  
Anna Bagorda ◽  
Antonia Bellizzi ◽  
Giovanni Busco ◽  
Lorenzo Guerra ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Leading Edge ◽  
Serum Deprivation ◽  
Breast Cancer Cell Lines ◽  
Kinase A

Metastasis results from a sequence of selective events often involving interactions with elements of the tumor-specific physiological microenvironment. The low-serum component of this microenvironment confers increased motility and invasion in breast cancer cells by activating the Na+/H+ exchanger isoform 1 (NHE1). The present study was undertaken to characterize the signal transduction mechanisms underlying this serum deprivation-dependent activation of both the NHE1 and the concomitant invasive characteristics such as leading edge pseudopodia development and penetration of matrigel in breast cancer cell lines representing different stages of metastatic progression. Using pharmacological and genetic manipulation together with transport and kinase activity assays, we observe that the activation of the NHE1 and subsequent invasion by serum deprivation in metastatic human breast cells is coordinated by a sequential RhoA/p160ROCK/p38MAPK signaling pathway gated by direct protein kinase A phosphorylation and inhibition of RhoA. Fluorescence resonance energy transfer imaging of RhoA activity and immunofluorescence analysis of phospho-RhoA and NHE1 show that serum deprivation dynamically remodels the cell, forming long, leading edge pseudopodia and that this signal module is preferentially compartmentalized in these leading edge pseudopodia, suggesting a tight topographic relation of the signaling module to an invasion-specific cell structure.

scholarly journals Spatial restriction of α4 integrin phosphorylation regulates lamellipodial stability and α4β1-dependent cell migration

2003 ◽  
Vol 162 (4) ◽  
pp. 731-741 ◽  
Author(s):  
Lawrence E. Goldfinger ◽  
Jaewon Han ◽  
William B. Kiosses ◽  
Alan K. Howe ◽  
Mark H. Ginsberg
Keyword(s):  
Cell Migration ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Control Cell ◽  
Adaptor Protein ◽  
Leading Edge ◽  
Dependent Cell ◽  
Kinase A ◽  
Migrating Cells

Întegrins coordinate spatial signaling events essential for cell polarity and directed migration. Such signals from α4 integrins regulate cell migration in development and in leukocyte trafficking. Here, we report that efficient α4-mediated migration requires spatial control of α4 phosphorylation by protein kinase A, and hence localized inhibition of binding of the signaling adaptor, paxillin, to the integrin. In migrating cells, phosphorylated α4 accumulated along the leading edge. Blocking α4 phosphorylation by mutagenesis or by inhibition of protein kinase A drastically reduced α4-dependent migration and lamellipodial stability. α4 phosphorylation blocks paxillin binding in vitro; we now find that paxillin and phospho-α4 were in distinct clusters at the leading edge of migrating cells, whereas unphosphorylated α4 and paxillin colocalized along the lateral edges of those cells. Furthermore, enforced paxillin association with α4 inhibits migration and reduced lamellipodial stability. These results show that topographically specific integrin phosphorylation can control cell migration and polarization by spatial segregation of adaptor protein binding.

scholarly journals Control of Adipogenesis by the SUMO-Specific Protease SENP2

2010 ◽  
Vol 30 (9) ◽  
pp. 2135-2146 ◽  
Author(s):  
Sung Soo Chung ◽  
Byung Yong Ahn ◽  
Min Kim ◽  
Hye Hun Choi ◽  
Ho Seon Park ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Marked Reduction ◽  
Adipocyte Differentiation ◽  
Marked Decrease ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Mrna Levels ◽  
Downstream Effectors ◽  
Specific Protease ◽  
Kinase A

ABSTRACT Here, we demonstrate that SENP2, a desumoylating enzyme, plays a critical role in the control of adipogenesis. SENP2 expression was markedly increased upon the induction of adipocyte differentiation, and this increase was dependent on protein kinase A activation. Remarkably, knockdown of SENP2 led to a dramatic attenuation of adipogenesis with a marked decrease in PPARγ and C/EBPα mRNA levels. Knockdown of SENP2 also caused a marked reduction in the level of C/EBPβ protein but not in that of C/EBPβ mRNA. Interestingly, sumoylation of C/EBPβ dramatically increased its ubiquitination and destabilization, and this increase could be reversed by SENP2. In addition, overexpression of C/EBPβ could overcome the inhibitory effect of SENP2 knockdown on adipogenesis. Furthermore, SENP2 was absolutely required for adipogenesis of preadipocytes implanted into mice. These results establish a critical role for SENP2 in the regulation of adipogenesis by desumoylation and stabilization of C/EBPβ and in turn by promoting the expression of its downstream effectors, such as PPARγ and C/EBPα.

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