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University of Montreal Researchers Identify 120-plus New ERK1/2 Substrate Candidates

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Researchers at the University of Montreal have completed a phosphoproteomic analysis of ERK1/2 signaling that significantly expands the number of potential substrates of these proteins.

In a study published last week in Molecular Systems Biology, the researchers identified 155 candidate ERK1/2 substrates, including 128 new targets, making it the most comprehensive investigation of ERK1/2 substrates done to date, University of Montreal researcher Pierre Thibault, leader of the effort, told ProteoMonitor.

The analysis suggests a role for ERK1/2 in a wide variety of cellular functions, including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions, and cell signaling, he noted.

Dysregulation of ERK1/2 is involved in a number of diseases including cancer, inflammatory disorders, and neurodegenerative disease, Thibault said. "So we were very interested in identifying substrates targeted by this kinase."

Several previous studies have also tackled this question, but limited overlap between the substrates identified by these efforts suggests, he said, that these analyses were far from comprehensive. Additionally, the Montreal team aimed to collect information on the dynamic changes in substrate phosphorylation over time – an aspect not included in previous work, Thibault said.

To identify the substrates, the researchers used the inhibitor PD184352 to inhibit MEK1/2. ERK1/2 are the only known physiological substrates of MEK1/2, allowing the researchers to block ERK1/2 by inhibiting MEK1/2. The use of the PD184352 agent was key, Thibault noted, because at the 2-micromolar concentration used in the study it is highly specific to MEK1/2, limiting the extent to which it might interfere with the activity of other kinases present.

Using mass spec analysis on a Thermo Fisher Scientific LTQ-Orbitrap XL instrument, the researchers compared inhibitor-treated IEC-6 rat intestinal epithelial cells to controls in biological triplicate, measuring phosphopeptide levels at time points of 0, 5, 15, and 60 minutes and filtering the results for consensus ERK1/2 substrate motifs. Via this analysis, they identified 7,936 unique phosphorylation sites on 1,861 proteins and 2,296 high-confidence phosphosites on 987 proteins containing the ERK1/2 consensus motif.

Of these, they identified 155 candidate substrates – phosphopeptides whose abundance increased in response to stimulation with serum and decreased in response to MEK1/2 inhibition – a list that included 12 ERK1/2 substrates identified and validated in previous experiments. Using gene ontology analysis, the researchers implicated the candidate substrates in a variety of functions including regulation of transcription, nucleic acid metabolism, signal transduction, RNA processing, cytoskeleton organization, chromatin organization, and apoptosis.

To validate their results, the researchers performed in vitro kinase assays on six of the candidate substrates, finding that all were efficiently phosphorylated in vitro by activated ERK1.

They also followed up with a more in-depth analysis of the candidate substrate AP-1 transcription factor JunB. In a series of in vitro and in vivo experiments, they validated the Ser256 of JunB as a physiological target of ERK1/2 and found that inhibiting ERK1/2 signaling eliminated the transcriptional activity of JunB/c-Fos heterodimers, suggesting that phosphorylation of the Ser256 site is key to regulating JunB's role in transcription.

The Montreal researchers significantly expanded the roster of known ERK1/2 substrates, nearly doubling the 80 substrates identified in the next largest effort – a 2011 study by a Massachusetts Institute of Technology team published in Science Signaling.

Thibault said, though, that despite the breadth of the study's findings, the researchers had likely identified only a fraction of existing ERK1/2 substrates.

"I think we are far from saturation," he said, noting that the study was limited by the technical means at the team's disposal. For instance, he said, improved enrichment and sample-preparation techniques would likely add to the number of identified substrates.

"Efficiency enrichment is important to the comprehensiveness" of analysis, he said. "We take care in enriching our extracts to get to at least the 90 percent phosphopeptide enrichment level." The researchers used homemade titanium dioxide affinity columns for enrichment.

Improved sample prep, meanwhile, would enable higher resolution studies of the kinetics of protein phosphorylation, Thibault said.

"To understand the kinetics of phosphorylation requires comprehensiveness" in terms of time points, he said. "We selected time points at 0, 5, 15, [and] 60 minutes... but I suspect that probably in coming years with more sophisticated sample treatment we will be able to sample the phosphoproteome at one minute intervals."

The researchers stopped their collection of phosphorylation kinetics data at an hour in order to limit the affect of changes in general protein abundance on their phosphorylation data – an important consideration for such work, Thibault said.

When looking for "change in the phosphoproteome [you want] to actually identify changes that are only modulated by cell signaling – that are not affected by protein synthesis – because obviously we don't want to confuse change in phosphorylation with change in protein abundance, for example," he said.

Most significantly, advances in mass spec will likely enable future research efforts to add to the list of ERK1/2 substrates, Thibault said.

"For instance, this [study] was done using a [Thermo Scientific] LTQ-Orbitrap XL," he said. "Now we can use the [Thermo Scientific] Orbitrap Elite, which is about 10 times more sensitive and dig to a deeper level in the phosphoproteome to make more identifications."

"I expect that within the next five years we will see the number of substrates expand significantly because of the technical advances" in mass spectrometry, he added.

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