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UPenn Team Develops Method to Monitor Cellular Phosphorylation Pathways

NEW YORK (GenomeWeb) – A team of scientists from the University of Pennsylvania has developed a method to monitor cellular phosphorylation pathways into chromatin and down to the gene level.

The new method, called phosphorylation-specific chromatin affinity purification (PS-ChAP), is expected to enable researchers to better understand how phosphorylation signaling dynamically interacts with chromatin.

In addition, when coupled with qPCR, the method may help elucidate how phosphorylation signaling in chromatin modifies gene expression under different stimuli, according to a paper published last week in the journal of Molecular and Cellular Proteomics.

Although it was postulated 10 years ago in the journal Cell that phosphorylation signaling might have a direct effect on chromatin and associated nucleosomes, no significant breakthroughs have been made in the area. "All the normal [phosphoproteomics] methods don't work on the intact nucleus level," said Benjamin Garcia, associate professor of biochemistry and molecular biophysics at the University of Pennsylvania and lead author on the study.

Specifically, common phosphopeptide enrichment tools that target phosphate-containing peptides aren't effective in work with chromatin-associated nucleosomes.

Consequently, Garcia and his colleagues turned to a different approach, labeling the phosphorylation sites of interest with the ATP analog ATP γ-S, which enables protein thiophosphorylation. As the authors noted, thiophosphorylation is useful for the study of protein phosphorylation due to the fact that it is irreversible, meaning the phosphorylation site will be maintained. Additionally, because it is not native to the body, it can be distinguished from previously occurring phosphorylation events. Also, it can be chemically modified to allow for better large-scale enrichment.

To determine the ideal enrichment workflow for the thiophosphorylated peptides, the researchers evaluated a variety of methods, including immunoaffinity chromatography and titanium dioxide chromatography, before ultimately settling on a selective chemistry-based enrichment method using iodacetyl beads. Combining ATP-γ-S labeling of nuclei with this enrichment protocol, they established their PS-ChAP workflow, which, followed by mass spec analysis of the captured peptides on Thermo Fisher Scientific LTQ-Orbitrap Elite mass spectrometer, provided a way to look at the intricacies of phosphorylation cell signaling inside the nucleus. In an initial test of the method, they found they were able to detect more than 100 proteins from mononucleosomes, including a number of chromatin-associated proteins like histone variants, DNA-binding proteins, and transcription factors.

Having established the utility of the method in mononucleosomes, Garcia and his colleagues used it to look at phosphorylation changes in chromatin that result from various stimuli. Using the method with SILAC labeling they studied the effect of α-amanitin treatment and cell starvation in HeLa cells, identifying a number of phosphorylation sites that appeared linked to transcriptional activation, notably on histone H3.

They then used the method to look at EGF-signaling to investigate how EGF-regulated phosphorylation changes impact the cell at the chromatin level, finding, the authors wrote, that, “a large proportion of cellular proteins are phosphorylated on chromatin and only a small subset of total phosphorylation sites are highly enriched in response to stimulus."

This work also identified proteins phosphorylated on genes during EGF stimulation and, using this information, Garcia and his team analyzed the linked nucleosomal DNA to characterize the genes associated with these proteins.

Non-stimulated and EGF-stimulated cells underwent qPCR using standard procedures on a Thermo Fisher Applied Biosystems 7900HT Fast Real-Time PCR platform. "We designed primers [for qPCR] we isolated out after we simulated the EGF path approach," said Garcia.

Looking at genes associated with the observed protein phosphorylation, the researchers found "significant enrichment of EGF-induced kinases at the promoter regions of EGF-responsive genes during ERG stimulation." They also found that this enrichment disappeared when the EGF-induced transcriptional response was blocked.

All of the data was analyzed using pFind Studio 2.8. The data were searched against the human IPI database (version 3.87, 91,464 sequences).

Now that they have proved the efficacy of their method to isolate chromatin and monitor its phosphorylation signaling pathways, the researchers' next plan to merge the proteomics and genomics research in this area to be able to understand what’s happening to kinases, Garcia said. One of the ways to do this is to explore analogue methods to detect the kinases.

For instance, Garcia told GenomeWeb, Kevan Shokat from the University of California, San Francisco has developed kinases that have been engineered to accept an ATP analogue. If researchers feed this analogue into cells, it will be specifically labeled and it will be easier to link the activity of a single kinase to a substrate and then to a particular gene, he said.

The authors also noted that combining PS-ChAP with next-generation sequencing methods like ChIP sequencing could allow them to identify "kinase substrate target genes in an unbiased and more high-throughput manner."

All of the UPenn team's data are accessible on the ProteomeXchange Consortium with the data set identifier PXD002436.