title: WNK4 inhibits plasma membrane targeting of NCC through regulation of syntaxin13 SNARE formation.
abstract: WNK4, a serine/threonine kinase, plays a critical role in the expression of membrane proteins in the cell surface; however, the underlying mechanism of WNK4 is not clear. Here, we demonstrate that WNK4 inhibits the fusion of plasma membrane delivering vesicle with sorting/recycling endosome through disrupting SNARE formation of syntaxin13, an endosomal t-SNARE and VAMP2, the v-SNARE in plasma membrane delivering vesicle. Their interaction and co-localization were enhanced by hyperosmotic stimulation which is known for WNK4 activation. The kinase domain of WNK4 interacts with the transmembrane domain (TM) of syntaxin13 and this interaction was abolished when the TM was replaced with that of syntaxin16. Interestingly, cell fractionation using sucrose gradients revealed that WNK4 inhibited the formation of the syntaxin13/VAMP2 SNARE complex in the endosomal compartment, but not syntaxin16/VAMP2 or syntaxin13/VAMP7. Syntaxin13 was not phosphorylated by WNK4 and WNK4KI also showed the same binding strength and similar inhibitory regulation on SNARE formation of syntaxin13. Physiological relevance of this mechanism was proved with the expression of NCC (Na(+) C1(-) co-transporter) in the cell surface. The inhibiting activity of WNK4 on surface expression of NCC was abolished by syntaxin13 siRNA transfection. These results suggest that WNK4 attenuates PM targeting of NCC proteins through regulation of syntaxin13 SNARE complex formation with VAMP2 in recycling and sorting endosome.
authors.affiliation: Department of Pharmacology and Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea.
authors.forename: Woo Young
authors.initials: WY
authors.lastname: Chung
authors.forename: Hyun Woo
authors.initials: HW
authors.lastname: Park
authors.forename: Jung Woo
authors.initials: JW
authors.lastname: Han
authors.forename: Min Goo
authors.initials: MG
authors.lastname: Lee
authors.forename: Joo Young
authors.initials: JY
authors.lastname: Kim
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D060066:Protein Interaction Maps; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins / Q000737:chemistry / Q000378:metabolism*; D049992:Sodium Chloride Symporters / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism*
chemical_list: D050765:Qa-SNARE Proteins; D049992:Sodium Chloride Symporters; C494807:VAMP2 protein, human; D050988:Vesicle-Associated Membrane Protein 2; D017346:Protein Serine-Threonine Kinases; C433806:WNK4 protein, human
keywords: NCC; NCC (Na(+) Cl(−) co-transporter); Na(+)Cl(−)co-transporter; OSR; PHAII; Recycling/sorting endosome; SAPK; SNARE; SNARE complex; STE20/SPS1-related proline/alanine-rich kinase; Syntaxin13; VAMP; VAMP2; WNK4; WNK4 kinase; oxidative stress responsive kinase; pseudohypoaldosteronism type II; soluble NSF attachment protein receptor; vesicle-associated membrane protein; with-no-lysine (K) kinase 4