Charakterisierung der molekularen Funktionen der WD-Repeat Proteine RACK1 und FAN und ihr Einfluss auf die TNF-vermittelte Signalweiterleitung
This thesis investigates the influence of the WD-Repeat proteins FAN (Factor associated with neutral Sphingomyelinase Activation) and RACK1 (Receptor for Activated C-Kinase 1) on TNF-Signalling. The structural resemblance of the protein CHS (Chediak-Higashi Symptom) to FAN, the mutated PKC (Protein-Kinase C) activity in CHS cells and the described interaction between RACK1 and FAN gave reason to take a closer look at the translocation and co-localization of RACK1 in murine cells after stimulation. No co-localization between RACK1 and PKC-beta II was seen in murine embryonic fibroblasts and murine NK-cells after PMA stimulation of PKC and antibody-incubation. After over-expression of PKC-beta-II-EGFP the translocation of PKC to the plasma membrane could be shown, but no co-localization of RACK1 was seen. TNF Stimulation did not seem to cause a change in the distribution of PKC or RACK1 no matter if FAN was present or not There was no unregulated phosphorylation of RACK1 after TNF-stimulation which is another indication that TNF-stimulation does not activate the PKC in murine fibroblasts. The interaction of RACK1 with Actin could be observed, after TNF-stimulation there is a different co-localization, this difference is not FAN dependent. The mapping of interacting domains of FAN and RACK1 as shown in the yeast-two-hybrid system and co-immunoprecipitation revealed the importance of WD7 of RACK1. In relation to preceding experiments of the group this shows the importance of WD 5-7 for the interaction of FAN and RACK1. Over expression of nSMase2 in murine Fibroblasts revealed a changed phenotype, but so far no hint of senescence could be observed. Interaction studies of FAN and nsMase2 using the yeast-two-hybrid system, co-immunoprecipitation and the laser scanning microscope showed no direct interaction of these two proteins in spite of the described influence of FAN on the activation of nSMase2. The missing link in this chain has yet to be found.
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