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

Abstract

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