Identifikace nových substrátů kinázy PKN3 a charakterizace úlohy fosforylace v regulaci aktivity Rho GAP proteinů

Dibus, Michal

Protein phosphorylation represents one of the most important posttranslational modifications in signal transduction and plays a crucial role in regulation of most of the cellular processes including cell cycle, communication with extracellular environment, cell migration or apoptosis. Phosphorylation is mediated by protein kinases, deregulation of which often negatively affects development and overall homeostasis and leads to development of several diseases, including cancer. In the first part of this work we focused on identification of new substrates of PKN3 kinase, which is a known player in regulation of cytoskeletal organization and pro-malignant tumor growth. Using an analog-sensitive mutant of PKN3 we performed a phosphoproteomic screen and identified 281 proteins that could potentially be phosphorylated by PKN3. Among these, we selected ARHGAP18, a protein from Rho GAP family, for further study. We confirmed PKN3 is able to phosphorylate ARHGAP18 on Thr154, Ser156 and Thr158 and that the two proteins are able to interact with one another in an ARHGAP18 isoform-specific manner. We further showed that substitution of the three candidate sites for phosphomimicking aspartate led to the activation of ARHGAP18 GAP domain which resulted in decreased levels of active RhoA, suggesting the existence...

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