Researchers at Milan's Institute of Molecular Oncology Foundation have done a proteomic analysis aimed at characterizing the effects of epidermal growth factor regulation on protein ubiquitination.
The study, which was published last month in the online edition of Molecular Systems Biology, showed that EGF signaling impacts ubiquitination across a wide range of protein networks and is one of the first to demonstrate cross-talk between ubiquitin-regulated and phospho-regulated signaling pathways, Simona Polo, an IFOM researcher and one of the paper's authors, told ProteoMonitor.
Ubiquitination is a post-translational modification involving the covalent attachment of one or more ubiquitin monomers or chains to target proteins. While the process has typically been tied to protein degradation, in recent years the function of ubiquitination in protein signaling has come to the fore, with mono-ubiquitination and the attachment of K63 ubiquitin chains being tied to this role.
Like phosphorylation, ubiquitination is initiated by and can be reversed by enzymes – E3 ligases and DUBs – suggesting, Polo said, that the modification "has the potential to form a network with signaling-related function."
"As with phosphorylation pathways, we thought that perhaps the identification of the substrates that are modified by ubiquitin could help us understand the entire network," she said.
The starting point for the IFOM study was an earlier discovery by Polo's team that "the EGF receptor is multiple mono-ubiquitinated and not ubiquitinated for degradation as thought before," she said. "[It] is actually modified by ubiquitin in a way that seems to be more important in the signaling network than for degradation."
To investigate the effects of EGF stimulation on ubiquitination the scientists compared the levels of ubiquitination in normal HeLa and B82-EGFR cells with that of cells that had undergone EGF stimulation. This analysis, which was done in the lab of Max Planck researcher Matthias Mann using SILAC labeling and high-resolution, high accuracy mass spec on a Thermo Fisher LTQ Orbitrap machine, identified 265 proteins – out of a total of 1,472 ubiquitinated proteins – whose regulation appeared to be regulated by activated EGFR.
Among the proteins regulated were a large number of ubiquitin effector enzymes, which suggests EGFR activation has a broad effect on the activity of the ubiquitin signaling machinery. The study similarly implicated EGFR activation in the ubiquitination of proteins key to a variety of aspects of cellular physiology like DNA repair, nuclear transport, mRNA processing, and ribosome biogenesis, as well as in ubiquitination involved in endocytic and cell signaling processes like intracellular signaling circuitries, cell-to-cell and cell-to-substrate adhesion, and actin remodeling.
As a proof of concept, the researchers examined the functional link between EGFR and EphA2 – a protein tied to development and often overexpressed in cancer and whose ubiquitination levels were affected by EGF stimulation. Knockdown of EphA2 significantly reduced EGF-induced cell proliferation and migration, suggesting that the protein is critically involved in these EGRF-associated processes and providing an example, Polo said, "of a specific target that we found downstream that has a profound effect on the biology of the EGF receptor."
That the study found ubiquitination to be such a wide-ranging process fits with past research on the modification, James Brownell, a scientific fellow at drugmaker Millennium Pharmaceuticals, told ProteoMonitor, saying that "at this point we've seen so many new aspects of what ubiquitin can do functionally that I suppose it's not much of a surprise. This is a fundamental signaling pathway. It is involved in virtually every biological pathway."
The IFOM study, though, was the first he'd come across attempting "to understand how a well-described signaling pathway that's driven by tyrosine phosphorylation interacts with the ubiquitin pathway," he noted.
"Traditionally the [ubiquitin] pathway was discovered as a [protein] degradation pathway, and that has been the overwhelming mindset of the research up until just the last couple of years," Brownell said.
The recent development by Genentech of antibodies capable of distinguishing K63 ubiquitinated proteins with high resolution have allowed people to investigate the modification's signaling function more easily than was previously possible, he said. A mass spec analysis by a group of Emory researchers published last month in the Journal of Biological Chemistry found that K63 chains comprise over a third of the ubiquitin linkages in mammalian cell lysate.
Within the pharmaceutical industry there is "rapidly growing interest in trying to figure out how to target specific aspects of the ubiquitin pathway," Brownell said. However, he noted, "the devil is in the details. Clearly there are disease-relevant targets that are modulated by K63 ubiquitination. But how do you go about selectively targeting those pathways?"
Ubiquitin's pervasiveness made study of the modification difficult, Polo said, because of fears that changing ubiquitination levels too much within a cell would make it impossible to get an unbiased look at the system.
"It's important not to disturb the system too much because ubiquitin homeostasis is very important to the entire cell," she said. "So whatever you change [in terms of ubiquitination levels] will affect the cell behavior and then your results won't be physiologically relevant."
Better enrichment tools are needed, Polo said, adding that because of these limitations it's unlikely that the study captured the complete EGF-induced ubiproteome.
"I'm pretty sure that our screening isn't complete," she said. "In the future when we have more tools available we will be able to add more proteins to this network for sure."
Looking forward, the researchers are planning to perform similar analysis of different signaling events, Polo noted.
"In principle we can compare the EGF-ubiproteome with the DNA-damage-ubiproteome and see what proteins are different and move ahead with the proteomics in that way," she said.
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