WNK4 diverts the thiazide-sensitive NaCl cotransporter to the lysosome and stimulates AP-3 interaction

Arohan R. Subramanya, Jie Liu, David Ellison, James B. Wade, Paul A. Welling

Research output: Contribution to journalArticle

83 Citations (Scopus)

Abstract

With-no-lysine kinase 4 (WNK4) inhibits electroneutral sodium chloride reabsorption by attenuating the cell surface expression of the thiazide-sensitive NaCl cotransporter (NCC). The underlying mechanism for this effect remains poorly understood. Here, we explore how WNK4 affects the trafficking of NCC through its interactions with intracellular sorting machinery. An analysis of NCC cell surface lifetime showed that WNK4 did not alter the net rate of cotransporter internalization. In contrast, direct measurements of forward trafficking revealed that WNK4 attenuated the rate of NCC surface delivery, inhibiting the anterograde movement of cotransporters traveling to the plasma membrane from the trans-Golgi network. The response was paralleled by a dramatic reduction in NCC protein abundance, an effect that was sensitive to the lysosomal protease inhibitor leupeptin, insensitive to proteasome inhibition, and attenuated by endogenous WNK4 knockdown. Subcellular localization studies performed in the presence of leupeptin revealed that WNK4 enhanced the accumulation of NCC in lysosomes. Moreover, NCC immunoprecipitated with endogenous AP-3 complexes, and WNK4 increased the fraction of cotransporters that associate with this adaptor, which facilitates cargo transport to lysosomes. WNK4 expression also increased LAMP-2-positive lysosomal content, indicating that the kinase may act by a general AP-3-dependent mechanism to promote cargo delivery into the lysosomal pathway. Taken together, these findings indicate that WNK4 inhibits NCC activity by diverting the cotransporter to the lysosome for degradation by way of an AP-3 transport carrier.

Original languageEnglish (US)
Pages (from-to)18471-18480
Number of pages10
JournalJournal of Biological Chemistry
Volume284
Issue number27
DOIs
StatePublished - Jul 3 2009

Fingerprint

Thiazides
Lysosomes
Lysine
Phosphotransferases
thiazide receptor
trans-Golgi Network
Carrier transport
Proteasome Endopeptidase Complex
Cell membranes
Protease Inhibitors
Sorting
Sodium Chloride
Machinery
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

WNK4 diverts the thiazide-sensitive NaCl cotransporter to the lysosome and stimulates AP-3 interaction. / Subramanya, Arohan R.; Liu, Jie; Ellison, David; Wade, James B.; Welling, Paul A.

In: Journal of Biological Chemistry, Vol. 284, No. 27, 03.07.2009, p. 18471-18480.

Research output: Contribution to journalArticle

Subramanya, Arohan R. ; Liu, Jie ; Ellison, David ; Wade, James B. ; Welling, Paul A. / WNK4 diverts the thiazide-sensitive NaCl cotransporter to the lysosome and stimulates AP-3 interaction. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 27. pp. 18471-18480.
@article{4f9e6dd5969c410eae45981d156e7a5b,
title = "WNK4 diverts the thiazide-sensitive NaCl cotransporter to the lysosome and stimulates AP-3 interaction",
abstract = "With-no-lysine kinase 4 (WNK4) inhibits electroneutral sodium chloride reabsorption by attenuating the cell surface expression of the thiazide-sensitive NaCl cotransporter (NCC). The underlying mechanism for this effect remains poorly understood. Here, we explore how WNK4 affects the trafficking of NCC through its interactions with intracellular sorting machinery. An analysis of NCC cell surface lifetime showed that WNK4 did not alter the net rate of cotransporter internalization. In contrast, direct measurements of forward trafficking revealed that WNK4 attenuated the rate of NCC surface delivery, inhibiting the anterograde movement of cotransporters traveling to the plasma membrane from the trans-Golgi network. The response was paralleled by a dramatic reduction in NCC protein abundance, an effect that was sensitive to the lysosomal protease inhibitor leupeptin, insensitive to proteasome inhibition, and attenuated by endogenous WNK4 knockdown. Subcellular localization studies performed in the presence of leupeptin revealed that WNK4 enhanced the accumulation of NCC in lysosomes. Moreover, NCC immunoprecipitated with endogenous AP-3 complexes, and WNK4 increased the fraction of cotransporters that associate with this adaptor, which facilitates cargo transport to lysosomes. WNK4 expression also increased LAMP-2-positive lysosomal content, indicating that the kinase may act by a general AP-3-dependent mechanism to promote cargo delivery into the lysosomal pathway. Taken together, these findings indicate that WNK4 inhibits NCC activity by diverting the cotransporter to the lysosome for degradation by way of an AP-3 transport carrier.",
author = "Subramanya, {Arohan R.} and Jie Liu and David Ellison and Wade, {James B.} and Welling, {Paul A.}",
year = "2009",
month = "7",
day = "3",
doi = "10.1074/jbc.M109.008185",
language = "English (US)",
volume = "284",
pages = "18471--18480",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "27",

