Investigating the Biological Functions of the Protein Kinase WNK1 in the Regulation of Cytoskeletal Structures and Membrane Trafficking

With No Lysine (WNK) 1, a serine/threonine kinase, is a unique kinase to its catalytic lysine residue at a non-canonical position relative to all other kinases. Characterization of endogenous WNK1 distribution by immunofluorescence reveals a perinuclear punctate pattern. I have investigated this perinuclear distribution and how it might relate to the biological functions of WNK1 from two aspects. First, I investigated cytoskeletal structures mainly focused on the microtubules. WNK1 localized on mitotic spindles during mitosis as well as interphase microtubules. Depletion of WNK1 caused aberrant mitotic spindles, chromosomes and defect of abscission. In interphase cells, disruption of radiating microtubules from microtubule organization center was observed. Centrosomal structure was impaired. Cells showed a migratory defect. Clues from a former student and my mass spectrometry data suggested that dynein and its associated protein-dynactin, centrosomal protein of 70 and 170 kDa might be potential interactors mediating microtubule related phenotypes. Second, I examined WNK1 and membrane trafficking events. Depletion of WNK1 caused higher amount of epidermal growth factor receptors remained at the later step of endocytosis. Lysosomes and lysosome-related organelles were disrupted. Biochemical assay suggested that WNK1 could associate with active Rab 6 or 7 effector complexes. I have identified that the homotypic fusion and vacuole protein sorting (HOPS) complex, one of Rab7 effector complexes could interact with WNK1. Mass spectrometry results showed that WNK1 could pull down clathrin heavy chain and adaptor protein complex-3 (AP-3) β subunit. AP-3 vesicles are also HOPS complex-mediated vesicular trafficking between the Trans-Golgi network and late endosomes. Co-localization analysis suggested that WNK1 co-localized with AP-3 in a high pearson correlation coefficient (0.53). Depletion of WNK1 showed defect of the maturation of autophagosomes. Taken together, WNK1 might affect membrane trafficking through HOPS complex-mediated homotypic (the assembly of phagophores) and heterotypic (late endosomes and lysosomes) membrane fusion.