Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Among water channel proteins (aquaporins), aquaporin-collecting duct (AQP-CD) is the vasopressin-regulated water channel. Vasopressin causes cAMP production in the renal collecting duct cells, and this is believed to lead to exocytic insertion of water channel into the apical membrane (shuttle hypothesis). AQP-CD contains a consensus sequence for cAMP-dependent protein kinase, residues at positions 253-256 (Arg-Arg-Gln-Ser). To determine the role of this site, Ser-256 was substituted for Ala, Leu, Thr, Asp, or Glu by site-directed mutagenesis. In Xenopus oocytes injected with wild-type or mutated AQP-CD cRNAs, osmotic water permeability (Pf) was 4.8-7.7 times higher than Pf of water-injected oocytes. Incubation with cAMP plus forskolin or direct cAMP injection into the oocytes increased Pf of wild-type, but not mutated, AQP-CD-expressing oocytes, whereas the amounts of AQP-CD expression were similar in wild and mutated types as identified by Western blot analysis. In vitro phosphorylation studies of AQP-CD proteins expressed in oocyte showed that cAMP-dependent protein kinase phosphorylated wild-type, but not mutated, AQP-CD proteins. Phosphoamino acid analysis revealed that this phosphorylation occurred at the serine residue. Moreover, phosphorylation of AQP-CD protein in intact rat kidney medulla tissues was stimulated by incubation with cAMP. Our data suggest that cAMP stimulates water permeability of AQP-CD by phosphorylation. This process may contribute to the vasopressin-regulated water permeability of collecting duct in addition to the apical insertion of AQP-CD by exocytosis.
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PMID:cAMP-dependent phosphorylation stimulates water permeability of aquaporin-collecting duct water channel protein expressed in Xenopus oocytes. 753 30

The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP. 754 85

Antidiuretic hormone modulates the water permeability (Pf) of epithelial cells in the rat kidney by vesicle-mediated insertion and removal of the aquaporin-2 (AQP-2) water channel. AQP-2 possesses a single consensus cAMP-dependent protein kinase A (PKA) phosphorylation site (Ser-256) hypothesized to regulate channel Pf(Kuwahara, M., Fushimi, K., Terada, Y., Bai, L., Sasaki, S., and Marumo, F. (1995) J. Biol. Chem. 270, 10384-10387). To test whether PKA phosphorylation of AQP-2 alters channel Pf, we compared the Pf values of purified AQP-2 endosomes after incubation with either PKA or alkaline phosphatase. Studies using [gamma-32P]ATP reveal that AQP-2 endosomes contain endogenous PKA and phosphatase activities that add and remove 32P label from AQP-2. However, the Pf (0.16 +/- 0.06 cm/s) of endosomes containing phosphorylated AQP-2 (0.7 +/- 0. 3 mol of PO4/mol of protein) is not significantly different from the same AQP-2 endosomes where 95 +/- 8% of the phosphate has been removed (Pf 0.14 +/- 0.06 cm/s). These data do not support a role for PKA phosphorylation in alteration of AQP-2's Pf. Instead, AQP-2 phosphorylation by PKA may modulate AQP-2's distribution between plasma membrane and intracellular vesicle compartments.
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PMID:Phosphorylation of aquaporin-2 does not alter the membrane water permeability of rat papillary water channel-containing vesicles. 862 14

The aquaporin-2 (AQP2) vasopressin water channel is translocated to the apical membrane upon vasopressin stimulation. Phosphorylation of serine 256 of AQP2 by cAMP-dependent protein kinase has been shown, but its relation to vasopressin-regulated translocation has not been elucidated. To address this question, wild type (WT) AQP2 and a mutant with alanine in place of serine 256 of AQP2 (S256A) were expressed in LLC-PK1 cells by electroporation. Measurements by a stopped-flow light-scattering method revealed that the osmotic water permeability (Pf) of LLC-PK1 cells transfected with WT was 69.6 +/- 6.5 microm/s (24.8 +/- 2.2 microm/s for mock-transfected), and stimulation by 500 microM 8-(4-chlorophenylthio)-cAMP increased the Pf by 85 +/- 12%. When S256A AQP2 was transfected, the cAMP-dependent increase in the Pf was only 8 +/- 5%. After cAMP stimulation, the increase in surface expression of AQP2 determined by surface biotin labeling was 4 +/- 10%, significantly less than that for WT (88 +/- 5%). In addition, an in vivo [32P]orthophosphate labeling assay demonstrated significant phosphorylation of WT AQP2 and only minimal phosphorylation of S256A AQP2 in LLC-PK1 cells. Our results indicated that serine 256 of AQP2 is necessary for regulatory exocytosis and that cAMP-responsive redistribution of AQP2 may be regulated by phosphorylation of AQP2.
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PMID:Phosphorylation of serine 256 is required for cAMP-dependent regulatory exocytosis of the aquaporin-2 water channel. 916 47

