Identification of Substrates and Pathways Regulated by WNK1

dc.contributor.advisorCobb, Melanie H.en
dc.creatorLee, Byung-Hoonen
dc.date.accessioned2010-07-12T18:33:27Z
dc.date.available2010-07-12T18:33:27Z
dc.date.issued2004-12-15
dc.description.abstractWNK (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 membrane events that regulate ion balance. The interaction with and phosphorylation of other molecules by WNK1 were also investigated here.en
dc.format.digitalOriginborn digitalen
dc.format.mediumElectronicen
dc.format.mimetypeapplication/pdfen
dc.identifier.oclc58973838
dc.identifier.urihttps://hdl.handle.net/2152.5/599
dc.language.isoenen
dc.subjectProtein-Serine-Threonine Kinasesen
dc.subjectMutagenesisen
dc.subjectHypertensionen
dc.titleIdentification of Substrates and Pathways Regulated by WNK1en
dc.typeThesisen
dc.type.genredissertationen
dc.type.materialTexten
thesis.date.available2005-12-15
thesis.degree.departmentGraduate School of Biomedical Sciencesen
thesis.degree.disciplineGenetics and Developmenten
thesis.degree.grantorUT Southwestern Medical Centeren
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophyen

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