Identification of Substrates and Pathways Regulated by WNK1
type: CreativeWork
description: WNK (With No lysine (K)), a serine/threonine protein kinase, is a unique molecule not belonging to any other canonical protein kinase family including mitogen-activated protein (MAP) kinases. The name of the WNK protein kinase family reflects the fact that a catalytic lysine lies in a position different in WNKs from that in all other protein kinases. The urgency of a mechanistic examination of the WNK family protein kinase was heightened by the discovery that mutations in at least two of the four human WNKs, WNK1 and 4, caused a heritable form of hypertension. My study focused on unveiling WNK1 substrates and interactors for a better understanding of the molecular pathways served by WNK kinases. Yeast two-hybrid screening was performed to identify the binding partners of WNK1 and yielded genuine interactors including synaptotagmin (Syt) isoforms, Smad2, and dynein light chain (LC8/PIN). WNK1, not WNK4, selectively binds to and phosphorylates Syt2 within its calcium binding C2 domains. Calcium strongly enhanced their binding in vitro. Essential Ca2+-binding residues in the Syt2 C2 domains were critical for formation of a WNK1-Syt2 complex and for Syt2 phosphorylation. WNK1 displayed specificity among Syt isoforms and mutational analysis implicated a hydrophobic residue on the WNK1 kinase domain surface as essential for the high affinity WNK1-Syt2 interaction and phosphorylation. Endogenous WNK1 and Syt2 coimmunoprecipitated and colocalized on a subset of secretory granules in the INS-1 cell line, a pancreatic beta cell model system. Importantly, phosphorylation by WNK1 increased the amount of Ca2+ required for Syt2 binding to phospholipid vesicles; mutation of Thr202, a WNK1 phosphorylation site identified from mass spectrometric analysis, partially prevented this change. These findings provide a biochemical scenario that could lead to the retention or insertion of proteins in the plasma membrane. WNK1 may serve as a molecular switch for vesicle trafficking and other…
abstract: WNK (With No lysine (K)), a serine/threonine protein kinase, is a unique molecule not belonging to any other canonical protein kinase family including mitogen-activated protein (MAP) kinases. The name of the WNK protein kinase family reflects the fact that a catalytic lysine lies in a position different in WNKs from that in all other protein kinases. The urgency of a mechanistic examination of the WNK family protein kinase was heightened by the discovery that mutations in at least two of the four human WNKs, WNK1 and 4, caused a heritable form of hypertension. My study focused on unveiling WNK1 substrates and interactors for a better understanding of the molecular pathways served by WNK kinases. Yeast two-hybrid screening was performed to identify the binding partners of WNK1 and yielded genuine interactors including synaptotagmin (Syt) isoforms, Smad2, and dynein light chain (LC8/PIN). WNK1, not WNK4, selectively binds to and phosphorylates Syt2 within its calcium binding C2 domains. Calcium strongly enhanced their binding in vitro. Essential Ca2+-binding residues in the Syt2 C2 domains were critical for formation of a WNK1-Syt2 complex and for Syt2 phosphorylation. WNK1 displayed specificity among Syt isoforms and mutational analysis implicated a hydrophobic residue on the WNK1 kinase domain surface as essential for the high affinity WNK1-Syt2 interaction and phosphorylation. Endogenous WNK1 and Syt2 coimmunoprecipitated and colocalized on a subset of secretory granules in the INS-1 cell line, a pancreatic beta cell model system. Importantly, phosphorylation by WNK1 increased the amount of Ca2+ required for Syt2 binding to phospholipid vesicles; mutation of Thr202, a WNK1 phosphorylation site identified from mass spectrometric analysis, partially prevented this change. These findings provide a biochemical scenario that could lead to the retention or insertion of proteins in the plasma membrane. WNK1 may serve as a molecular switch for vesicle trafficking and other…
author: Lee, Byung-Hoon
language: en
keywords: Protein-Serine-Threonine Kinases, Mutagenesis, Hypertension
date published: 2010-07-12T18:33:27Z
date modified: 2026-07-24T05:52:24Z
provider: University of Texas Southwestern Medical Center
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Texas Southwestern Medical Center

PropertyValue
property ID: Handle
value: https://hdl.handle.net/2152.5/599