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Kinases regulating AP2 complex phosphorylation in Caenorhabditis elegans
Zounarová, Apolena ; Macůrková, Marie (advisor) ; Vinopal, Stanislav (referee)
Heterotetrameric adaptor protein 2 complex (AP2) is a fundamental component of clathrin-coated pits playing a part in every step of clathrin-coated vesicle generation. Although the mechanism of AP2 function has been extensively studied for no less than 20 years, the exact role of the regulatory phosphorylation on T156 of its μ2 subunit remains unclear. The main kinase responsible for the T156 phosphorylation in mammals is AAK1; however, many observations suggest that other kinases collaborate on this event. The aim of this project is to shed light on the importance of AP2 phosphorylation in Caenorhabditis elegans and elucidate the function of SEL-5/AAK1 kinase. To determine the relationship between SEL-5/AAK1, AP2 phosphorylation, and the function of endocytosis, we used a combination of phenotype analysis of C. elegans transgenic lines and in vitro assays. We showed that DPY-23, the C. elegans μ2 subunit, is phosphorylated analogously to its human orthologue. We confirmed that DPY-23 phosphorylation depends on SEL-5, yet we were not able to prove the kinase activity of SEL-5 directly. Interestingly, our results further revealed that DPY-23 phosphorylation is dispensable for the endocytosis of a model cargo, and we also showed that interaction motifs located outside the kinase domain of SEL-5 are...
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Mechanisms regulating the function of adaptor protein 2 complex during endocytosis
Zounarová, Apolena ; Macůrková, Marie (advisor) ; Pleskot, Roman (referee)
Adaptor protein complex 2 (AP2) mediates the interaction of clathrin with the plasma membrane and thus enables the clathrin-coated vesicle formation. AP2 is also responsible for cargo recognition and it recognizes cargo either directly using endocytic motifs YxxΦ or [DE]xxxL[LI] in the cytosolic domains of cargo or indirectly via additional adaptor proteins from which β-arrestin and ARH are the best-known. The binding sites for endocytic motifs are located in the core of AP2 complex and, similarly to the clathrin-binding site, they are blocked by autoinhibitory mechanism in the inactive cytosolic form of AP2. Therefore, binding of endocytic motifs and clathrin must be preceded by conformational change of AP2 complex which is triggered by membrane-bound phosphatidylinositol-4,5-bisphosphates and greatly facilitated by phosphorylation at Thr156 by AAK1 kinase. AP2 is also important for later stages of endocytosis during which it recruits proteins responsible for membrane curvature, fission, and eventual disassembly of clathrin coat. Repeated association of AP2 with the plasma membrane is prevented by the protein NECAP, but the mechanism of inactivation is still poorly understood.
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