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David I Cook

Publications and source records attributed to David I Cook.

15 recordsLinked to original sources
PubMed2006

Scoring of predicted GRK2 phosphorylation sites in Nedd4-2.

MOTIVATION: Epithelial Na(+) channels (ENaC) mediate the transport of sodium (Na) across epithelia in the kidney, gut and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4-2. These ligases bind to proline-rich motifs (PY motifs) present in the C-termini of ENaC subunits. Loss of this inhibition leads to hypertension. We have previously reported that ENaC channels are maintained in the active state by the G protein coupled receptor kinase, GRK2. The enzyme has been implicated in the development of essential hypertension [R. D. Feldman (2002) Mol. Pharmacol., 61, 707-709]. Additional findings in our lab pointed towards a possible role for GRK2 in the phosphorylation and inactivation of Nedd4-2. RESULTS: We have predicted GRK2 phosphorylation sites on Nedd4-2 by combining sequence analysis, homology modeling and surface accessibility calculations. A total of 24 potential phosphorylation sites were predicted by sequence analysis. Of these, 16 could be modeled using homology modeling and 6 of these were found to have sufficient surface exposure to be accessible to the GRK2 enzyme responsible for the phosphorylation of Nedd4-2. The method provides an ordered list of the most probable GRK2 phosphorylation sites on Nedd4-2 providing invaluable guidance to future experimental studies aimed at mutating certain Nedd4-2 residues in order to prevent phosphorylation by GRK2. The method developed could be applied in a wide variety of biological applications involving the binding of one molecule to a protein. The relative effectiveness of the technique is determined mainly by the quality of the homology model built for the protein of interest. CONTACT: jarthur@med.usyd.edu.au

Algorithms↗
PubMed2006

Stimulation of the epithelial sodium channel (ENaC) by the serum- and glucocorticoid-inducible kinase (Sgk) involves the PY motifs of the channel but is independent of sodium feedback inhibition.

The epithelial sodium channel (ENaC) is the major mediator of sodium transport across the apical membranes of the distal nephron, the distal colon, the respiratory tract and the ducts of exocrine glands. It is subject to feedback inhibition by increased intracellular Na+, a regulatory system wherein the ubiquitin protein ligases, Nedd4 and Nedd4-2, bind to conserved PY motifs in the C-termini of ENaC and inactivate the channel. It has been proposed recently that the kinase Sgk activates the channel as a consequence of phosphorylating Nedd4-2, thus preventing it from inhibiting the channels. This proposal predicts that Sgk should interfere with Na+ feedback regulation of ENaC. We have tested this prediction in Xenopus laevis oocytes and in mouse salivary duct cells and found that in neither system did increased activity of Sgk interrupt Na+ feedback inhibition of ENaC. We found, however, that Sgk stimulation was largely abolished in oocytes expressing ENaC channels with C-terminal truncations or mutated PY motifs. We were also unable to confirm that Sgk directly interacts with Nedd4-2 in vitro. We conclude that the stimulatory effect of Sgk on ENaC requires the presence of the channel's PY motifs, but it is not due to the interruption of Na+ feedback regulation.

Amino Acid Motifs↗
PubMed2005

The ubiquitin-protein ligases Nedd4 and Nedd4-2 show similar ubiquitin-conjugating enzyme specificities.

Nedd4 and Nedd4-2 are closely related HECT-type ubiquitin-protein ligases (E3) implicated in the regulation of a number of proteins and pathways. Given the close homology between these E3 enzymes it would be predicted that a conserved ubiquitin-conjugating enzyme (E2) specificity exists between the two proteins. However, E2 specificities for Nedd4 and Nedd4-2 are not well established. In the present studies we aimed at clarifying the E2-specificities of Nedd4 and Nedd4-2 using in vitro ubiquitination assays. We demonstrate strong substrate ubiquitination in the presence of UbcH5b by both Nedd4 and Nedd4-2. We also found that Ube2e3, an E2 previously shown to be used by Nedd4-2, is used less efficiently than UbcH5b. Our results suggest that for optimal ubiquitination Nedd4 and Nedd4-2 require the same E2 enzymes.

Amino Acid Sequence↗
PubMed2004

Nedd4-2 functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells.

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl(-) channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.

Albumins↗
PubMed2004

Acute effects of parainfluenza virus on epithelial electrolyte transport.

Parainfluenza viruses are important causes of respiratory disease in both children and adults. In particular, they are the major cause of the serious childhood illness croup (laryngotracheobronchitis). The infections produced by parainfluenza viruses are associated with the accumulation of ions and fluid in the respiratory tract. It is not known, however, whether this accumulation is because of a direct effect of the viruses on ion and fluid transport by the respiratory epithelium. Here we show that a model parainfluenza virus (the Sendai virus), in concentrations observed during respiratory infections, activates Cl- secretion and inhibits Na+ absorption across the tracheal epithelium. It does so by binding to a neuraminidase-insensitive glycolipid, possibly asialo-GM1, triggering the release of ATP, which then acts in an autocrine fashion on apical P2Y receptors to produce the observed changes in ion transport. These findings indicate that fluid accumulation in the respiratory tract associated with parainfluenza virus infection is attributable, at least in part, to direct effects of the virus on ion transport by the respiratory epithelium.

