Browsing by Subject "Protein Kinases"
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Item A MYC-PROTEIN KINASE A SIGNALING LOOP REGULATES MYC IN PROSTATE CANCER CELLS(2012-01-01) PADMANABHAN, ACHUTH; Bieberich, Charles J.; Biological Sciences; Biological SciencesMYC is frequently overexpressed in many human malignancies including prostate cancer. MYC accumulation is tightly regulated through transcriptional and post-translational mechanisms. Here, we demonstrate that a complex MYC/Protein Kinase A (PKA) signaling loop functions in prostate cancer cells. We show that MYC up-regulates Protein Kinase A Catalytic subunit � (PKAC�) transcription in human prostate cancer cells and mouse prostate cancers. We further demonstrate that PKA positively regulates MYC by phosphorylating Ser279, thereby priming phosphorylation by Polo-like kinase 1 at Ser281 and protecting MYC from degradation. Consistent with this, brief pharmacologic pan-PKA inhibition diminishes MYC level. Paradoxically however, prolonged Protein Kinase A Catalytic subunit ? (PKAC?), but not PKAC�, knockdown increases MYC mRNA and protein levels, linking transcriptional repression of MYC with PKAC? activity, and revealing distinct functional roles among PKA catalytic isoforms in MYC regulation. We demonstrate that this effect stems from differential substrate selectivity among PKA isoforms, providing a mechanistic understanding of differential MYC regulation by PKA. Our observations establish PKA as a viable therapeutic target in MYC-overexpressing cancers, but highlight the risk of global PKA inhibition, underscoring the need to develop advanced inhibition strategies to disrupt specific kinase/substrate interactions.Item PHOSPHOPROTEOMIC AND TRANSCRIPTOMIC-DRIVEN STUDY OF THE CELL WALL INTEGRITY SIGNALING PATHWAY (CWIS) IN THE MODEL FUNGUS, ASPERGILLUS NIDULANS(2020-01-20) Chelius, Cynthia Louise; Marten, Mark R; Chemical, Biochemical & Environmental Engineering; Engineering, Chemical and BiochemicalFilamentous fungi are industrially relevant and medically significant. In both cases the cell wall plays a critical role in protecting fungi from physical and environmental stress. To increase our general understanding of cell-wall repair, we focused on the model species Aspergillus nidulans studying the regulation of biosynthetic proteins responsible for wall maintenance and repair via the cell wall integrity signaling pathway (CWIS). The CWIS contains a mitogen-activated protein kinase (MAPK) cascade which becomes activated upon physical or environmental stress. MpkA, the final kinase in this cascade, controls expression of some key wall biosynthesis genes (?-1,3-glucan synthases). However, the transcription of many other wall related genes (e.g., ?-1-3-glucan and chitin synthase genes) is regulated in an unknown, MpkA-independent, manner. To better understand the various cellular roles MpkA plays during steady-state growth, we compared global changes in protein phosphorylation and gene expression between an mpkA deletion mutant (?mpkA) and its isogenic parent. We found strong evidence suggesting MpkA is involved in maintaining cell-wall strength, branching regulation, and response to iron starvation. Next, we sought to characterize the CWIS pathway and its downstream effectors. Because cellular signaling is a dynamic process, we used a multi-omic approach employing quantitative, label-free, mass spectrometry (short timeframe; 10 min) to assess protein phosphorylation and RNA-sequencing (long timeframe; 120 min) to assess gene-expression levels. To differentiate statistically-significant dynamic behavior from noise, a multivariate adaptive regression splines (MARS) model was applied to both data sets. Overall, we identified 794 phosphorylation sites and 1800 genes dynamically phosphorylated and expressed, respectively, upon cell wall perturbation. Several of these phosphorylation sites belong to kinases, and 12 of these kinases were tested for their involvement in CWIS using deletion strains to determine cell-wall strength and micafungin sensitivity. Combining -omic data with characterization studies reveals putative, new connections between the CWIS pathway and calcium signaling, the high osmolarity-glycerol (HOG) pathway, and the septation initiation network (SIN). This multi-omic experiment was replicated in the ?mpkA strain to develop hypotheses about which signaling events are MpkA-independent. This work demonstrates the capacity of a multi-omics approach to study signaling networks.