Monitoring Cellular Phosphorylation Signaling Pathways into Chromatin and Down to the Gene Level

Abstract

Protein phosphorylation, one of the most common and important modifications of acute and reversible regulation of protein function, plays a dominant role in almost all cellular processes. These signaling events regulate cellular responses, including proliferation, differentiation, metabolism, survival, and apoptosis. Several studies have been successfully used to identify phosphorylated proteins and dynamic changes in phosphorylation status after stimulation. Nevertheless, it is still rather difficult to elucidate precise complex phosphorylation signaling pathways. In particular, how signal transduction pathways directly communicate from the outer cell surface through cytoplasmic space and then directly into chromatin networks to change the transcriptional and epigenetic landscape remains poorly understood. Here, we describe the optimization and comparison of methods based on thiophosphorylation affinity enrichment, which can be utilized to monitor phosphorylation signaling into chromatin by isolation of phosphoprotein containing nucleosomes, a method we term phosphorylation-specific chromatin affinity purification (PS-ChAP). We utilized this PS-ChAP 1 approach in combination with quantitative proteomics to identify changes in the phosphorylation status of chromatin-bound proteins on nucleosomes following perturbation of transcriptional processes. We also demonstrate that this method can be employed to map phosphoprotein signaling into chromatin containing nucleosomes through identifying the genes those phosphorylated proteins are found on via thiophosphate PS-ChAP-qPCR. Thus, our results showed that PS-ChAP offers a new strategy for studying cellular signaling and chromatin biology, allowing us to directly and comprehensively investigate phosphorylation signaling into chromatin to investigate if these pathways are involved in altering gene expression. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD002436. Molecular & Cellular Proteomics

Glycosylation and other PTMs alterations in neurodegenerative diseases: Current status and future role in neurotrauma

“…Approximately 80% of mammalian proteins are modified posttranslationally. For instance, phosphorylation is implicated in cell signaling , acetylation in epigenetics and NDs , ubiquitination in regulation of cellular protein homeostasis , hydroxylation in collagen formation , and glycosylation in the pathophysiology of neurotrauma . The basic findings from studies that demonstrated the role of PTMs in the relevant diseases are summarized in Table .…”

The Contribution of Mass Spectrometry-Based Proteomics to Understanding Epigenetics

“…Although very promising, ChEP also has limitations, such as the inability to follow local changes in the protein abundance as a consequence of protein re-localization in specific chromatin regions and the fact that not all chromatin components may be fixed by formaldehyde. Chromatin constituents can also be characterized at the PTM level, as recently done by the phosphorylation specific-chromatin affinity purification (PS-ChAP) to monitor phosphorylation changes following activation or inhibition of global transcriptional patterns [75].…”

“…Chromatin constituents Mononucleosome and thiophosphorylation affinity enrichment [75] Peptide pull-down Chromatin readers Bait: peptides bearing a specific hPTM/sequence DNA reader affinity purification Chromatin readers Bait: DNA sequences [85,86] mChIP (modified chromatin immunoprecipitation)…”