Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells

Katsura, Toshiya; Gustafson, Corinne E.; Ausiello, Dennis A.; Brown, Dennis

doi:10.1152/ajprenal.1997.272.6.f816

Cited by 238 publications

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“…Like rat counterpart, human B/K protein was highly expressed in the kidney (Figure 3). Moreover, in vivo phosphorylation experiments, B/K protein was very efficiently and specifically phosphorylated by vasopressin in a porcine kidney cell line LLC-PK1 (Figure 4) which has been used for studying PKA-dependent signal mechanisms such as the exocytosis of aquaporin-2 (Katsura et al, 1997). Our findings showing the type-2 vasopressin receptor-specific phosphorylation of B/K protein more clearly demonstrated the PKA-dependency of B/K protein phosphorylation.…”

Section: Discussionsupporting

confidence: 55%

Protein kinase A-dependent phosphorylation of B/K protein

Chin

Choi²,

Jang³

et al. 2006

Exp Mol Med

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We have previously isolated a novel protein "B/K" that contains two C2-like domains. Here, we report the isolatioin and mRNA distribution of a human B/K isoform, and protein kinase A (PKA)-dependent phosphorylation of the B/K protein. The 1.5 kb human B/K cDNA clone exhibits 89% and 97% identities with rat B/K in the sequences of nucleotide and amino acid, respectively. Human B/K isoform encodes a 474 amino acid protein and shows structural features similar to the rat counterpart including two C2 domains, three consensus sequences for PKA, absence of a transmembrane region, and conservation of the N-terminal cysteine cluster. On Northern and dot blot analyses, a 3.0 kb B/K transcript was abundantly present in human brain, kidney, and prostate. Among the brain regions, strong signals were observed in the frontal and temporal lobes, the hippocampus, the hypothalamus, the amygdala, the substantia nigra, and the pituitary. Recombinant B/K proteins containing three consensus sites for PKA was very efficiently phosphorylated in vitro by PKA catalytic subunit. B/K protein which was overexpressed in LLC-PK1 cells was also strongly phosphorylated in vivo by vasopressin analog DDAVP, and PKA-specific inhibitor H89 as well as type 2 vasopressin receptor antagonist specifically suppressed DDAVP-induced B/K phosphorylation. These results suggest that B/K proteins play a role as potential substrates for PKA in the area where they are expressed.

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Section: Discussionsupporting

confidence: 55%

Protein kinase A-dependent phosphorylation of B/K protein

Chin

Choi²,

Jang³

et al. 2006

Exp Mol Med

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“…The production of stably transfected LLC-PK 1 cells expressing an AQP2 S256A mutant that mimics nonphosphorylated AQP2 (LLC-AQP2 (S256A)) was previously described (10). Following the same procedure, LLC-PK 1 cells were stably transfected with c-myc-tagged AQP2 inserted in the pcDNA1/Neo vector in which Ser 256 was replaced with aspartate (LLC-AQP2 (S256D)) in order to mimic phosphorylated AQP2.…”

Section: Methodsmentioning

confidence: 99%

“…Phosphorylation of AQP2 at Ser 256 is essential but not sufficient for steady-state expression of AQP2 at the cell surface (10,12,25,38). We investigated the Ser 256 phosphorylation state of AQP2 that accumulates at the cell surface and in the trans-Golgi region following hypertonic challenge by comparing hypertonicity-induced accumulation of AQP2 in both regions of LLC-PK 1 cells stably expressing mutants that either mimic Ser 256 -phosphorylated (LLC-AQP2 (S256D)) or nonphosphorylated (LLC-AQP2 (S256A)) AQP2 (Fig.…”

Section: Acute Hypertonicity Induces Segregation Of Ser 256 -Phosphormentioning

confidence: 99%

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Acute Hypertonicity Alters Aquaporin-2 Trafficking and Induces a MAPK-dependent Accumulation at the Plasma Membrane of Renal Epithelial Cells