}

TY - JOUR

T1 - WNK4 diverts the thiazide-sensitive NaCl cotransporter to the lysosome and stimulates AP-3 interaction

AU - Subramanya, Arohan R.

AU - Liu, Jie

AU - Ellison, David

AU - Wade, James B.

AU - Welling, Paul A.

PY - 2009/7/3

Y1 - 2009/7/3

N2 - With-no-lysine kinase 4 (WNK4) inhibits electroneutral sodium chloride reabsorption by attenuating the cell surface expression of the thiazide-sensitive NaCl cotransporter (NCC). The underlying mechanism for this effect remains poorly understood. Here, we explore how WNK4 affects the trafficking of NCC through its interactions with intracellular sorting machinery. An analysis of NCC cell surface lifetime showed that WNK4 did not alter the net rate of cotransporter internalization. In contrast, direct measurements of forward trafficking revealed that WNK4 attenuated the rate of NCC surface delivery, inhibiting the anterograde movement of cotransporters traveling to the plasma membrane from the trans-Golgi network. The response was paralleled by a dramatic reduction in NCC protein abundance, an effect that was sensitive to the lysosomal protease inhibitor leupeptin, insensitive to proteasome inhibition, and attenuated by endogenous WNK4 knockdown. Subcellular localization studies performed in the presence of leupeptin revealed that WNK4 enhanced the accumulation of NCC in lysosomes. Moreover, NCC immunoprecipitated with endogenous AP-3 complexes, and WNK4 increased the fraction of cotransporters that associate with this adaptor, which facilitates cargo transport to lysosomes. WNK4 expression also increased LAMP-2-positive lysosomal content, indicating that the kinase may act by a general AP-3-dependent mechanism to promote cargo delivery into the lysosomal pathway. Taken together, these findings indicate that WNK4 inhibits NCC activity by diverting the cotransporter to the lysosome for degradation by way of an AP-3 transport carrier.

AB - With-no-lysine kinase 4 (WNK4) inhibits electroneutral sodium chloride reabsorption by attenuating the cell surface expression of the thiazide-sensitive NaCl cotransporter (NCC). The underlying mechanism for this effect remains poorly understood. Here, we explore how WNK4 affects the trafficking of NCC through its interactions with intracellular sorting machinery. An analysis of NCC cell surface lifetime showed that WNK4 did not alter the net rate of cotransporter internalization. In contrast, direct measurements of forward trafficking revealed that WNK4 attenuated the rate of NCC surface delivery, inhibiting the anterograde movement of cotransporters traveling to the plasma membrane from the trans-Golgi network. The response was paralleled by a dramatic reduction in NCC protein abundance, an effect that was sensitive to the lysosomal protease inhibitor leupeptin, insensitive to proteasome inhibition, and attenuated by endogenous WNK4 knockdown. Subcellular localization studies performed in the presence of leupeptin revealed that WNK4 enhanced the accumulation of NCC in lysosomes. Moreover, NCC immunoprecipitated with endogenous AP-3 complexes, and WNK4 increased the fraction of cotransporters that associate with this adaptor, which facilitates cargo transport to lysosomes. WNK4 expression also increased LAMP-2-positive lysosomal content, indicating that the kinase may act by a general AP-3-dependent mechanism to promote cargo delivery into the lysosomal pathway. Taken together, these findings indicate that WNK4 inhibits NCC activity by diverting the cotransporter to the lysosome for degradation by way of an AP-3 transport carrier.

UR - http://www.scopus.com/inward/record.url?scp=67650533826&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=67650533826&partnerID=8YFLogxK

U2 - 10.1074/jbc.M109.008185

DO - 10.1074/jbc.M109.008185

M3 - Article

C2 - 19401467

AN - SCOPUS:67650533826

VL - 284

SP - 18471

EP - 18480

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 27

ER -