The KCNQ1 potassium channel is down-regulated by ubiquitylating enzymes of the Nedd4/Nedd4-like family

Jespersen, Thomas; Membrez, Mathieu; Nicolas, Céline S.; Pitard, Bruno; Staub, Olivier; Olesen, Søren‐Peter; Baró, Isabelle; Abriel, Hugues

doi:10.1016/j.cardiores.2007.01.008

Cited by 128 publications

(125 citation statements)

References 26 publications

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“…A major player in Kv7.1 endocytosis and turnover appears to be the ubiquitinylation enzyme Nedd4-2 (14), and interestingly PKA can inhibit Nedd4-2 by direct phosphorylation (28). To investigate if the observed intracellular accumulation of Kv7.1 involves Nedd4-2, we subjected polarized MDCK cells stably expressing a Nedd4-2 interaction-deficient Kv7.1 mutant (Kv7.1-YA) to treatment with H-89 for 3-5 h. The Kv7.1-YA mutant has a Y to A mutation in the Nedd4-2 interaction domain (LPxY), which inhibits its interaction with Nedd4-2 (14). In contrast to wildtype channels, Kv7.1-YA mutant channels remained strongly associated with the basolateral membrane throughout the treatment with no detectable intracellular accumulation (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

Andersen

Hefting

Steffensen

et al. 2015

American Journal of Physiology-Cell Physiology

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The potassium channel Kv7.1 plays critical physiological roles in both heart and epithelial tissues. In heart, Kv7.1 and the accessory subunit KCNE1 forms the slowly activating delayed-rectifier potassium current current, which is enhanced by protein kinase A (PKA)-mediated phosphorylation. The observed current increase requires both phosphorylation of Kv7.1 and the presence of KCNE1. However, PKA also stimulates Kv7.1 currents in epithelial tissues, such as colon, where the channel does not coassemble with KCNE1. Here, we demonstrate that PKA activity significantly impacts the subcellular localization of Kv7.1 in Madin-Darby canine kidney cells. While PKA inhibition reduced the fraction of channels at the cell surface, PKA activation increased it. We show that PKA inhibition led to intracellular accumulation of Kv7.1 in late endosomes/lysosomes. By mass spectroscopy we identified eight phosphorylated residues on Kv7.1, however, none appeared to play a role in the observed response. Instead, we found that PKA acted by regulating endocytic trafficking involving the ubiquitin ligase Nedd4-2. We show that a Nedd4-2-resistant Kv7.1-mutant displayed significantly reduced intracellular accumulation upon PKA inhibition. Similar effects were observed upon siRNA knockdown of Nedd4-2. However, although Nedd4-2 is known to regulate Kv7.1 by ubiquitylation, biochemical analyses demonstrated that PKA did not influence the amount of Nedd4-2 bound to Kv7.1 or the ubiquitylation level of the channel. This suggests that PKA influences Nedd4-2-dependent Kv7.1 transport though a different molecular mechanism. In summary, we identify a novel mechanism whereby PKA can increase Kv7.1 current levels, namely by regulating Nedd4-2-dependent Kv7.1 transport.

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Section: Resultsmentioning

confidence: 99%

“…The plasmids pXOOM-hKv7.1, pXOOM-hKv7.1-YA, pXOOM-hNedd4-2, pXOOM-hNedd4-2-CS, and pEGFP-N2-hKv7.1 have been described (4,14,15,17). eGFP-Rab7 was a kind gift from Guiscard Seebohm.…”

Section: Chemical Compounds N-[2-(p-bromocinnamylamino)ethyl]-5-isoqmentioning

confidence: 99%

Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

Andersen

Hefting

Steffensen

et al. 2015

American Journal of Physiology-Cell Physiology

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“…E3 typically determines substrate specificity of the ubiquitination reaction. Interestingly, two highly homologous E3 enzymes, NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) and to a lesser extent NEDD4-1, have been implicated in ubiquitination of many mammalian transporters and channels (21)(22)(23)(24)(25)(26). These enzymes contain the catalytic HECT (homologous E6-AP carboxyl-terminal) domain.…”

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confidence: 99%

Protein Kinase C-dependent Ubiquitination and Clathrin-mediated Endocytosis of the Cationic Amino Acid Transporter CAT-1

