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Browsing by Author "Domènech Moreno, Eva"

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  • Domènech Moreno, Eva (2017)
    In this Master’s project, I have studied a mammalian serine-threonine kinase NUAK2 implicated in human disease but whose molecular functions and interacting proteins are as of yet poorly characterized. The goal was to identify new interacting proteins to increase understanding of the molecular functions and potentially link to human physiology and disease. Recent work from the host lab shows NUAK2 loss in cultured primary cells mimics loss of the tumor suppressor LKB1 which also acts upstream of NUAK2, together suggesting NUAK2 could be involved in tumor suppression. Currently, only two protein-protein interacting proteins with NUAK2 have been identified: NUAK2 is targeted to actin stress fibers by the myosin phosphatase Rho-interacting protein (MRIP), and it is involved in regulating cell contractility by affecting indirectly the phosphorylation cycle of the myosin light chain through inactivation of the myosin phosphatase target subunit 1 (MYPT1). In this project, I utilized a novel protein-protein interaction screening method that utilizes proximity-dependent biotin labeling to identify new interacting proteins with NUAK2 in human embryonic kidney cells (HEK 293). This method is based on fusing an E.Coli promiscuous biotin ligase, BirA*(R118G), to the investigated protein. The BirA*(R118G) ligase biotinylates all the proteins in close proximity of the fusion protein creating a history of protein-protein associations over time. Afterwards, the biotinylated proteins can be isolated by affinity purification methods and identified by mass-spectrometry. The screening identified the previously known interaction partners of NUAK2 indicating it was technically successful. In addition, I also identified in total 108 novel potential protein interaction partners for NUAK2. One of the top hits was Cytospin-A, a cross-linking protein between microtubules and actin cytoskeleton, supporting a role of NUAK2 as regulator of cytoskeleton. Supporting the validity of our finding, Cytospin-A depletion in mammalian cells causes defective actin-cytoskeleton reorganization, a very similar phenotype seen with NUAK2 depletion. In future studies, I will continue to investigate the specific role of NUAK2 and Cytospin-A aiming for detailed information on the function of NUAK2 in regulation of microtubules and actin cytoskeleton. Validation of some of the other identified interactions is expected to provide novel insights to the biology and role of NUAK2 in LKB1 tumor suppressor functions.