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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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