WNK4 inhibits plasma membrane targeting of NCC through regulation of syntaxin13 SNARE formation.
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.
['Chung|Woo Young|WY|', 'Park|Hyun Woo|HW|', 'Han|Jung Woo|JW|', 'Lee|Min Goo|MG|', 'Kim|Joo Young|JY|']
D002462:Cell Membrane D011992:Endosomes D057809:HEK293 Cells D006801:Humans D060066:Protein Interaction Maps D017346:Protein Serine-Threonine Kinases D017434:Protein Structure, Tertiary D050765:Qa-SNARE Proteins D049992:Sodium Chloride Symporters D050988:Vesicle-Associated Membrane Protein 2
WNK4 NCC Syntaxin13 SNARE VAMP2 SNARE complex NCC (Na(+) Cl(−) co-transporter) Na(+)Cl(−)co-transporter OSR PHAII Recycling/sorting endosome SAPK STE20/SPS1-related proline/alanine-rich kinase VAMP WNK4 kinase oxidative stress responsive kinase pseudohypoaldosteronism type II soluble NSF attachment protein receptor vesicle-associated membrane protein with-no-lysine (K) kinase 4