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Regulatory Proteins Affect Signaling in Cancer-Associated EGFR-MAPK Pathway

NEW YORK (GenomeWeb) – Mutations in the cancer-associated epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway lead to variations in the amount of regulatory adaptor proteins, rather than an increase in the main EGFR-MAPK pathway proteins themselves, according to a new study.

As they wrote in Science Signalling yesterday, researchers led by first author Tujin Shi and senior author Steven Wiley of the Pacific Northwest National Laboratory quantified protein abundance using mass spectrometry-based proteomics in both cancer and normal cell lines.

"We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species," they said in their paper. "In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations."

The team found core pathway proteins in amounts of between 50,000 to 70,000 copies per cell, and adaptor proteins — such as SOS1, SOS2, and GAB1 — in amounts of an order of magnitude lower. These regulatory proteins can modify the activity of the EGFR-MAPK pathway by forming complexes or performing posttranslational modifications, the authors said.

"Differences in the abundance of pathway regulators could have a marked effect on signaling, but identifying which are functionally active in a specific cell type is rarely done," they wrote. "There are also technological problems in reliably quantifying protein abundance in cells."

The study eschewed antibody assays, but mass spec-based approaches did not guarantee the authors would be able to detect the low levels of adaptor proteins. The team noted that the accuracy for quantifying proteins can be improved with selected reaction monitoring and heavy isotope labels for standard peptides, but using those techniques requires prior knowledge of which proteins in the pathway are considered important.

To get around this problem, the PNNL researchers first characterized the EGFR-MAPK pathway in a model cell line, identifying the core pathway proteins as well as the regulatory proteins with transcriptional profiling and shotgun proteomics. Then, using RNA sequencing and targeted proteomics, they quantified protein abundance across the different cell lines.

"We initiated this study to determine whether cancer cells displayed quantitative differences in the abundance of proteins that could dysregulate the EGFR-MAPK pathway," the authors wrote. "Our results suggest that the relative abundance and stoichiometry of most core EGFR-MAPK pathway proteins are highly conserved, indicating that relative abundance and stoichiometry are important for pathway function. Variability in receptor abundance and the feedback regulators that modulate the activity of core pathway proteins, rather than differences in expression of the core proteins themselves, is most likely responsible for cell type–specific responses to EGF."