Investigation of novel PKC targets in the regulation of autophagy
Kaleli, Hümeyra Nur (2019) Investigation of novel PKC targets in the regulation of autophagy. [Thesis]
Protein Kinase C (PKC) isozymes are serine/threonine kinases that are important for activation and/or inactivation of intracellular signaling pathways and therefore regulate cellular metabolism. Autophagy is a degradation mechanism functioning under basal conditions and activating under cellular stress including nutrient limitation, oxidative stress or abnormal protein accumulation. It is initiated by formation of double or multi-membrane vesicles in the cytoplasm. These vesicles engulf the cargo and carry it to the lysosome. After the fusion of autophagic vesicle with lysosomes, the cargo is degraded, and its constituents are recycled. The signaling pathways regulated by PKC isozymes are also involved in autophagy mechanism. However, the interaction between autophagy and PKC isozymes is still unclear. The aim of the study is to find novel proteins targeted by PKC isozymes during autophagy mechanism. For this aim, lentiviral shRNA library system was used for silencing of the genes in GFP-LC3 stably expressing mouse embryonic fibroblast (MEF) transgenic cells (MEF GFP-LC3). Upon activation of PKC isozymes, autophagic machinery was examined by GFP-LC3 puncta count, LC3 shift assay and p62 accumulation. Then the positive clones were selected, and their genomic DNA was isolated for target gene sequencing. The genes were identified with Sanger sequence analysis and their relationship with PKC isozymes were analyzed by using RT-q-PCR. Consequently, the role of target gene in the regulation of autophagy was determined by commonly used autophagy techniques.
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