Wojtal, Kacper Andrze

English

cAMP-dependent protein kinase A is one of the most important enzymes in the eukaryotic cell. The function of this protein is strictly in a close relation to the signaling pathways, which trigger the production of intracellular secondary messenger –cAMP. As a consequence of PKA activation numerous protein substrates are being phosphorylated and consequently signal is propagated downstream to the effector proteins. PKA-mediated phosphorylation is implicated in many physiological processes like intracellular transport, gene expression and/or cytoskeleton dynamics. Many of these processes are mediated by direct phosphorylation by PKA but recently a number of publications indicated the involvement of anchoring of PKA holoenzymes as the important factor. The work presented in this thesis answers some of the questions related to the involvement of cAMP/PKA signaling pathway in the process of development of polarity of hepatocytes. In this thesis we have demonstrated that anchoring of one of subunits of PKA (RIIα) is regulating the appearance of one of the apical proteins (MDR1) in a manner that is dependent of the presence of glucosylceramide (Chapter 2). Although, the exact mechanism of this dependency has not been revealed yet, we may speculate that PKA RIIα is required at the level of membrane microdomains located at the sorting center such as Golgi complex. Interestingly, the affected sorting pathway did not have any impact on other apical proteins such as DPP4 or MRP2 suggesting that anchoring of PKA RII is necessary for trafficking/sorting of some but not all apical proteins. Moreover, the perturbation of PKA RII anchoring at the Golgi resulted in missorting of glucosylceramide to the basolateral plasma membrane, which in normal conditions travels directly to the apical surface. Interestingly, we have shown that anchoring of PKA RII subunits at Golgi located AKAP (A Kinase Anchoring Protein) such as BIG2 is crucial for maintaining proper Golgi morphology (Chapter 3). We have observed that this interaction is important for vesiculation/fusion events at the Golgi apparatus, and as a consequence controls formation of organized stacked cisternae. Importantly, the organization of MT network appears to be not affected by displacement of PKA RII from BIG2. Scattering of Golgi stacks was observed also when cells were incubated with BFA, but this mechanism did not affect interaction between BIG2 and PKA RII. Interestingly, downregulation of BIG2 using siRNA approach triggered scattering of Golgi complex, which was in concert with our earlier observations, that BIG2 and its anchored PKA RII is crucial for maintenance integrity of Golgi complexes in hepatocytes. In Chapter 4 we demonstrate that OSM, but not cAMP stimulate polarity development in a molecular mechanism that is dependent on apical positioning of centrosomes. Interestingly, again anchoring of PKA RII seems to play crucial role for this process, and we propose that pericentrin is an AKAP responsible for those events. Mispositioning of centrosomes did not affect ability of centrosomes to nucleate MT’s and displacement of PKA RIIα from centrosomal AKAP’s do not affect MT network. We propose that OSM mediated elevation of polarity requires repositioning of PKA RII positive centrosomes in the vicinity of apical plasma membrane, in order to utilize the signaling scaffolds located at the centrosome. Chapter 5 of this thesis contains data about OSM and db-cAMP mediated polarity development in relation to PKA activity. Importantly, out data indicate that activity of PKA type I, but not of type II seems to play a crucial role in stimulatory effects on polarity of both OSM and db-cAMP. Moreover, we show that OSM signaling pathway does not directly affect cAMP/PKA cascade, but it seems that OSM requires PKA (type I) activity. Interestingly, we have observed that stimulation with OSM increases PKA-dependent phosphorylation of some protein substrates. Although this observation is still preliminary, we may speculate that PKA phosphorylate some protein substrates which can be important in the process of development of polarity in hepatocytes

