Activation of PI3-kinase stimulates endocytosis of ROMK via Akt1/SGK1-dependent phosphorylation of WNK1

Chih-Jen Cheng; Chou-Long Huang

doi:10.1681/ASN.2010060681

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Activation of PI3-kinase stimulates endocytosis of ROMK via Akt1/SGK1-dependent phosphorylation of WNK1

Journal article

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Peer reviewed

Activation of PI3-kinase stimulates endocytosis of ROMK via Akt1/SGK1-dependent phosphorylation of WNK1

Chih-Jen Cheng and Chou-Long Huang

Journal of the American Society of Nephrology, Vol.22(3), pp.460-471

03/2011

DOI: 10.1681/ASN.2010060681

PMCID: PMC3060440

PMID: 21355052

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Abstract

WNK kinases stimulate endocytosis of ROMK channels to regulate renal K+ handling. Phosphatidylinositol 3-kinase (PI3K)-activating hormones, such as insulin and IGF 1, phosphorylate WNK1, but how this affects the regulation of ROMK abundance is unknown. Here, serum starvation of ROMK-transfected HEK cells led to an increase of ROMK current density; subsequent addition of insulin or IGF1 inhibited ROMK currents in a PI3K-dependent manner. Serum and insulin also increased phosphorylation of the downstream kinases Akt1 and SGK1 as well as WNK1. A biotinylation assay suggested that insulin and IGF1 inhibit ROMK by enhancing its endocytosis, a process that WNK1 may mediate. Knockdown of WNK1 with siRNA or expression of a phospho-deficient WNK1 mutant (T58A) both prevented insulin-induced inhibition of ROMK currents, suggesting that phosphorylation at Threonine-58 of WNK1 is important to mediate the inhibition of ROMK by PI3K-activating hormones or growth factors. In vitro and in vivo kinase assays supported the notion that Akt1 and SGK1 can phosphorylate WNK1 at this site, and we established that Akt1 and SGK1 synergistically inhibit ROMK through WNK1. We used dominant-negative intersectin and dynamin constructs to show that SGK1-mediated phosphorylation of WNK1 inhibits ROMK by promoting its endocytosis. Taken together, these results suggest that PI3K-activating hormones inhibit ROMK by enhancing its endocytosis via a mechanism that involves phosphorylation of WNK1 by Akt1 and SGK1.

Phosphorylation

Endocytosis - drug effects

Endocytosis - physiology

Gene Expression Regulation - drug effects

HEK293 Cells

Humans

Immediate-Early Proteins - metabolism

Insulin - pharmacology

Insulin-Like Growth Factor I - pharmacology

Intracellular Signaling Peptides and Proteins

Kidney - cytology

Kidney - drug effects

Kidney - metabolism

Minor Histocompatibility Antigens

Patch-Clamp Techniques

Phosphatidylinositol 3-Kinases - drug effects

Phosphatidylinositol 3-Kinases - metabolism

Potassium Channels, Inwardly Rectifying - metabolism

Protein-Serine-Threonine Kinases - genetics

Protein-Serine-Threonine Kinases - metabolism

Proto-Oncogene Proteins c-akt - metabolism

RNA, Small Interfering - pharmacology

Signal Transduction - physiology

WNK Lysine-Deficient Protein Kinase 1

Details

Title: Subtitle: Activation of PI3-kinase stimulates endocytosis of ROMK via Akt1/SGK1-dependent phosphorylation of WNK1
Creators: Chih-Jen Cheng - The University of Texas Southwestern Medical Center
Chou-Long Huang - Internal Medicine
Resource Type: Journal article
Publication Details: Journal of the American Society of Nephrology, Vol.22(3), pp.460-471
DOI: 10.1681/ASN.2010060681
PMID: 21355052
PMCID: PMC3060440
ISSN: 1046-6673
eISSN: 1533-3450
Grant note: R01 DK059530 / NIDDK NIH HHS P30DK079328 / NIDDK NIH HHS R01DK-59530 / NIDDK NIH HHS P30 DK079328 / NIDDK NIH HHS
Language: English
Date published: 03/2011
Academic Unit: Nephrology; Internal Medicine
Record Identifier: 9984359936002771

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