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Non-enzymatic roles of kinases and phosphatases - the case of MTMR9 and AAK1
Knop, Filip ; Macůrková, Marie (advisor) ; Harnoš, Jakub (referee) ; Soukup, Vladimír (referee)
Enzymatic roles of kinases and phosphatases in almost every aspect of cellular life are well described in a wide variety of examples. Lately the role of the same proteins independent of their catalytic activity is being increasingly appreciated. In this work, we focus on two proteins, mammalian MTMR9, and Caenorhabditis elegans SEL-5/AAK1. MTMR9 belongs to the myotubularin-related family of lipid phosphatases (MTMR) and is known to be a pseudophosphatase, a catalytically inactive member of the MTMR group. SEL-5/AAK1, on the other hand, is characterized by its kinase activity with at least two putative substrates identified so far. We described the localization of MTMR9 to early secretory pathway and its colocalization with known ER-to-GA compartment (ERGIC) markers. We also identified several possible MTMR9-interacting partners, such as RAB1 and MTMR6, whose localization and/or activity could be potentially regulated by MTMR9 binding. Disruption of proper MTMR9 levels led to an alteration in WNT3A secretion and subsequently to a reduced activity of the Wnt signaling pathway. Similarly, we identified SEL-5/AAK1 role in two separate Wnt-regulated developmental processes in C. elegans. Firstly, SEL-5 along with other members of the retromer complex regulate a proper QL.d migration. Secondly, excretory...
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Vesicular roles of Arp2/3 nucleation-promoting factors
Dostál, Vojtěch ; Libusová, Lenka (advisor) ; Malínský, Jan (referee) ; Befekadu, Asfaw (referee)
F-actin is involved in key aspects of vesicular traffic, such as membrane deformation, tubulation and vesicle motion. Branching of F-actin is mediated by Arp2/3 but this complex must first be activated by so-called nucleation-promoting factors (NPFs). These factors play an essential role in the decision where and when branched actin should form on the membrane surface. The thesis focuses on the mechanisms which underlie localization and activation of NPFs, especially in terms of the phosphoinositide composition of the vesicle membranes. I show that one of the NPFs, the WASH complex, does not exclusively depend on the retromer complex for its membrane anchoring, as previously theorized. Rather, its understudied subunit SWIP enables the complex to independently bind to the membrane. I also present data showing that the WASH complex has essential roles in maintaining lysosomal function. Additionally, I elucidate the function of another NPF known as WHAMM in the ERGIC compartment, showing that it depends on the presence of myotubularin 9 for its ability to form membrane tubules. The thesis improves our understanding of the interface between the actin cytoskeleton and intracellular membrane system.
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Binding proteins of MTMR9
Holšteinová, Aneta ; Doubravská, Lenka (advisor) ; Cebecauer, Marek (referee)
Myotubularins are lipid phosphatases that dephosphorylate phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate the position three of the inositol ring. This allows them to regulate the structure of the lipid layer of the membrane compartment. The first member of the family was described in association with a severe hereditary myopathy. From that point on, another thirteen members have been added to the family. The catalytically inactive MTMR9 carrying the conserved mutation in the phosphatase domain regulates the localization of the marker of the early secretory pathway, RAB1A, the cis-Golgi structure and the secretion. MTMR9 interacts with the catalytically active MTMR6 and MTMR8 that specifically localizes and increases their phophatase activity. The aim of this diploma thesis was to find out whether the phenotype observed in cells with altered MTMR9 levels is dependent on the catalytically active phosphatases MTMR6 and MTMR8. We proved the influence of MTMR6 and MTMR8 on the distribution of tranfected RAB1A between the intermediate compartment and the Golgi apparatus. MTMR6 and MTMR8 also take part in regulating the cis-Golgi structure. By the use of two different approaches we did not manage to clarify the influence of MTMR6 and MTMR8 on secretion. Changes in the catalytic...
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