Hasler

Nunes²,

Bouley³

et al. 2008

Journal of Biological Chemistry

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The unique phenotype of renal medullary cells allows them to survive and functionally adapt to changes of interstitial osmolality/tonicity. We investigated the effects of acute hypertonic challenge on AQP2 (aquaporin-2) water channel trafficking. In the absence of vasopressin, hypertonicity alone induced rapid (<10 min) plasma membrane accumulation of AQP2 in rat kidney collecting duct principal cells in situ, and in several kidney epithelial lines. Confocal microscopy revealed that AQP2 also accumulated in the trans-Golgi network (TGN) following hypertonic challenge. AQP2 mutants that mimic the Ser 256 -phosphorylated and -nonphosphorylated state accumulated at the cell surface and TGN, respectively. Hypertonicity did not induce a change in cytosolic cAMP concentration, but inhibition of either calmodulin or cAMP-dependent protein kinase A activity blunted the hypertonicity-induced increase of AQP2 cell surface expression. Hypertonicity increased p38, ERK1/2, and JNK MAPK activity. Inhibiting MAPK activity abolished hypertonicity-induced accumulation of AQP2 at the cell surface but did not affect either vasopressin-dependent AQP2 trafficking or hypertonicity-induced AQP2 accumulation in the TGN. Finally, increased AQP2 cell surface expression induced by hypertonicity largely resulted from a reduction in endocytosis but not from an increase in exocytosis. These data indicate that acute hypertonicity profoundly alters AQP2 trafficking and that hypertonicity-induced AQP2 accumulation at the cell surface depends on MAP kinase activity. This may have important implications on adaptational processes governing transcellular water flux and/or cell survival under extreme conditions of hypertonicity.Most mammalian cells are exposed to an extracellular environment that remains isotonic under normal physiological conditions, largely as a result of renal regulatory mechanisms that maintain water and electrolyte body fluid composition within a very narrow range. This process relies on the countercurrent concentration mechanism that occurs along the loop of Henle and which, in turn, depends on the generation of a NaCl and urea concentration gradient along the cortico-papillary axis. As a consequence, renal medullary cells are physiologically exposed to changing conditions of variable interstitial osmolality/tonicity that can reach up to 1200 mosmol/kg in human inner medulla and up to 4500 mosmol/kg in some remarkable rodent species that are adapted to life in arid desert conditions (1). The unique phenotype of these cells allows them to survive and function under these "hostile" conditions and to rapidly adapt to recurrent variations in their environmental tonicity that follow physiological and behavioral changes. In addition to the promotion of intracellular events that ensure cell survival, hypertonicity also promotes events that mediate changes in cell function in various physiological settings. Both rapid and slow responses mediate cell adaptation to increased extracellular tonicity. Rapid responses include actin and micr...

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“…7 The expression and activity of AQP2 are regulated by the antidiuretic hormone (vasopressin, AVP) via the G-protein coupled AVP-2 receptor (V2R). 8 The binding of AVP to the V2R leads to the activation of the G-protein Gs, followed by activation of adenylate cyclase (AC), increased cyclic adenosine monophosphate (cAMP) levels, activation of protein kinase A (PKA), and finally phosphorylation of AQP2 9 and translocation to the luminal membrane. 10 AVP also induces mRNA and protein expression of AQP2.…”

mentioning

confidence: 99%

Acute Rejection Modulates Gene Expression in the Collecting Duct

Edemir

Reuter²,

Borgulya³

et al. 2008

Journal of the American Society of Nephrology

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Kidney transplantation, especially when associated with acute rejection, leads to changes in the expression of many genes, including those encoding solute transporters and water channels. In a rat model of acute rejection after allogeneic renal transplantation, impaired renal function, increased urine volume, and increased fractional excretion of sodium were observed. Gene array analysis revealed that these findings were associated with significant downregulation of water channels (aquaporin-1, -2, -3, and -4) and transporters of sodium, glucose, urea, and other solutes. In addition, changes in expression of various receptors, kinases, and phosphatases that modulate the expression or activity of renal transport systems were observed. Syngeneic transplantation or treatment with cyclosporine A following allogeneic transplantation did not impair graft function but did lead to the downregulation of aquaporin-1, -3, and -4 and several solute transporters. However, expression of aquaporin-2 and the epithelial sodium channel did not change, suggesting that the downregulation of these transporters following allogeneic transplantation is rejection-dependent. In conclusion, changes in gene expression may explain the impaired handling of solute and water after allogeneic transplantation, especially during acute rejection. Treatment with cyclosporine A improves the regulation of solute and water by preventing the downregulation of aquaporin-2 and epithelial sodium channel, even though many other transporter genes remain downregulated. Renal transplantation (TX) in humans is often accompanied by increased transport capacities of the graft and disturbances in salt and water homeostasis. 1-3 Half-life of grafts with normal function after TX is 11.5 yr compared with 7.2 yr for those with impaired renal function. 4 A better understanding of the underlying cellular and molecular mechanisms leading to such changes may help to increase longterm graft survival.Previously, using an allogeneic rat renal TX (aTX) model, we have demonstrated acute changes in expression and function of several transporters and receptors after aTX. 5,6 For example, the expression of Na ϩ /H ϩ -exchanger-3 (NHE3), aquaporin-2 (AQP2), and the epithelial Na ϩ channel (ENaC) were downregulated at the protein and mRNA level. Major transporters for water and Na ϩ reabsorption in the collecting duct (CD) are ENaC and AQP2. 7 The expression and activity of AQP2 are regulated by the antidiuretic hormone (vasopressin, AVP) via the G-protein coupled AVP-2 receptor (V2R). 8 The binding of AVP to the V2R leads to the activation of the G-protein Gs, followed by activation of adenylate cyclase (AC), increased cyclic adenosine monophosphate (cAMP) levels, activation of protein kinase A (PKA), and finally phosphorylation of AQP2 9 and translocation to the

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Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells

Cited by 238 publications

References 0 publications

Protein kinase A-dependent phosphorylation of B/K protein

Protein kinase A-dependent phosphorylation of B/K protein

Acute Hypertonicity Alters Aquaporin-2 Trafficking and Induces a MAPK-dependent Accumulation at the Plasma Membrane of Renal Epithelial Cells

Acute Rejection Modulates Gene Expression in the Collecting Duct

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