Viña-Vilaseca

Bender-Sigel

Sorkina

et al. 2011

Journal of Biological Chemistry

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Cationic amino acid transporter 1 (CAT-1) is responsible for the bulk of the uptake of cationic amino acids in most mammalian cells. Activation of protein kinase C (PKC) leads to downregulation of the cell surface CAT-1. To examine the mechanisms of PKC-induced down-regulation of CAT-1, a functional mutant of CAT-1 (CAT-1-HA-GFP) was generated in which a hemagglutinin antigen (HA) epitope tag was introduced into the second extracellular loop and GFP was attached to the carboxyl terminus. CAT-1-HA-GFP was stably expressed in porcine aorthic endothelial and human epithelial kidney (HEK) 293 cells. Using the HA antibody internalization assay we have demonstrated that PKC-dependent endocytosis was strongly inhibited by siRNA depletion of clathrin heavy chain, indicating that CAT-1-HA-GFP internalization requires clathrin-coated pits. Internalized CAT-1-HA-GFP was accumulated in early, recycling, and late endosomes. PKC activation also resulted in ubiquitination of CAT-1. CAT-1 ubiquitination and endocytosis in phorbol ester-stimulated porcine aorthic endothelial and HEK293 cells were inhibited by siRNA knockdown of NEDD4-2 and NEDD4-1 E3 ubiquitin ligases, respectively. In contrast, ubiquitination and endocytosis of the dopamine transporter was dependent on NEDD4-2 in all cell types tested. Altogether, our data suggest that ubiquitination mediated by NEDD4-2 or NEDD4-1 leading to clathrin-mediated endocytosis is the common mode of regulation of various transporter proteins by PKC. Cationic amino acid transporter (CAT)-12 is a member of the SLC7 gene family of membrane transport proteins. The SLC7 family is divided into two groups, glycosylated (CAT) and nonglycosylated transporters (reviewed in Ref. 1). The latter associate with a glycosylated protein from the SLC3 family to form heteromeric amino acid transporters. There are four proven members in the CAT subfamily: CAT-1, CAT-2A, CAT-2B, and CAT-3 (reviewed in Ref. 2). All CATs have a predicted topology of 14 transmembrane domains with cytoplasmic amino and carboxyl termini. The largest, third extracellular loop is glycosylated in all CATs. CAT-2 proteins are also glycosylated in the second loop. CAT-1 is expressed ubiquitously in all adult tissues except the liver (reviewed in Ref.3). The transport properties of CAT-1 resemble those of system y ϩ , defined by the selectivity for cationic amino acids, K m of 0.1-0.2 mM, Na ϩ and pH independence, and strong trans-stimulation. CAT-1 is ubiquitously expressed and the main portal of entry for cationic amino acids into mammalian cells. Homozygous knockout of CAT-1 in mice is postnatally lethal (4). CAT-1 activity has been reported to be regulated through activation of protein kinase C (PKC) (5, 6). Even though there are some discrepancies, most studies conclude that activation of PKC leads to inhibition of L-Arg uptake due to the decreased activity of CAT-1. Studies of CAT-1 phosphorylation and mutations of the PKC phosphorylation consensus sites in CAT-1 suggested that CAT-1 inhibition by PKC is not due to CAT-1 phosp...

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“…In line with its distribution, it has a variety of membrane proteins as substrates. The ubiquitination mediated by Nedd4-2 is required for the down-regulation of many membrane proteins, such as voltage-gated Na ϩ channel (25), neurotrophin receptor TrkA (26), potassium channel KCNQ (27,28), Cl Ϫ channel ClC-2 (29), Na ϩ -glucose co-transporter SGLT1 (30), and Na ϩ -phosphate co-transporter NaPi IIb (24). The list of ion channels/transporters regulated by Nedd4-2 has grown longer since the identification of ENaC as a substrate of Nedd4-2.…”

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confidence: 99%

Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2

Zhang¹,

Na²,

et al. 2010

Journal of Biological Chemistry

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Nedd4-2 is an archetypal HECT ubiquitin E3 ligase that disposes target proteins for degradation. Because of the proven roles of Nedd4-2 in degradation of membrane proteins, such as epithelial Na ؉ channel, we examined the effect of Nedd4-2 on the apical Ca 2؉ channel TRPV6, which is involved in transcellular Ca 2؉ transport in the intestine using the Xenopus laevis oocyte system. We demonstrated that a significant amount of Nedd4-2 protein was distributed to the absorptive epithelial cells in ileum, cecum, and colon along with TRPV6. When coexpressed in oocytes, Nedd4-2 and, to a lesser extent, Nedd4 down-regulated the protein abundance and Ca 2؉ influx of TRPV6 and TRPV5, respectively. TRPV6 ubiquitination was increased, and its stability was decreased by Nedd4-2. The Nedd4-2 inhibitory effects on TRPV6 were partially blocked by proteasome inhibitor MG132 but not by the lysosome inhibitor chloroquine. The rate of TRPV6 internalization was not significantly altered by Nedd4-2. The HECT domain was essential to the inhibitory effect of Nedd4-2 on TRPV6 and to their association. The WW1 and WW2 domains interacted with TRPV6 terminal regions, and a disruption of the interactions by D204H and D376H mutations in the WW1 and WW2 domains increased TRPV6 ubiquitination and degradation. Thus, WW1 and WW2 may serve as a molecular switch to limit the ubiquitination of TRPV6 by the HECT domain. In conclusion, Nedd4-2 may regulate TRPV6 protein abundance in intestinal epithelia by controlling TRPV6 ubiquitination.

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The KCNQ1 potassium channel is down-regulated by ubiquitylating enzymes of the Nedd4/Nedd4-like family

Cited by 128 publications

References 26 publications

Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

Protein Kinase C-dependent Ubiquitination and Clathrin-mediated Endocytosis of the Cationic Amino Acid Transporter CAT-1

Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2

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