The goal of this study was to compare single channel water and glycerol permeabilities of mammalian aquaporins (AQP) 1-5 and the major intrinsic protein of lens fiber (MIP). Each of the six cloned cDNAs from rat was left untagged or was epitope-tagged with c-Myc or FLAG at either the N or C terminus so that results would not depend on epitope identity or location. The constructs were expressed in Xenopus oocytes for measurement of osmotic water permeability (Pf), [3H]glycerol uptake, and protein expression. Each of the 30 epitope-tagged constructs was expressed strongly at the oocyte plasma membrane. The 10-min uptake of [3H]glycerol was increased significantly (range of 4.5-8-fold over control) in oocytes expressing untagged AQP3 (GLIP) and each of the four tagged AQP3 constructs; [3H]glycerol uptake was not increased in oocytes expressing AQP1, AQP2, AQP4, AQP5, or MIP. In oocytes microinjected with 5 ng of cRNA, average Pf values (in cm/s x 10(-3)) were 0.67 +/- 0.06 (control), 19 +/- 2 (AQP1), 10 +/- 1 (AQP2), 8 +/- 2 (AQP3), 29 +/- 1 (AQP4), 10 +/- 1 (AQP5), and 1.3 +/- 0.2 (MIP), and they were relatively insensitive to the presence, identity, or location of the epitope tag. Pf values were not affected by protein kinase A or C activation. After normalization for plasma membrane expression by immunoprecipitation of microdissected plasma membranes, single channel water permeabilities (pf, referenced to the AQP1 pf of 6 x 10(-14) cm3/s) were (in cm3/s x 10(-14)) 3.3 +/- 0.2 (AQP2), 2.1 +/- 0.3 (AQP3), 24 +/- 0.6 (AQP4), 5.0 +/- 0.4 (AQP5), and 0.25 +/- 0.05 (MIP); pf values were insensitive to epitope identity and location. These results indicate very different intrinsic water permeabilities for the mammalian aquaporin homologs, with the pf value for AQP4 remarkably higher than those for the others. The pf values establish limits on aquaporin tissue densities required for physiological function and suggest significant structural and functional differences among the aquaporins.
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PMID:Water and glycerol permeabilities of aquaporins 1-5 and MIP determined quantitatively by expression of epitope-tagged constructs in Xenopus oocytes. 919 10

Vasopressin-dependent translocation of aquaporin-2 (AQP2) between intracellular vesicles and the plasma membrane has been demonstrated in vivo and in vitro. Furthermore, the vasopressin-induced increase in apical membrane water permeability of renal principal cells is dependent on a rise in intracellular adenosine 3',5'-cyclic monophosphate and activation of protein kinase A (PKA). To determine whether trafficking of AQP2 is dependent on PKA phosphorylation, we first examined the effect of the PKA-inhibitor N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89) on AQP2 translocation in transfected LLC-PK1 cells. Vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of the cells for 60 min with H-89. This reagent also caused a dense accumulation of AQP2 in the Golgi region. Next, LLC-PK1 cells were stably transfected with AQP2 cDNA in which the PKA phosphorylation site, Ser256, was replaced with alanine (S256A). S256A-AQP2 was not phosphorylated in vitro by PKA, and S256A-AQP2 was mainly localized to intracellular vesicles in the basal condition, similar to wild-type AQP2. However, after stimulation with vasopressin or forskolin, the cellular distribution of S256A-AQP2 remained unchanged. In addition, the usual vasopressin-induced increase in endocytosis seen in AQP2-transfected cells was not observed in S256A-AQP2-transfected cells. These results demonstrate that the Ser256 PKA phosphorylation site is possibly involved in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the plasma membrane and in the subsequent stimulation of endocytosis.
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PMID:Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells. 922 44

In a systematic analysis of genes expressed in human adipose tissue, we detected a novel gene that is expressed uniquely in adipose tissue. The sequence showed that it encodes a 342-amino-acid protein containing six putative transmembrane domains, and is a new member of the aquaporin family of water-selective membrane channels. We named this gene aquaporin 9. It features a cyclic-AMP protein kinase phosphorylation consensus site in the NH3-terminal domain. Expression of the cRNA in Xenopus oocytes yielded a 7-fold increase in osmotic water permeability blocked by 0.3 mM HgCl2, and also facilitated the uptake of glycerol. Northern blot analysis demonstrated that the mRNA is abundant in adipose tissue, but not in other tissues. Thus, this gene product may participate in glycerol transport in adipocytes.
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PMID:Molecular cloning and expression of a novel human aquaporin from adipose tissue with glycerol permeability. 940 33

