Skip to main content
Premium Trial:

Request an Annual Quote

Pawson Lab Uses Targeted Proteomics to Unravel Role of Shc1 in EGF Signaling

Premium

A team led by researchers at Toronto's Mount Sinai Hospital has completed a proteomic analysis of the scaffold protein Shc1 and its role in regulating EGF signaling.

The study, published this week in Nature, offers insights into the processes and protein interactions through which Shc1 modulates EGF network activity. It also provides a demonstration of targeted proteomics' potential as a tool for unraveling complex cellular processes.

As the authors note, a primary role of scaffold proteins like Shc1 is to enhance cell surface receptor protein signaling by providing "docking sites for downstream effectors." This, however, said Mount Sinai researcher Anthony Pawson, leader of the study, raises the question of why that function hasn't simply been taken over by the receptor protein itself.

"If [the scaffold] is just sort of an intermediate coupling the receptor to downstream targets," he noted, "why don't you just incorporate bits of the scaffold into the receptor and be done with it?"

To explore this question, the researchers combined affinity purification mass spectrometry with multiple-reaction monitoring, allowing them to characterize temporal changes in Shc1 phosphorylation status and protein-binding partners upon EGF stimulation.

Ultimately, they demonstrated that Shc1 goes through several distinct rounds of protein interactions and phosphorylation events that drive a shift in EGF signaling from an initial stage focused on cell survival and division, to an intermediate state involved in functions like vesicle trafficking, to a final point characterized by a focus on cytoskeletal organization and the termination of the original mitogenic signal.

"All of that makes [Shc1] a pretty complex beast," Pawson told ProteoMonitor. "It's not just a scaffold, but rather a dynamic entity that is mediating a series of signaling events downstream of the receptor dependent, to an extent, on a sort of feedback."

In addition, Pawson noted, the researchers found that the later rounds of interactions took place at different locations within the cell, "so there is probably a certain amount of spatial information as well as temporal information."

While scaffold proteins were known to be subject to feedback phosphorylation affecting their signaling properties, what "hadn't been appreciated," Pawson said, is “that there is a much wider range of associated proteins and that there is this sort of temporal aspect to the signaling so that the scaffold has sort of multiple functions."

The researchers arrived at their finding by combining high-resolution mass spec on an AB Sciex TripleTOF 5600 with MRM on an AB Sciex 5500 QTRAP, using the TripleTOF 5600 to perform mapping of Shc1 protein partners over time and following that with targeted MRM assays to quantify these proteins as well as Shc1 phosphorylation events.

Using MRM, Pawson and his colleagues quantified 41 Sch1 binding partners and Shc1 phosphorylation at 16 time points over the first 90 minutes after EGF stimulation, finding initial rapid phosphorylation of tyrosine phosphosites followed by later, separate waves of serine/threonine phosphorylation.

To attain high-quality quantitative data on the multiple analytes they were interested in monitoring, the researchers used AB Sciex's Scheduled MRM, or sMRM, feature, in which the mass spec schedules its cycles according to the LC elution times of the analytes being measured.

The technique provided a roughly three- to four-fold improvement in signal to noise, said Mount Sinai researcher Lorne Taylor, co-author on the study and leader of the project's mass spec efforts. However, he noted, the technique requires careful attention to all stages of the process – and the LC stability in particular – to make it work reproducibly.

"Anyone who does a lot of scheduled MRM lies wide awake at night worrying about their LC retention times," he told ProteoMonitor, adding that in the future, the researchers would likely try to switch over from nanoflow LC to a more robust standard flow approach. Similar methods are offered by other vendors including Agilent and Thermo Fisher Scientific.

In significant part, Pawson said, the success of the project hinged on the sensitivity and precision of the sMRM technology. "Even if we had been able to identify a bunch of binding proteins, that in itself wouldn't have been that significant," he said. "It was the ability to really sort of quantitate the precise dynamics" that provided the key insights.

Moving forward, Pawson said, the researchers plan to apply the mass spec workflows used in the Nature paper for similar studies of additional scaffold proteins. They are also interested in using it to investigate cancer signaling and response to various therapeutics.

In fact, Pawson noted, he and his team are currently collaborating with an unnamed industrial partner on research using their assays to profile signaling in breast cancer cells and changes in response to treatment with therapeutic antibodies.

The partnership is also looking at drug development, Taylor said.

"As signaling feedback loops become amplified or attenuated we may be able to find that using this sort of technology," he said. "Oncology drugs typically rewire cellular processes, and maybe you can see this rewiring happening and you might be able to use it to make a better [drug] or companion diagnostic."

"We don't know," he added, "but we hope [the methodology] has a lot of legs that we can apply to this."

Thus far, this cancer research has focused on analysis of cell lines. But, Pawson said, they are interested in applying it to actual clinical samples, as well.

A common question with regard to such efforts is whether large enough clinical samples will be available to perform the mass spec analysis with the required sensitivity. Taylor suggested that the heterogeneity of a clinical sample could offer challenges not presented by analyses of cell lines, but the workflow's sensitivity, he said, "is in the right ballpark for us to have enough clinical material."

The Scan

Shape of Them All

According to BBC News, researchers have developed a protein structure database that includes much of the human proteome.

For Flu and More

The Wall Street Journal reports that several vaccine developers are working on mRNA-based vaccines for influenza.

To Boost Women

China's Ministry of Science and Technology aims to boost the number of female researchers through a new policy, reports the South China Morning Post.

Science Papers Describe Approach to Predict Chemotherapeutic Response, Role of Transcriptional Noise

In Science this week: neural network to predict chemotherapeutic response in cancer patients, and more.