Adenosine Triphosphate↗
PubMed2004

The kinase Grk2 regulates Nedd4/Nedd4-2-dependent control of epithelial Na+ channels.

Epithelial Na(+) channels mediate the transport of Na across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, with loss of this inhibition leading to hypertension. Here, we report that these channels are maintained in the active state by the G protein-coupled receptor kinase, Grk2, which has been previously implicated in the development of essential hypertension. We also show that Grk2 phosphorylates the C terminus of the channel beta subunit and renders the channels insensitive to inhibition by Nedd4-2. This mechanism has not been previously reported to regulate epithelial Na(+) channels and provides a potential explanation for the observed association of Grk2 overactivity with hypertension. Here, we report a G protein-coupled receptor kinase regulating a membrane protein other than a receptor and provide a paradigm for understanding how the interaction between membrane proteins and ubiquitin protein ligases is controlled.

Animals↗
PubMed2004

Regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases Nedd4 and Nedd4-2.

Nedd4 and Nedd4-2 are ubiquitin-protein ligases known to regulate a number of membrane proteins including receptors and ion transporters. Regulation of the epithelial Na(+) channel by Nedd4 and Nedd4-2 is mediated via interactions between the PY motifs of the epithelial sodium channel subunits and the Nedd4/Nedd4-2 WW domains. This example serves as a model for the regulation of other PY motif-containing ion channels by Nedd4 and Nedd4-2. We found that the carboxyl termini of the six voltage-gated Na(+) (Na(v)) channels contain typical PY motifs (PPXY), and a further Na(v) contains a PY motif variant (LPXY). Not only did we demonstrate by Far-Western analysis that Nedd4 and Nedd4-2 interact with the PY motif-containing Na(v) channels, but we also showed that these channels have conserved WW domain binding specificity. We further showed that the carboxyl termini fusion proteins of one central nervous system and one peripheral nervous system-derived Na(+) channel (Na(v)1.2 and Na(v)1.7, respectively) are readily ubiquitinated by Nedd4-2. In Xenopus oocytes, Nedd4-2 strongly inhibited the activities of all three Na(v)s (Na(v)1.2, Na(v)1.7, and Na(v)1.8) tested. Interestingly, Nedd4 suppressed the activity of Na(v)1.2 and Na(v)1.7 but was a poor inhibitor of Na(v)1.8. Our results provide evidence that Nedd4 and Nedd4-2 are likely to be key regulators of specific neuronal Na(v) channels in vivo.

Amino Acid Sequence↗
PubMed2003

Insulin secretagogues, but not glucose, stimulate an increase in [Ca2+]i in the fetal human and porcine beta-cell.

Fetal pancreatic beta-cells release insulin poorly in response to glucose; however, the cellular mechanism for this is unknown. By using fura-2 to measure changes in the cytoplasmic free Ca(2+) concentration in beta-cells, we examined human/porcine fetal islet-like cell clusters (ICCs) and human adult islets for the presence of functional K(+)(ATP) and voltage-activated Ca(2+) ion channels. The effects of glucose, glyceraldehyde, leucine, KCl, and the channel effectors glipizide and BAY K8644 were studied. In fetal human/porcine ICCs and adult islets, KCl, glipizide, and BAY K8644 increased [Ca(2+)](i). Both glucose and glyceraldehyde increased [Ca(2+)](i) in islets but had no effect on ICCs. Leucine increased [Ca(2+)](i) in islets and porcine but not human ICCs. We hypothesize that the beneficial effect of leucine in fetal porcine, but not human ICCs, is attributable to time-dependent maturation of the beta-cells, because porcine ICCs examined were at 87% of the gestational period, and human ICCs were at 42%. Our data demonstrate that both K(+)(ATP) and voltage-activated Ca(2+) channels, required for glucose-stimulated increase in [Ca(2+)](i), are functional early in gestation. This suggests that the cause of the immaturity of fetal human/porcine beta-cells is at a more proximal step of glucose-induced metabolism than the channels on the cell surface.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2002

The role of individual Nedd4-2 (KIAA0439) WW domains in binding and regulating epithelial sodium channels.

The amiloride-sensitive epithelial sodium channel (ENaC) is essential for fluid and electrolyte homeostasis. ENaC consists of alpha, beta, and gamma subunits, each of which contains a PPxY motif that interacts with the WW domains of the ubiquitin-protein ligases Nedd4 and Nedd4-2. Disruption of this interaction, as in Liddle's syndrome in which mutations delete or alter the PPxY motif of either the beta or the gamma subunits, results in increased ENaC activity. We report here that Nedd4-2 has two major isoforms that show tissue-specific expression; however, both isoforms can inhibit ENaC in Xenopus oocytes. Because there are four WW domains in Nedd4-2, we analyzed binding kinetics and affinity between individual WW domains and ENaC subunits. Using whole cell patch-clamp techniques, we studied the role of individual WW domains in the regulation of ENaC in mammalian cells. We report here that unlike Nedd4, only two of the Nedd4-2 WW domains, WW3 and WW4, are required for both the binding to ENaC subunits and the regulation of Na+ feedback control of ENaC. Although both WW3 and WW4 individually can interact with all three ENaC subunits in vitro, both domains together are essential for in vivo function of Nedd4-2 in ENaC regulation. These data suggest that Nedd4-2 WW3 and WW4 interact with distinct, noninterchangeable sites in ENaC and that to prevent Na+ feedback control of ENaC it is necessary to occlude both sites.