Mutations in the aquaporin-2 (AQP2) water channel gene cause autosomal recessive nephrogenic diabetes insipidus (NDI). Here we report the first patient with an autosomal dominant form of NDI, which is caused by a G866A transition in the AQP2 gene of one allele, resulting in a E258K substitution in the C-tail of AQP2. To define the molecular cause of NDI in this patient, AQP2-E258K was studied in Xenopus oocytes. In contrast to wild-type AQP2, AQP2-E258K conferred a small increase in water permeability, caused by a reduced expression at the plasma membrane. Coexpression of wild-type AQP2 with AQP2-E258K, but not with an AQP2 mutant in recessive NDI (AQP2-R187C), revealed a dominant-negative effect on the water permeability conferred by wild-type AQP2. The physiologically important phosphorylation of S256 by protein kinase A was not affected by the E258K mutation. Immunoblot and microscopic analyses revealed that AQP2-E258K was, in contrast to AQP2 mutants in recessive NDI, not retarded in the endoplasmic reticulum, but retained in the Golgi compartment. Since AQPs are thought to tetramerize, the retention of AQP2-E258K together with wild-type AQP2 in mixed tetramers in the Golgi compartment is a likely explanation for the dominant inheritance of NDI in this patient.
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PMID:An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex. 964 57

T1alpha is a protein of unknown function that is expressed at the plasma membrane in epithelia involved in fluid transport, including type I alveolar epithelial cells, choroid plexus, and ciliary epithelium. The purpose of this study was to test the hypothesis that T1alpha functions as a water channel or a regulator of aquaporin-type water channels that are coexpressed with T1alpha. Two complementary DNAs (cDNAs) (hT1alpha-1 and hT1alpha-2) encoding human isoforms of T1alpha were cloned by homology to the rat T1alpha coding sequence. The cDNAs encoded 164 (hT1alpha-1) and 162 (hT1alpha-2) amino acid proteins with high homology to rat T1alpha in a putative membrane-spanning domain. hT1alpha-1 transcripts of 2. 6 and 1.4 kb were detected in human lung, heart, and skeletal muscle, and a single hT1alpha-2 transcript of 1.2 kb was detected in human lung. Rat and mouse T1alpha were isolated by reverse transcription-polymerase chain reaction and confirmed by DNA sequence analysis. Expression of mouse, rat, and human T1alpha isoforms in Xenopus oocytes did not increase osmotic water permeability (Pf) above that in water-injected oocytes, nor was there an effect of protein kinase A or C activation; Pf was increased > 10-fold in positive control oocytes expressing aquaporin (AQP)1 or AQP5. Coexpression of AQP1 or AQP5 with excess T1alpha gave Pf not different from that in oocytes expressing AQP1 or AQP5 alone. Oocyte plasma membrane localization of epitope-tagged T1alpha protein was confirmed and quantified by immunoprecipitation of microdissected plasma membranes. Quantitative densitometry indicated that the single-channel water permeability of T1alpha is under 2 x 10(-16) cm3/s, suggesting that T1alpha is not involved in the high transalveolar water permeability in intact lung. The cloning of hT1alpha isoforms may permit the development of an assay of type I cell antigen in airspace fluid as a marker of human lung injury.
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PMID:Evidence against a role of mouse, rat, and two cloned human t1alpha isoforms as a water channel or a regulator of aquaporin-type water channels. 965 Nov 90

The antidiuretic hormone arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells by inducing a cAMP-dependent translocation of water channels (aquaporin-2, AQP-2) from intracellular vesicles into the apical cell membranes. In subcellular fractions from primary cultured rat inner medullary collecting duct (IMCD) cells, enriched for intracellular AQP-2-bearing vesicles, catalytic protein kinase A (PKA) subunits and several protein kinase A anchoring proteins (AKAPs) were detected. In nonstimulated IMCD cells the majority of AQP-2 staining was detected intracellularly but became mainly localized within the cell membrane after stimulation with AVP or forskolin. Quantitative analysis revealed that preincubation of the cells with the synthetic peptide S-Ht31, which prevents the binding between AKAPs and regulatory subunits of PKA, strongly inhibited AQP-2 translocation in response to forskolin. Preincubation of the cells with the PKA inhibitor H89 prior to forskolin stimulation abolished AQP-2 translocation. In contrast to H89, S-Ht31 did not affect the catalytic activity of PKA. These data demonstrate that not only the activity of PKA, but also its tethering to subcellular compartments, are prerequisites for cAMP-dependent AQP-2 translocation.
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PMID:Protein kinase A anchoring proteins are required for vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells. 998 36


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