Dissertations of the University of Groningen

   University of GroningenThe role of cAMP-dependent protein kinase A in bile canalicular plasma membranebiogenesis in hepatocytesWojtal, Kacper AndrzeIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Wojtal, K. A. (2007). The role of cAMP-dependent protein kinase A in bile canalicular plasma membranebiogenesis in hepatocytes. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 11-10-2022Acknowledgements  202I would like to thank my promoter Prof. Dick Hoekstra and co-promoter Dr. Sven C.D. van IJzendoorn for making it happen. I would like to thank also the members of the reading committee: Prof. Han Moshage, Prof. Edo Vellenga and Prof. Maikel Peppelenbosch for revision of this manuscript. I would like to thank also my paranimfs: Krzysztof and Andrzej for assisting me to the “last moment” ;)  I would like to thank Prof. J.D. Scott who kindly provided me with AKAP-IS and SCRAMBLE constructs, Dr. Hauri for the DPP IV antibody, Dr. J. Goldenring for the AKAP350 antibody, Dr. M. Vaughan for the BIG2 antibody, and Karin Klappe for the synthesis of C6-NBD-sphingolipids. Dr. E. Klussmann for AKAP7-derived peptide, Prof. H. Kampinga for -tubulin antibodies, Prof. F. Herberg and M. Diskar from University of Kassel for the successful collaboration and reagents. I would like to also thank Prof. J.J.L. van der Want and D. Kalicharan from the Section of Electron Microscopy for interesting collaboration.   I would like to thank the entire group of Membrane Cell Biology, also including former members: Dick Hoekstra, Sven van IJzendoorn, Wia Baron, Jan Willem Kok, Hans de Vries, Inge Zuhorn, Olaf Maier, Tiemen van der Heide, Joke van der Wouden, Joanna Rejman, Tounsia Aït Slimane, Sandrine Audouy, Donatienne Tyteca, Delphine Theard, Luc Wasungu, Zuzana Siskova, Anne-Jan Dijkhuis, Bert Klunder, Fuxin Shi, Magdalena Golachowska, John Hinrichs, Ina Hummel, Sai Uppalapati, Karin Klappe, Jenny Dallinga-de Jonge, Anita Nomden, Willy Wisser, Tini Roek, Gerry Hoogenberg and all the students who passed through the MCB during my stay there. Big thanks go to Jan Wijbenga for being a source of helpful information and for being a member of “the UMCG lunch team”. Special thanks to Volker Oberle, who was my supervisor during my student times.  I would like to acknowledge also the entire GUIDE office for helping me with forms and courses along the entire PhD programme.   203I would like to also acknowledge all my friends and colegues who made Groningen my second home: Danny for being a friend and for taking care of my physical condition, Delphine for the “shoulder”, friendship and for trying to keep the ZEN-thing around, Luc for being the office mate, for guitar sessions and all the spare time fun, Koen for the friendship and for introducing me to the wonderful world of Belgian beer, Annemieke for being a unique Dutch girlfriend and for getting me addicted to the indoor climbing, Saravanan for your everlasting enthusiasm, Zuzana for all the fun in the office and interesting discussion about Western culture, Magda for talking me into the scuba diving and Argentinian tango.  I would like to thank my polish friends and colleagues for being much more than only compatriots: Andrzej, Marta and Jakub Lulkowie, Krzysztof Rembacz, Joanna Adrian, Joanna Krzeslak, Magdalena Golachowska, Ewa Polek, Agnieszka Rarok, Jacek Lubelski and all the others.  Very special thanks go also to the Sunday Basketball Team: Saravanan, Erik, Tibor, Kaushal, Andrzej, Grzegorz, Orest, Peter, Anton, Jacek, Rafal, and the others.  I would like to give my credits to all the Bleekerslaaners in the international student house, who made my first days in Groningen truly unforgettable (Jan –Jul 2002).  Last but not least, I would like to acknowledge my parents and my family, who gave me all their support and attention during my stay in Groningen. Special thanks to Magdalena Obtulowicz.   204  

The role of cAMP-dependent protein kinase A in bile canalicular plasma membrane biogenesis in hepatocytes

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The role of cAMP-dependent protein kinase A in bile canalicular plasma membrane biogenesis in hepatocytes

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