Alternative Splicing↗
PubMed2002

Regulation of the epithelial sodium channel by N4WBP5A, a novel Nedd4/Nedd4-2-interacting protein.

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPXY motif that is believed to be important for interaction with the WW domains of the ubiquitin-protein ligases, Nedd4 and Nedd4-2. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PPXY motif of either the beta or gamma subunits, has been shown to result in increased ENaC activity and arterial hypertension. Here we present evidence that N4WBP5A, a novel Nedd4/Nedd4-2-binding protein, is a potential regulator of ENaC. In Xenopus laevis oocytes N4WBP5A increases surface expression of ENaC by reducing the rate of ENaC retrieval. We further demonstrate that N4WBP5A prevents sodium feedback inhibition of ENaC possibly by interfering with the xNedd4-2-mediated regulation of ENaC. As N4WBP5A binds Nedd4/Nedd4-2 via PPXY motif/WW domain interactions and appears to be associated with specific intracellular vesicles, we propose that N4WBP5A functions by regulating Nedd4/Nedd4-2 availability and trafficking. Because N4WBP5A is highly expressed in native renal collecting duct and other tissues that express ENaC, it is a likely candidate to modulate ENaC function in vivo.

Animals↗
PubMed2002

Patch-clamp studies on epithelial sodium channels in salivary duct cells.

Mouse mandibular salivary duct cells contain an amiloride-sensitive Na+ current and express all three subunits of the epithelial Na+ channel, ENaC. This amiloride-sensitive Na+ current is subject to feedback regulation by intracellular Na+ and we have previously demonstrated that this regulation is mediated by an ubiquitin-protein ligase, which we identified as Nedd4. The evidence supporting this identification is as follows: (1) antibodies raised against murine Nedd4 block Na+ feedback inhibition; (2) a mutant of murine Nedd4 containing the WW domains but no HECT domain (ubiquitin-protein ligase) blocks Na+ feedback inhibition; and (3) Nedd4 is expressed in mouse mandibular salivary duct cells. In the present studies, we have used whole-cell patch-clamp methods to further investigate the mechanisms by which ubiquitin-protein ligases regulate the amiloride-sensitive Na+ conductance in mouse salivary duct cells. In particular, we have examined the possibility that the ubiquitin-protein ligase, KIAA0439, which is closely related to Nedd4, may mediate Na+ feedback control of amiloride-sensitive Na+ channels. Furthermore, we have attempted to define the mechanism by which ubiquitin-protein ligases inhibit Na+ channels. We have found that KIAA0439 is expressed in mouse mandibular ducts and interacts with the PY motifs of the alpha-, beta-, and gamma-subunits of ENaC in vitro. Furthermore, in whole-cell patch-clamp studies, a glutathione-S-transferase (GST)-fusion protein containing the WW motifs of human KIAA0439 was able to inhibit feedback regulation of the amiloride-sensitive Na+ current by intracellular Na+. We also examined whether GST-fusion proteins containing the C-termini of the alpha-, beta-, and gamma-subunits of ENaC are able to interrupt Na+ feedback regulation of the amiloride-sensitive Na+ current. We found that the C-termini of the beta- and gamma-subunits were able to do so, whereas the C-terminus of the alpha-subunit was not. We conclude that KIAA0439 is, together with Nedd4, a potential mediator of the control of epithelial Na+ channels in salivary duct cells by intracellular Na+. We further conclude that ubiquitin-protein ligases interact with the Na+ channels through the C-termini of the beta- and gamma-subunits of the Na+ channels.

Amiloride↗
PubMed2002

Use of adenoviruses to study isoform specificity of G-protein-receptor-coupled Ca2+ signaling in intact epithelial cells.

It is firmly established that the activation of many heptahelical receptors by extracellular agonists leads to the activation of effectors such as phospholipase Cbeta (PLCbeta), the subsequent production of inositol-1,4,5-trisphosphate (IP3), and a resultant increase in intracellular free Ca2+. Heterotrimeric G-proteins have a critical role in transducing the signal from the heptahelical receptor to PLCbeta and in determining the specificity and duration of the cellular responses. There remain, however, a number of areas of uncertainty regarding the exact mechanisms involved in regulating G-protein-mediated receptor-effector coupling in different cell types. For example, the molecular identity of the G-protein involved and the degree of isoform specificity among G-proteins of the same family and their receptors remains unclear. It is also not known in many cell types whether it is the alpha- or the betagamma-subunits of these G-proteins that activate PLCbeta. In order to address these issues, we have used replication-deficient adenoviruses as a tool to deliver, into intact epithelial cells, transgenes coding for proteins involved in G-protein-coupled receptor signaling pathways.

Adenoviridae↗