Browsing by Subject "Phosphoproteomics"
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Item Open Access Detection of phosphorylation signatures specific to cancer-related PI3-Kinase isoforms p110α and p110β(2023-01) Sulaiman, MahnoorThe PI3K signaling pathway is required for many physiological activities, but it is commonly disrupted during cancer formation. The PI3K p110α and βisoforms, encoded by the PIK3CA and PIK3CB genes, are lipid kinases that phosphorylate PIP2 to PIP3 to activate the PI3K pathway. However, the distinct molecular targets of these isoforms have yet to be discovered, making targeted treatment problematic. According to cancer genomics research, the PIK3CA gene is commonly altered in cancers, but the PIK3CB gene is frequently amplified. The clinical usage of Pan-PI3K inhibitors has resulted in significant side effects, prompting the development of isoform-specific inhibitors. However, it has been shown that these drugs trigger alternate signaling systems downstream, leading in resistance to single-agent treatment. Our research intends to uncover distinctive protein-protein interactions of PI3K isoforms, as well as the consequent different phospho-proteomic signatures, which might be crucial determinants of specific cellular activities. This will be accomplished by using isogenic MEF cells that are only dependent on the p110α or p110β isoforms, isoform-specific pharmacological inhibitors BYL-719 and KIN 193, and a high-resolution mass spectrometry-based method to determine the phosphorylation levels of these protein samples. The predictive biomarkers discovered in this study can be utilized to identify patients who will benefit from PI3K-targeted drugs and to better understand the resistance mechanisms that may arise in response to pathway inhibition.Item Open Access Phosphoproteomic analysis of class IA P110β isoform-specific signal transducers upon PI3K pathway activation(2023-08) Daloğlu, BerilPI3K pathway activation is a common event observed in various human cancers. As it regulates cell proliferation, migration and metabolism, it has been widely targeted for anticancer therapies and alteration of drug resistance. Studies focusing on PI3K Class IA isoform-specific inhibition has become critical to achieve alternative targeted treatment methods. Although previous findings show that Class IA isoforms differ in their routes of activation, the downstream targets specific to the isoforms have not been fully profiled yet. This study focuses on the phosphoproteomic analysis of p110 isoform-specific downstream molecules upon PI3K pathway activation. We have created a doxycycline-inducible system in Mouse Embryonic Fibroblasts (MEFs) to express wildtype PIK3CB gene encoding p110β or its catalytic and helical domain over-activating mutants, E1051K and N553S. By immunoblotting, the changes in downstream phosphorylation events were analyzed for MEF p110β mutants or for MEF p110β WT cells upon mitogenic PI3K stimulation. These results were collaborated with the comparative phosphoproteomic data obtained from LC-MS/MS Mass Spectrometry analysis. Our findings show that upon short-term stimulation of the PI3K pathway, proteins associated with transcriptional regulation, cytoskeletal rearrangement, cellular signalling, migration and metabolism are differentially phosphorylated. After longer stimulations on the other hand, proteins involved in cell cycle progression, phosphatase activity, nucleocytoplasmic transport and RNA metabolism become more prominent in the comparative phosphoproteome. Similar to early response proteins in β WT cells, the phosphoproteome of β E1051K cells were associated with cytoskeletal rearrangement and cellular migration. Aligning with the morphological changes observed, proteins involved in anatomical structure regulation were found. Additionally, some tumour suppressors and oncogene proteins were found among the differentially regulated phosphoproteins. On the other hand, the phosphoproteome of β N553S cells were enriched for functions of RNA metabolism, nuclear import and apoptotic regulation. Proteins involved in structural organization and microtubule assembly were also observed. 37 kDa Akt substrate Poly(rC) Binding Protein 1 (Pcbp1) was found as a common differentially phosphorylated protein in the phosphoproteomes of mutant and WT p110β expressing cells. It is an RNA-binding protein associated with metastasis and EMT, and has a critical role in gene expression by coordinating RNA stability and processing. As our results have shown that p110β isoform has a major role in cytoskeletal reorganization, cell migration and transcriptional regulation, studying the regulation of p110β-Pcbp1 axis can pave the way to understand the mechanisms of increased tumour progression in p110β-dependent cells.