Browsing by Subject "Kinase substrate screening"

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    Elucidating the Role of Protein Kinase D in Osteoclasts
    (2022-11) Kazemi, Parandis
    The Protein Kinase D (PRKD) family of serine/threonine kinases have been shown to contribute to a complex set of biological functions such as signal transduction, cell proliferation, differentiation, cell vesicle trafficking, secretion, polarity, and locomotion. Our lab has demonstrated that PRKD affects multiple aspects of osteoclast formation, such as cell-to-cell fusion, migration, and differentiation. Nevertheless, the specific targets of PRKD phosphorylation in osteoclasts remain poorly characterized. In this thesis, we aim to identify the targets of PRKD in osteoclast cell culture and to study the network effect of dephosphorylating those identified targets in osteoclasts. We utilized two complementary phospho-proteomic approaches: 1) Quantitative phospho-proteomics approach to establish a global view of changes in thousands of phospho-sites upon PRKD inhibition, followed by bioinformatic analysis of the functional enrichment clusters and phospho-motif enrichment analysis to identify candidate targets, 2) Targeted phospho-proteomics using PRKD recombinant kinase, followed by bioinformatic analysis of the phospho-motif enrichment and assessment of candidate targets in the context of known cellular signaling. Using the quantitative proteomics analysis of the PRKD inhibitor-treated osteoclasts cell culture, we showed that the main cellular pathways influenced by PRKD inhibition are mRNA processing, transcription, and regulation of transcription, which corresponded to the candidate targets: SRRM2, HDAC5, and JUND. Additionally, by using the targeted phospho-proteomics method, we identified two novel PRKD targets: SIPA1L3 and LSP1 which are associated with the actin cytoskeleton and cell membrane and contribute to cell motility. Subsequent validations of these findings would play an important role in better understanding the cellular regulators of osteoclast formation and bone resorption and establishing new therapeutic modalities to limit bone resorption in large numbers of patients afflicted with osteolytic conditions.